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Sample records for delta-opioid receptor endocytosis

  1. Structure of the [delta]-opioid receptor bound to naltrindole

    SciTech Connect

    Granier, Sébastien; Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Weis, William I.; Kobilka, Brian K.

    2012-07-11

    The opioid receptor family comprises three members, the {mu}-, {delta}- and {kappa}-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The {delta}-opioid receptor ({delta}-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the {mu}-OR and {kappa}-OR have recently been solved. Here we report the crystal structure of the mouse {delta}-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the {mu}-OR and {kappa}-OR, the {delta}-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the {delta}-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

  2. Effects of imipramine treatment on delta-opioid receptors of the rat brain cortex and striatum.

    PubMed

    Varona, Adolfo; Gil, Javier; Saracibar, Gonzalo; Maza, Jose Luis; Echevarria, Enrique; Irazusta, Jon

    2003-01-01

    Imipramine (CAS 113-52-0) is being utilized widely for the treatment of major depression. In recent years, there has been evidence of the involvement of the endogenous opioid system in major depression and its treatment. There is some evidence indicating that opioid receptors could be involved in the antidepressant mechanism of action. Regarding this topic, mood-related behavior of endogenous enkephalins seems to be mediated by delta-opioid receptors. In this work, the effects of subacute (5 day) and chronic (15 day) treatments of imipramine on the density and the affinity of the delta-receptors in the striatum and in the parietal and frontal cortices of the rat brain are described. Studied parameters (Bmax and Kd) were calculated by a saturation binding assay with the delta-opioid agonists [3H]-DPDPE (tyrosyl-2,6-3H(N)-(2-D-penicillamine-5-D-penicillamine)-enkephalin) as specific ligand and DSLET ([D-serine2]-D-leucine-enkephalin-threonine) as non-radioactive competing ligand. It was found that 15 days treatment significantly decreased the delta-opioid receptor density,without changing the affinity, in the frontal cortex of the rat brain. That decrease was confirmed by delta-opioid receptor immunostaining. These results suggest that delta-opioid receptors could play a role in the chronic action mechanism of imipramine. PMID:12608010

  3. Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors.

    PubMed

    Chaturvedi, K; Bandari, P; Chinen, N; Howells, R D

    2001-04-13

    This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors. PMID:11152677

  4. Interaction of co-expressed mu- and delta-opioid receptors in transfected rat pituitary GH(3) cells.

    PubMed

    Martin, N A; Prather, P L

    2001-04-01

    mu- and delta-Opioid agonists interact in a synergistic manner to produce analgesia in several animal models. Additionally, receptor binding studies using membranes derived from brain tissue indicate that interactions between mu- and delta-opioid receptors might be responsible for the observation of multiple opioid receptor subtypes. To examine potential interactions between mu- and delta-opioid receptors, we examined receptor binding and functional characteristics of mu-, delta-, or both mu- and delta-opioid receptors stably transfected in rat pituitary GH(3) cells (GH(3)MOR, GH(3)DOR, and GH(3)MORDOR, respectively). Saturation and competition binding experiments revealed that coexpression of mu- and delta-opioid receptors resulted in the appearance of multiple affinity states for mu- but not delta-opioid receptors. Additionally, coadministration of selective mu- and delta-opioid agonists in GH(3)MORDOR cells resulted in a synergistic competition with [(3)H][D-Pen(2,5)]enkephalin (DPDPE) for delta-opioid receptors. Finally, when equally effective concentrations of [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) and two different delta-opioid agonists (DPDPE or 2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydroquinolino-[2,3,3-g]-isoquinoline; TAN67) were coadministered in GH(3)MORDOR cells, a synergistic inhibition of adenylyl cyclase activity was observed. These results strongly suggest that cotransfection of mu- and delta-opioid receptors alters the binding and functional characteristics of the receptors. Therefore, we propose that the simultaneous exposure of GH(3)MORDOR cells to selective mu- and delta-opioid agonists produces an interaction between receptors resulting in enhanced receptor binding. This effect is translated into an augmented ability of these agonists to inhibit adenylyl cyclase activity. Similar interactions occurring in neurons that express both mu- and delta-opioid receptors could explain observations of multiple

  5. Delta opioid receptors presynaptically regulate cutaneous mechanosensory neuron input to the spinal cord dorsal horn.

    PubMed

    Bardoni, Rita; Tawfik, Vivianne L; Wang, Dong; François, Amaury; Solorzano, Carlos; Shuster, Scott A; Choudhury, Papiya; Betelli, Chiara; Cassidy, Colleen; Smith, Kristen; de Nooij, Joriene C; Mennicken, Françoise; O'Donnell, Dajan; Kieffer, Brigitte L; Woodbury, C Jeffrey; Basbaum, Allan I; MacDermott, Amy B; Scherrer, Grégory

    2014-03-19

    Cutaneous mechanosensory neurons detect mechanical stimuli that generate touch and pain sensation. Although opioids are generally associated only with the control of pain, here we report that the opioid system in fact broadly regulates cutaneous mechanosensation, including touch. This function is predominantly subserved by the delta opioid receptor (DOR), which is expressed by myelinated mechanoreceptors that form Meissner corpuscles, Merkel cell-neurite complexes, and circumferential hair follicle endings. These afferents also include a small population of CGRP-expressing myelinated nociceptors that we now identify as the somatosensory neurons that coexpress mu and delta opioid receptors. We further demonstrate that DOR activation at the central terminals of myelinated mechanoreceptors depresses synaptic input to the spinal dorsal horn, via the inhibition of voltage-gated calcium channels. Collectively our results uncover a molecular mechanism by which opioids modulate cutaneous mechanosensation and provide a rationale for targeting DOR to alleviate injury-induced mechanical hypersensitivity. PMID:24583022

  6. Delta Opioid Receptors Presynaptically Regulate Cutaneous Mechanosensory Neuron Input to the Spinal Cord Dorsal Horn

    PubMed Central

    Bardoni, Rita; Tawfik, Vivianne L.; Wang, Dong; François, Amaury; Solorzano, Carlos; Shuster, Scott A.; Choudhury, Papiya; Betelli, Chiara; Cassidy, Colleen; Smith, Kristen; de Nooij, Joriene C.; Mennicken, Françoise; O’Donnell, Dajan; Kieffer, Brigitte L.; Woodbury, C. Jeffrey; Basbaum, Allan I.; MacDermott, Amy B.; Scherrer, Grégory

    2014-01-01

    SUMMARY Cutaneous mechanosensory neurons detect mechanical stimuli that generate touch and pain sensation. Although opioids are generally associated only with the control of pain, here we report that the opioid system in fact broadly regulates cutaneous mechanosensation, including touch. This function is predominantly subserved by the delta opioid receptor (DOR), which is expressed by myelinated mechanoreceptors that form Meissner corpuscles, Merkel cell-neurite complexes, and circumferential hair follicle endings. These afferents also include a small population of CGRP-expressing myelinated nociceptors that we now identify as the somatosensory neurons that coexpress mu and delta opioid receptors. We further demonstrate that DOR activation at the central terminals of myelinated mechanoreceptors depresses synaptic input to the spinal dorsal horn, via the inhibition of voltage-gated calcium channels. Collectively our results uncover a molecular mechanism by which opioids modulate cutaneous mechanosensation and provide a rationale for targeting DOR to alleviate injury-induced mechanical hypersensitivity. PMID:24583022

  7. Autoradiographic localization of mu and delta opioid receptors in the mesocorticolimbic dopamine system

    SciTech Connect

    Dilts, R.P. Jr.

    1989-01-01

    In vitro autoradiographic techniques were coupled with selective chemical lesions of the A10 dopamine cells and intrinsic perikarya of the region to delineate the anatomical localization of mu and delta opioid receptors, as well as, neurotensin receptors. Mu opioid receptors were labeled with {sup 125}I-DAGO. Delta receptors were labeled with {sup 125}I-DPDPE. Neurotensin receptors were labeled with {sup 125}I-NT3. Unilateral lesions of the dopamine perikarya were produced by injections of 6-OHDA administered in the ventral mesencephalon. Unilateral lesions of intrinsic perikarya were induced by injections of quinolinic acid in to the A10 dopamine cell region. Unilateral lesions produced with 6-OHDA resulted in the loss of neurotensin receptors in the A10 region and within the terminal fields. Mu opioid receptors were unaffected by this treatment, but delta opioid receptors increased in the contralateral striatum and nucleus accumbens following 6-OHDA administration. Quinolinic acid produced a reduction of mu opioid receptors within the A10 region with a concomitant reduction in neurotensin receptors in both the cell body region and terminal fields. These results are consistent with a variety of biochemical and behavioral data which suggest the indirect modulation of dopamine transmission by the opioids. In contrast these results strongly indicate a direct modulation of the mesolimbic dopamine system by neurotensin.

  8. Delta Opioid Receptors: The Link between Exercise and Cardioprotection

    PubMed Central

    Borges, Juliana P.; Verdoorn, Karine S.; Daliry, Anissa; Powers, Scott K.; Ortenzi, Victor H.; Fortunato, Rodrigo S.; Tibiriçá, Eduardo; Lessa, Marcos Adriano

    2014-01-01

    This study investigated the role of opioid receptor (OR) subtypes as a mechanism by which endurance exercise promotes cardioprotection against myocardial ischemia-reperfusion (IR) injury. Wistar rats were randomly divided into one of seven experimental groups: 1) control; 2) exercise-trained; 3) exercise-trained plus a non-selective OR antagonist; 4) control sham; 5) exercise-trained plus a kappa OR antagonist; 6) exercise-trained plus a delta OR antagonist; and 7) exercise-trained plus a mu OR antagonist. The exercised animals underwent 4 consecutive days of treadmill training (60 min/day at ∼70% of maximal oxygen consumption). All groups except the sham group were exposed to an in vivo myocardial IR insult, and the myocardial infarct size (IS) was determined histologically. Myocardial capillary density, OR subtype expression, heat shock protein 72 (HSP72) expression, and antioxidant enzyme activity were measured in the hearts of both the exercised and control groups. Exercise training significantly reduced the myocardial IS by approximately 34%. Pharmacological blockade of the kappa or mu OR subtypes did not blunt exercise-induced cardioprotection against IR-mediated infarction, whereas treatment of animals with a non-selective OR antagonist or a delta OR antagonist abolished exercise-induced cardioprotection. Exercise training enhanced the activities of myocardial superoxide dismutase (SOD) and catalase but did not increase the left ventricular capillary density or the mRNA levels of HSP72, SOD, and catalase. In addition, exercise significantly reduced the protein expression of kappa and delta ORs in the heart by 44% and 37%, respectively. Together, these results indicate that ORs contribute to the cardioprotection conferred by endurance exercise, with the delta OR subtype playing a key role in this response. PMID:25415192

  9. The pharmacological profile of delta opioid receptor ligands, (+) and (-) TAN-67 on pain modulation.

    PubMed

    Nagase, H; Yajima, Y; Fujii, H; Kawamura, K; Narita, M; Kamei, J; Suzuki, T

    2001-04-01

    We designed the nonpeptidic highly selective delta opioid receptor agonist on the basis of message address concept and the accessory site theory and synthesized (+/-) TAN-67. In spite of highly potent agonistic activity in in vitro assay, (+/-) TAN-67 (racemate) afforded a weak antinociceptive effect in the mouse tail-flick test. This result led us to separate (+/-) TAN-67 to optical pure compounds, (+) and (-) TAN-67. An i.t.-treatment with (-) TAN-67 produced profound antinociceptive effects through specifically acting on delta1 receptors. Unlike (-) TAN-67, i.t.-administered (+) TAN-67 displayed dose-related nociceptive behaviors such as scratching, biting and licking. The effect of (+) TAN-67 was blocked by i.t.-treatment with NTI (delta receptor antagonist) and (-) TAN-67 (delta1 receptor agonist), but not by morphine (mu receptor agonist). The mechanisms involved in spinal pain modulation induced by (+) and (-) TAN-67 were also described. PMID:11358331

  10. Effect of delta opioid receptor activation on spatial cognition and neurogenesis in cerebral ischemic rats.

    PubMed

    Wang, Shu-Yan; Duan, Ya-Le; Zhao, Bing; Wang, Xiang-Rui; Zhao, Zheng; Zhang, Guang-Ming

    2016-05-01

    This study aimed to investigate whether a selective delta opioid receptor agonist, [D-Ala2, D-Leu5]-Enkephalin (DADLE), regulates neurogenesis in the hippocampus of ischemic rats. Using an intracerebral cannula, rats were subjected to cerebral ischemia using the standard four-vessel occlusion. DADLE (2.5nmol), DADLE (2.5nmol) with naltrindole (NAL) (2.5nmol), or vehicle was administered at the onset of reperfusion. Bromodeoxyuridine (BrdU, 100mg/kg, intraperitoneal) was used to label newly formed cells from days 1 to 7 after ischemia. Immunohistochemistry was used to evaluate cell proliferation and apoptosis and differentiation 7days 28 days, respectively, after ischemia. Morris water maze test was conducted to test spatial learning and memory 23-27 days after ischemia. We found that DADLE treatment improved performance in the Morris water maze test, promoted proliferation and differentiation of newly formed neurons, and inhibited differentiation into astrocytes in a rat model of cerebral ischemia. Furthermore, the protective effects of DADLE were significantly reversed by co-administration of NAL (P<0.05), a highly potent and selective delta opioid receptor antagonist. Our findings suggest that DADLE promotes spatial cognitive function recovery and regulates neurogenesis after ischemia, which may provide a promising therapeutic strategy for cerebral ischemia. PMID:27016387

  11. Differential desensitization of mu- and delta- opioid receptors in selected neural pathways following chronic morphine treatment.

    PubMed Central

    Noble, F.; Cox, B. M.

    1996-01-01

    1. Morphine produces a plethora of pharmacological effects and its chronic administration induces several side-effects. The cellular mechanisms by which opiates induce these side-effects are not fully understood. Several studies suggest that regulation of adenylyl cyclase activity by opioids and other transmitters plays an important role in the control of neural function. 2. The aim of this study was to evaluate desensitization of mu- and delta- opioid receptors, defined as a reduced ability of opioid agonists to inhibit adenylyl cyclase activity, in four different brain structures known to be involved in opiate drug actions: caudate putamen, nucleus accumbens, thalamus and periaqueductal gray (PAG). Opiate regulation of adenylyl cyclase in these regions has been studied in control and morphine-dependent rats. 3. The chronic morphine treatment used in the present study (subcutaneous administration of 15.4 mg morphine/rat/day for 6 days via osmotic pump) induced significant physical dependence as indicated by naloxone-precipitated withdrawal symptoms. 4. Basal adenylyl cyclase in the four brain regions was not modified by this chronic morphine treatment. In the PAG and the thalamus, a desensitization of mu- and delta-opioid receptors was observed, characterized by a reduced ability of Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO; mu), Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE; delta) and [D-Ala2]-deltorphin-II (DT-II; delta) to inhibit adenylyl cyclase, activity following chronic morphine treatment. 5. The opioid receptor desensitization in PAG and thalamus appeared to be heterologous since the metabotropic glutamate receptor agonists, L-AP4 and glutamate, and the 5-hydroxytryptamine (5-HT)1A receptor agonist, R(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), also showed reduced inhibition of adenylyl cyclase activity following chronic morphine treatment. 6. In the nucleus accumbens and the caudate putamen, desensitization of delta-opioid receptor

  12. RAPID HETEROLOGOUS DESENSITIZATION OF ANTINOCICEPTIVE ACTIVITY BETWEEN MU OR DELTA OPIOID RECEPTORS AND CHEMOKINE RECEPTORS IN RATS

    PubMed Central

    Chen, Xiaohong; Geller, Ellen B.; Rogers, Thomas J.; Adler, Martin W.

    2007-01-01

    Previous studies have shown pretreatment with chemokines CCL5/RANTES (100 ng) or CXCL12/SDF-1alpha (100 ng) injected into the periaqueductal grey (PAG) region of the brain, 30 minutes (min) before the mu opioid agonist DAMGO (400 ng), blocked the antinociception induced by DAMGO in the in vivo cold water tail-flick (CWT) antinociceptive test in rats. In the present experiments, we tested whether the action of other agonists at mu and delta opioid receptors is blocked when CCL5/RANTES or CXCL12/SDF-1alpha is administered into the PAG 30 min before, or co-administered with, opioid agonists in the CWT assay. The results showed that (1) CXCL12/SDF-1alpha (100 ng, PAG) or CCL5/RANTES (100 ng, PAG), given 30 min before the opioid agonist morphine, or selective delta opioid receptor agonist DPDPE, blocked the antinociceptive effect of these drugs; (2) CXCL12/SDF-1alpha (100 ng, PAG) or CCL5/RANTES (100 ng, PAG), injected at the same time as DAMGO or DPDPE, significantly reduced the antinociceptive effect induced by these drugs. These results demonstrate that the heterologous desensitization is rapid between the mu or delta opioid receptors and either CCL5/RANTES receptor CCR5 or CXCL12/SDF-1alpha receptor CXCR4 in vivo, but the effect is greater if the chemokine is administered before the opioid. PMID:17049756

  13. Potent cyclic enkephalin analogues for delta opioid receptors in the rat brain

    SciTech Connect

    Lui, G.; Kao, J.; Hruby, V.; Morelli, M.; Gulya, K.; Yamamura, H.I.

    1986-03-01

    (/sup 3/H) (D-Pen/sup 2/,D-Pen/sup 5/) enkephalin ((/sup 3/H)DPDPE) and (/sup 3/H) (D-Pen/sup 2/, L-Pen/sup 5/) enkephalin ((/sup 3/H)DPLPE) characterization studies showed high affinity binding of these radioligands to rat brain membranes with dissociation constants of 1.8 and 1.0 nM, respectively, while a similar number of receptor density was found with both radiolabeled ligands (77 fmoles/mg protein). Unlabeled DPDPE inhibited both radioligands with high affinity (IC50 = 7 nM0 while morphine (IC50 = 80 nM), DAGO (IC50 = 250 nM) and PLO17 (no inhibition at 1000 nM) were less effective in inhibiting the binding, thus, illustrating the selective action of these radiolabeled ligands at the delta opioid receptor. A series of conformationally restricted D-penicillamine containing cyclic enkephalin analogues were synthesized using standard solid phase methods and their ability to inhibit (/sup 3/H)DPDPE and (/sup 3/H)DPLPE were examined in rat brain radioreceptor assays. Substitutions in the DPDPE molecule were made in phe/sup 4/. These substitutions were pNO/sub 2/-phe/sup 4/, beta-methyl-phe/sup 4/, pNO/sub 2/-beta-methyl-phe/sub 4/, pNO/sub 2/-beta-methyl-phe/sup 4/ (three isomeric forms: A,B,D). The IC50 values for the above enkephalin analogues were 3.7, 16, 7, 7, 200 nM, respectively. Thus, these potent analogues of DPDPE should be useful in determining the structure activity relationships of the delta opioid receptor in rat brain.

  14. Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin.

    PubMed

    Christoffers, Keith H; Khokhar, Arshia; Chaturvedi, Kirti; Howells, Richard D

    2002-04-15

    We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions. PMID:11853866

  15. Lack of renal tubular and hemodynamic effects of non-selective and delta-opioid receptor antagonism.

    PubMed

    Barrett, R J; Turpin, J A; McGuirk, B A; Kau, S T

    1985-01-01

    The renal pharmacological actions of the non-selective opioid receptor antagonist naloxone and the selective delta (delta)-opioid receptor antagonist ICI 154,129 were examined in conscious dogs. Neither naloxone nor ICI 154,129 altered glomerular filtration rate, renal blood flow, blood pressure, heart rate, or renal excretion of water, Na+, K+, or Cl-. In addition, urine and plasma osmolality and electrolyte concentrations and hematocrit were unchanged, suggesting that neither agent produced physiologically significant alteration in plasma vasopressin levels. These data suggest that (a) naloxone and ICI 154,129 exert no renal pharmacological effects in dogs and (b) under resting physiological conditions, delta-opioid receptors, as well as other opioid receptor subtypes, probably are not involved in the tonic regulation of renal hemodynamics or tubular function. PMID:3983226

  16. Quantitative evaluation of human delta opioid receptor desensitization using the operational model of drug action.

    PubMed

    Navratilova, Edita; Waite, Sue; Stropova, Dagmar; Eaton, Miriam C; Alves, Isabel D; Hruby, Victor J; Roeske, William R; Yamamura, Henry I; Varga, Eva V

    2007-05-01

    Agonist-mediated desensitization of the opioid receptors is thought to function as a protective mechanism against sustained opioid signaling and therefore may prevent the development of opioid tolerance. However, the exact molecular mechanism of opioid receptor desensitization remains unresolved because of difficulties in measuring and interpreting receptor desensitization. In the present study, we investigated deltorphin II-mediated rapid desensitization of the human delta opioid receptors (hDOR) by measuring guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding and inhibition of cAMP accumulation. We developed a mathematical analysis based on the operational model of agonist action (Black et al., 1985) to calculate the proportion of desensitized receptors. This approach permits a correct analysis of the complex process of functional desensitization by taking into account receptor-effector coupling and the time dependence of agonist pretreatment. Finally, we compared hDOR desensitization with receptor phosphorylation at Ser363, the translocation of beta-arrestin2, and hDOR internalization. We found that in Chinese hamster ovary cells expressing the hDOR, deltorphin II treatment leads to phosphorylation of Ser363, translocation of beta-arrestin2 to the plasma membrane, receptor internalization, and uncoupling from G proteins. It is noteworthy that mutation of the primary phosphorylation site Ser363 to alanine had virtually no effect on agonist-induced beta-arrestin2 translocation and receptor internalization yet significantly attenuated receptor desensitization. These results strongly indicate that phosphorylation of Ser363 is the primary mechanism of hDOR desensitization. PMID:17322005

  17. Ethanol Disinhibits Dorsolateral Striatal Medium Spiny Neurons Through Activation of A Presynaptic Delta Opioid Receptor.

    PubMed

    Patton, Mary H; Roberts, Bradley M; Lovinger, David M; Mathur, Brian N

    2016-06-01

    The dorsolateral striatum mediates habit formation, which is expedited by exposure to alcohol. Across species, alcohol exposure disinhibits the DLS by dampening GABAergic transmission onto this structure's principal medium spiny projection neurons (MSNs), providing a potential mechanistic basis for habitual alcohol drinking. However, the molecular and circuit components underlying this disinhibition remain unknown. To examine this, we used a combination of whole-cell patch-clamp recordings and optogenetics to demonstrate that ethanol potently depresses both MSN- and fast-spiking interneuron (FSI)-MSN GABAergic synaptic transmission in the DLS. Concentrating on the powerfully inhibitory FSI-MSN synapse, we further show that acute exposure of ethanol (50 mM) to striatal slices activates delta opioid receptors that reside on FSI axon terminals and negatively couple to adenylyl cyclase to induce a long-term depression of GABA release onto both direct and indirect pathway MSNs. These findings elucidate a mechanism through which ethanol may globally disinhibit the DLS. PMID:26758662

  18. Protecting motor networks during perinatal ischemia: the case for delta opioid receptors

    PubMed Central

    Johnson, Stephen M.; Freiberg, Sara M.

    2011-01-01

    Perinatal ischemia is a common clinical problem with few successful therapies to prevent neuronal damage. Delta-opioid receptor (DOR) activation is a versatile, evolutionarily-conserved, endogenous neuroprotective mechanism that blocks several steps in the deleterious cascade of neurological events during ischemia. DOR activation prior to ischemia or severe hypoxia is neuroprotective in spinal motor networks, as well as cortical, cerebellar, and hippocampal neural networks. In addition to providing acute and long-lasting neuroprotection against ischemia, DOR activation appears to provide neuroprotection when given before, during, or following the onset of ischemia. Finally, DORs can be upregulated by several physiological and experimental perturbations. Potential adverse side effects affecting motor control, such as respiratory depression and seizures, are not well established in young mammals and may be mitigated by altering drug choice and method of drug administration. The unique features of DOR-dependent neuroprotection make it an attractive potential therapy that may be given to at-risk pregnant mothers shortly before delivery to provide long-lasting neuroprotection against unpredictable perinatal ischemic events. PMID:20536941

  19. delta-Opioid receptors are more efficiently coupled to adenylyl cyclase than to L-type Ca(2+) channels in transfected rat pituitary cells.

    PubMed

    Prather, P L; Song, L; Piros, E T; Law, P Y; Hales, T G

    2000-11-01

    Opioid receptors often couple to multiple effectors within the same cell. To examine potential mechanisms that contribute to the specificity by which delta-receptors couple to distinct intracellular effectors, we stably transfected rat pituitary GH(3) cells with cDNAs encoding for delta-opioid receptors. In cells transfected with a relatively low delta-receptor density of 0.55 pmol/mg of protein (GH(3)DOR), activation of delta-receptors produced inhibition of adenylyl cyclase activity but was unable to alter L-type Ca(2+) current. In contrast, activation of delta-receptors in a clone that contained a higher density of delta-receptors (2.45 pmol/mg of protein) and was also coexpressed with mu-opioid receptors (GH(3)MORDOR), resulted in not only the expected inhibition of adenylyl cyclase activity but also produced inhibition of L-type Ca(2+) current. The purpose of the present study was to determine whether these observations resulted from differences in delta-opioid receptor density between clones or interaction between delta- and mu-opioid receptors to allow the activation of different G proteins and signaling to Ca(2+) channels. Using the delta-opioid receptor alkylating agent SUPERFIT, reduction of available delta-opioid receptors in GH(3)MORDOR cells to a density similar to that of delta-opioid receptors in the GH(3)DOR clone resulted in abolishment of coupling to Ca(2+) channels, but not to adenylyl cyclase. Furthermore, although significantly greater amounts of all G proteins were activated by delta-opioid receptors in GH(3)MORDOR cells, delta-opioid receptor activation in GH(3)DOR cells resulted in coupling to the identical pattern of G proteins seen in GH(3)MORDOR cells. These findings suggest that different threshold densities of delta-opioid receptors are required to activate critical amounts of G proteins needed to produce coupling to specific effectors and that delta-opioid receptors couple more efficiently to adenylyl cyclase than to L-type Ca(2

  20. Role of Mu and Delta Opioid Receptors in the Nucleus Accumbens in Cocaine-Seeking Behavior

    PubMed Central

    Simmons, Diana; Self, David W.

    2009-01-01

    Previous studies suggest that opioid receptors in the ventral tegmental area (VTA), but not the nucleus accumbens (NAc), play a role in relapse to drug-seeking behavior. However, environmental stimuli that elicit relapse also release the endogenous opioid β-endorphin in the NAc. Using a within–session extinction/reinstatement paradigm in rats that self-administer cocaine, we found that NAc infusions of the mu opioid receptor (MOR) agonist DAMGO moderately reinstated responding on the cocaine-paired lever at low doses (1.0–3.0 ng/side), whereas the delta opioid receptor (DOR) agonist DPDPE induced greater responding at higher doses (300–3000 ng/side) that also enhanced inactive lever responding. Using doses of either agonist that induced responding on only the cocaine-paired lever, we found that DAMGO-induced responding was blocked selectively by pretreatment with the MOR antagonist CTAP, while DPDPE-induced responding was selectively blocked by the DOR antagonist naltrindole. Cocaine-primed reinstatement was blocked by intra-NAc CTAP but not naltrindole, indicating a role for endogenous MOR-acting peptides in cocaine-induced reinstatement of cocaine-seeking behavior. In this regard, intra-NAc infusions of β-endorphin (100–1000 ng/side) induced marked cocaine-seeking behavior, an effect blocked by intra-NAc pretreatment with the MOR but not DOR antagonist. Conversely, cocaine seeking elicited by the enkephalinase inhibitor thiorphan (1–10 μg/side) was blocked by naltrindole but not CTAP. MOR stimulation in more dorsal caudate-putamen sites was ineffective, while DPDPE infusions induced cocaine seeking. Together, these findings establish distinct roles for MOR and DOR in cocaine relapse, and suggest that NAc MOR could be an important therapeutic target to neutralize the effects of endogenous β-endorphin release on cocaine relapse. PMID:19279569

  1. Delta opioid receptors expressed in forebrain GABAergic neurons are responsible for SNC80-induced seizures

    PubMed Central

    CHUNG, Paul CHU SIN; BOEHRER, Annie; STEPHAN, Aline; MATIFAS, Audrey; SCHERRER, Gregory; DARCQ, Emmanuel; BEFORT, Katia; KIEFFER, Brigitte L.

    2014-01-01

    The delta opioid receptor (DOR) has raised much interest for the development of new therapeutic drugs, particularly to treat patients suffering from mood disorders and chronic pain. Unfortunately, the prototypal DOR agonist SNC80 induces mild epileptic seizures in rodents. Although recently developed agonists do not seem to show convulsant properties, mechanisms and neuronal circuits that support DOR-mediated epileptic seizures remain to be clarified. DORs are expressed throughout the nervous system. In this study we tested the hypothesis that SNC80-evoked seizures stem from DOR activity at the level of forebrain GABAergic transmission, whose inhibition is known to facilitate the development of epileptic seizures. We generated a conditional DOR knockout mouse line, targeting the receptor gene specifically in GABAergic neurons of the forebrain (Dlx-DOR). We measured effects of SNC80 (4.5, 9, 13.5 and 32 mg/kg), ARM390 (10, 30 and 60 mg/kg) or ADL5859 (30, 100 and 300 mg/kg) administration on electroencephalograms (EEGs) recorded in Dlx-DOR mice and their control littermates (Ctrl mice). SNC80 produced dose-dependent seizure events in Ctrl mice, but these effects were not detected in Dlx-DOR mice. As expected, ARM390 and ADL5859 did not trigger any detectable change in mice from both genotypes. These results demonstrate for the first time that SNC80-induced DOR activation induces epileptic seizures via direct inhibition of GABAergic forebrain neurons, and supports the notion of differential activities between first and second-generation DOR agonists. PMID:25447299

  2. delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains.

    PubMed

    Bourova, Lenka; Kostrnova, Alexandra; Hejnova, Lucie; Moravcova, Zuzana; Moon, Hyo-Eun; Novotny, Jiri; Milligan, Graeme; Svoboda, Petr

    2003-04-01

    Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor

  3. Delta opioid receptors colocalize with corticotropin releasing factor in hippocampal interneurons

    PubMed Central

    Williams, Tanya J.; Milner, Teresa A.

    2011-01-01

    The hippocampal formation (HF) is an important site at which stress circuits and endogenous opioid systems intersect, likely playing a critical role in the interaction between stress and drug addiction. Prior study findings suggest that the stress-related neuropeptide corticotropin releasing factor (CRF) and the delta opioid receptor (DOR) may localize to similar neuronal populations within HF lamina. Here, hippocampal sections of male and cycling female adult Sprague-Dawley rats were processed for immunolabeling using antisera directed against the DOR and CRF peptide, as well as interneuron subtype markers somatostatin or parvalbumin, and analyzed by fluorescence and electron microscopy. Both DOR- and CRF-labeling was observed in interneurons in the CA1, CA3, and dentate hilus. Males and normal cycling females displayed a similar number of CRF immunoreactive neurons co-labeled with DOR and a similar average number of CRF-labeled neurons in the dentate hilus and stratum oriens of CA1 and CA3. In addition, 70% of DOR/CRF dual-labeled neurons in the hilar region co-labeled with somatostatin, suggesting a role for these interneurons in regulating perforant path input to dentate granule cells. Ultrastructural analysis of CRF-labeled axon terminals within the hilar region revealed that proestrus females have a similar number of CRF-labeled axon terminals that contain DORs compared to males but an increased number of CRF-labeled axon terminals without DORs. Taken together, these findings suggest that while DORs are anatomically positioned to modulate CRF immunoreactive interneuron activity and CRF peptide release, their ability to exert such regulatory activity may be compromised in females when estrogen levels are high. PMID:21277946

  4. Hormonal regulation of delta opioid receptor immunoreactivity in interneurons and pyramidal cells in the rat hippocampus

    PubMed Central

    Williams, Tanya J.; Torres-Reveron, Annelyn; Chapleau, Jeanette D.; Milner, Teresa A.

    2011-01-01

    Clinical and preclinical studies indicate that women and men differ in relapse vulnerability to drug-seeking behavior during abstinence periods. As relapse is frequently triggered by exposure of the recovered addict to objects previously associated with drug use and the formation of these associations requires memory systems engaged by the hippocampal formation (HF), studies exploring ovarian hormone modulation of hippocampal function are warranted. Previous studies revealed that ovarian steroids alter endogenous opioid peptide levels and trafficking of mu opioid receptors in the HF, suggesting cooperative interaction between opioids and estrogens in modulating hippocampal excitability. However, whether ovarian steroids affect the levels or trafficking of delta opioid receptors (DORs) in the HF is unknown. Here, hippocampal sections of adult male and normal cycling female Sprague-Dawley rats were processed for quantitative immunoperoxidase light microscopy and dual label fluorescence or immunoelectron microscopy using antisera directed against the DOR and neuropeptide Y (NPY). Consistent with previous studies in males, DOR-immunoreactivity (-ir) localized to select interneurons and principal cells in the female HF. In comparison to males, females, regardless of estrous cycle phase, show reduced DOR-ir in the granule cell layer of the dentate gyrus and proestrus (high estrogen) females, in particular, display reduced DOR-ir in the CA1 pyramidal cell layer. Ultrastructural analysis of DOR-labeled profiles in CA1 revealed that while females generally show fewer DORs in the distal apical dendrites of pyramidal cells, proestrus females, in particular, exhibit DOR internalization and trafficking towards the soma. Dual label studies revealed that DORs are found in NPY-labeled interneurons in the hilus, CA3, and CA1. While DOR colocalization frequency in NPY-labeled neuron somata was similar between animals in the hilus, proestrus females had fewer NPY-labeled neurons that

  5. Central delta-opioid receptor interactions and the inhibition of reflex urinary bladder contractions in the rat.

    PubMed

    Dray, A; Nunan, L; Wire, W

    1985-07-01

    The in vivo effects of a number of opioid agonists and antagonists were studied on the spontaneous reflex contractions of the urinary bladder recorded isometrically in the rat anesthetized with urethane. All substances were administered into the central nervous system by the intracereboventricular (i.c.v.) or spinal intrathecal (i.t.) route. The conformationally restricted enkephalin analogues [2-D-penicillamine, 5-L-cysteine] enkephalin (DPLCE), [2-D-penicillamine, 5-L-penicillamine] enkephalin (DPLPE) and [2-D-penicillamine, 5-D-penicillamine] enkephalin (DPDPE) produced dose-related inhibition of reflex bladder contractions when administered by the i.c.v. or i.t. route. Both the novel delta-opioid receptor antagonist ICI 154,129 (200-600 micrograms) [N,N-bisallyl-Tyr-Gly-Gly-Psi-(CH2S)-Phe-Leu-OH) and ICI 174,864 (1-3 micrograms) [N,N-dially-Tyr-Aib-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid] attenuated or abolished the effects of DPLCE, DPLPE and DPDPE when administered by the i.c.v. or i.t. route. The antagonism observed was selective since the equipotent inhibition produced by the mu-opioid receptor agonist [D-Ala2, Me-Phe4, Gly(ol)5] enkephalin (DAGO) was unaffected. Overall, ICI 154,129 was considerably weaker than ICI 174,864 and both antagonists inhibited bladder activity at doses higher than those required to demonstrate delta-receptor antagonism. Further studies of the agonistic effect of ICI 174,864 showed that it was insensitive to low doses of naloxone (2 micrograms, i.c.v. or i.t.) but could be abolished by higher (10-15 micrograms) doses of naloxone. These observations suggested that the agonistic effect of ICI 174,864 was not mediated by mu-opioid receptor. beta-Endorphin (0.2-1.0 micrograms, i.c.v.) inhibited bladder contractions but following recovery from this effect, appeared to prevent the expression of delta-receptor antagonism by ICI 174,864. In addition a previously subthreshold dose of ICI 174,864 now exhibited marked agonistic

  6. [Cys(O2NH2)2]enkephalin analogues and dalargin: selectivity for delta-opioid receptors.

    PubMed

    Pencheva, N; Bocheva, A; Dimitrov, E; Ivancheva, C; Radomirov, R

    1996-05-23

    To investigate the structure-activity relationships for potent and selective action of enkephalins at the delta-opioid receptors, two newly synthesized analogues, [Cys(O2NH2)2,Leu5]enkephalin and [Cys(O2NH2)2, Met5] enkephalin and the hexapeptide [D-Ala2,Leu5]enkephalyl-Arg (dalargin) were tested and compared with [Leu5]enkephalin and [Met5]enkephalin, for their effectiveness to inhibit electrically evoked contractions of the mouse vas deferens (predominantly enkephalin-selective delta-opioid receptors) and the guinea pig ileum (mu- and kappa-opioid receptors). The mouse vas deferens assays included evaluation of the effects of opioid agonists on the first, purinergic, and the second, adrenergic, components of electrically evoked biphasic responses (10 Hz and 20 Hz) and on ATP- or noradrenaline-evoked, tetrodotoxin-resistant responses. The opioids tested inhibited in a similar manner: (i) the purinergic and the adrenergic components of the electrically evoked contractions; and (ii) the ATP- and noradrenaline-induced postjunctional responses of the mouse vas deferens. Extremely low IC50 values (of 2-5 orders) were found for [Cys(O2NH2)2,Leu5] enkephalin, whose relative potency was between 239 and 1316 times higher than that of [Leu5]enkephalin. The order of potency for the other peptides in this tissue was: [Cys(O2NH2)2,Met5]enkephalin > [Leu5]enkephalin > dalargin > [Met5]enkephalin. The highest IC50 values in the guinea pig ileum assays, indicating the lowest affinity for mu-/kappa-opioid receptors, were obtained for the cysteine sulfonamide analogues, while dalargin showed a potency four times higher than that of [Met5]enkephalin. The order of potency in this tissue was: dalargin > [Met5]enkephalin > [Leu5]enkephalin > [Cys(O2NH2)2,Met5]enkephalin > [Cys(O2NH2)2,Leu5]enkephalin. The ratio, IC50 in guinea pig ileum: IC50 in mouse vas deferens, indicating selectivity of the respective peptide for delta-opioid receptors, was extremely high for [Cys(O2NH2)2,Leu5

  7. Autoradiographic comparison of the distribution of the neutral endopeptidase enkephalinase and of. mu. and delta opioid receptors in rat brain

    SciTech Connect

    Waksman, G.; Hamel, E.; Fournie-Zaluski, M.C.; Roques, B.P.

    1986-03-01

    The neutral endopeptidase EC 3.4.24.11, also designated enkephalinase, has been visualized by in vitro autoradiography using the tritiated inhibitor (/sup 3/H)-N-((2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl)glycine, ((/sup 3/H)HACBO-Gly). Specific binding of (/sup 3/H)HACBO-Gly corresponding to 85% of the total binding to brain slices was inhibited by 1 ..mu..M thiorphan, a selective inhibitor of enkephalinase, but remained unchanged in the presence of captopril, a selective inhibitor of angiotensin-converting enzyme. Very high levels of (/sup 3/H)HACBO-Gly binding were found in the choroid plexus and the substantia nigra. High levels were present in the caudate putamen, globus pallidus, nucleus accumbens, olfactory tubercle, and in the substantia gelatinosa of the spinal cord. The distribution of enkephalinase was compared to that of ..mu.. and delta opioid receptors, selectively labeled with (/sup 3/H)Tyr-D-Ala-Gly-MePhe-glycinol and (/sup 3/H)Try-D-Thr-Gly-Phe-Leu-Thr, respectively. In the caudate putamen, (/sup 3/H)HACBO-Gly binding overlapped the clustered ..mu.. sites but appeared more closely related to the diffusely distributed delta sites. The association of enkephalinase with delta and ..mu.. opioid receptors in these areas is consistent with the observed role of the enzyme in regulating the effects of opioid peptides in striatal dopamine release and analgesia, respectively. Except for the choroid plexus and the cerebellum, the close similarity observed in numerous rat brain areas between the distribution of enkephalinase and that of ..mu.. and/ or delta opioid binding sites could account for most of the pharmacological effects elicited by enkephalinase inhibitors.

  8. Preferential cytoplasmic localization of delta-opioid receptors in rat striatal patches: comparison with plasmalemmal mu-opioid receptors.

    PubMed

    Wang, H; Pickel, V M

    2001-05-01

    The activation of delta-opioid receptors (DORs) in the caudate-putamen nucleus (CPN) produces regionally distinct changes in motor functions, many of which are also influenced by opioids active at micro-opioid receptors (MORs). These actions most likely occur in MOR-enriched patch compartments in the CPN. To determine the functional sites for DOR activation and potential interactions involving MOR in these regions, immunoperoxidase and immunogold-silver labeling methods were applied reversibly for the ultrastructural localization of DOR and MOR in single rat brain sections containing patches of the CPN. DOR immunoreactivity was commonly seen within the cytoplasm of spiny and aspiny neurons, many of which also expressed MOR. In dendrites and spines, DOR labeling was preferentially localized to membranes of the smooth endoplasmic reticulum and spine apparatus, whereas MOR showed a prominent plasmalemmal distribution. DOR- and/or MOR-labeled spines received asymmetric, excitatory synapses, some of which showed notable perforations, suggesting the involvement of these receptors in activity-dependent synaptic plasticity. DORs were more frequently detected than were MORs within axon terminals that formed either asymmetric synapses with spine heads or symmetric synapses with spine necks. Our results suggest that in striatal patches, DORs, often in cooperation with MORs, play a direct modulatory role in controlling the postsynaptic excitability of spines, whereas presynaptic neurotransmitter release onto spines is mainly influenced by DOR activation. In comparison with MOR, the prevalent association of DOR with cytoplasmic organelles that are involved in intracellular trafficking of cell surface proteins suggests major differences in availability of these receptors to extracellular opioids. PMID:11312309

  9. Exploratory synthesis of peptide-alpha-thioester segments spanning the polypeptide sequence of the delta-opioid receptor, a G protein-coupled receptor.

    PubMed

    Johnson, Erik C B; Kent, Stephen B H

    2007-01-01

    We have decided to use the delta-opioid receptor (372 residues) as a model system to develop methods for the total chemical synthesis of G protein-coupled receptors. The most important feature of this receptor from a chemical synthesis perspective is the wealth of cysteines spread throughout its sequence, which are required for native chemical ligation. A total of 13 cysteines are located in the the delta-opioid receptor polypetide chain in both loop and putative transmembrane (TM) regions. We envisioned a synthesis of the polypeptide that would make use of peptide-alpha-thioesters ranging from 37 to 63 residues in length. Here, we report data from an exploratory synthesis of such a set of peptide-alpha-thioesters. For all seven peptides, the crude material approximately 30 residues into the synthesis was sufficiently homogeneous to make isolation and purification straightforward. Extension of the peptides to between 40 and 50 residues in length generally produced a significant decrease in the quality of the crude products, although in most cases, we judged that high purity peptides could probably be isolated. By 60 residues, however, the crude peptide product mixtures are probably too heterogeneous to purify to homogeneity by reversed-phase HPLC. In general, delta-opioid receptor peptides with a single predicted TM domain were sufficiently soluble to handle postcleavage and to analyze by reversed-phase HPLC, whereas 1.5 TM domains rendered the peptides too hydrophobic to handle or analyze by standard protocols. Given the challenges of chain assembly, handling, and purification of these peptides, a synthetic strategy that uses approximately 12 or 13 shorter peptide segments of 20-40 residues each is probably a more feasible approach. PMID:17120238

  10. Role of kappa and delta opioid receptors in mediating morphine-induced antinociception in morphine tolerant infant rats

    PubMed Central

    Stoller, Dawn C.; Sim-Selley, Laura J.; Smith, Forrest L.

    2011-01-01

    We have previously noted that the antinociceptive efficacy of morphine was significantly decreased in rat pups chronically infused with morphine from implanted osmotic minipumps. In this study, morphine was fully efficacious (i.e., 100% maximum possible effect, %MPE) in the 52 ºC tail-immersion test after a 72-h infusion from implanted saline-filled osmotic minipumps. However, administration of up to 1000 mg/kg s.c. morphine failed to elicit greater than a 27% MPE in rats infused with morphine at 2 mg/kg/h. Morphine was more efficacious when the water bath temperature was decreased to 49 ºC. Experiments were conducted to determine the mechanisms whereby chronic morphine administration leads to a decrease in antinociceptive efficacy. The kappa-opioid antagonist nor-binalorphimine completely blocked the antinociceptive effects of morphine in morphine-infused rat pups. The kappa agonist U50,488 elicited antinociception however, the requirement to use higher doses in morphine- than saline-infused rats indicates that kappa cross-tolerance was present. Thus, in tolerant rats the antinociceptive effects of high doses of morphine appear to be mediated through kappa-opioid receptors. The delta-opioid antagonist naltrindole was inactive in both treatment groups. DAMGO-stimulated [35S]GTPγS and [3H]naloxone binding reveal that the anatomical distribution of the mu-opioid receptor was consistent with that of the adult rat brain. In adult rats, the mu-opioid receptor is desensitized during morphine tolerance. However, desensitization was not evident in P17 rats based on the lack of significant decreases in [35S]GTPγS binding. Furthermore, [3H]naloxone binding indicated a lack of mu receptor downregulation in morphine-tolerant rat pups. PMID:17300766

  11. Delta Opioid activation of the Mitogen-activated protein kinase cascade does not require transphosphorylation of Receptor Tyrosine Kinases

    PubMed Central

    Kramer, H Kenneth; Onoprishvili, Irma; Andria, Matthew L; Hanna, Kayane; Sheinkman, Karina; Haddad, Lisa B; Simon, Eric J

    2002-01-01

    Background In this study, we investigated the mechanism(s) by which delta opioids induce their potent activation of extracellular signal-regulated protein kinases (ERKs) in different cell lines expressing the cloned δ-opioid receptor (δ-OR). While it has been known for some time that OR stimulation leads to the phosphorylation of both ERK isoforms, the exact progression of events has remained elusive. Results Our results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed δ-ORs are stimulated by the δ-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET). Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET. These results appear to rule out any structural or catalytic role for the EGFR in the δ-opioid-mediated MAPK cascade. To confirm these results, we used C6 glioma cells, a cell line devoid of the EGFR. In δ-OR-expressing C6 glioma cells, opioids produce a robust phosphorylation of ERK 1 and 2, whereas EGF has no stimulatory effect. Furthermore, antagonists to the RTKs that are endogenously expressed in C6 glioma cells (insulin receptor (IR) and platelet-derived growth factor receptor (PDGFR)) were unable to reduce opioid-mediated ERK activation. Conclusion Taken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated δ-OR, itself, is likely to act as its own signalling scaffold. PMID:11897012

  12. Activation of peripheral delta opioid receptors eliminates cardiac electrical instability in a rat model of post-infarction cardiosclerosis via mitochondrial ATP-dependent K+ channels.

    PubMed

    Maslov, L N; Lishmanov, Yu B; Solenkova, N V; Gross, G J; Stefano, G B; Tam, S W

    2003-07-01

    The effects of the selective delta-1 (delta(1)) opioid receptor agonist, DPDPE, and the selective delta(2) opioid receptor agonist, DSLET, have been studied on the ventricular fibrillation threshold (VFT) in rats with an experimental post-infarction cardiosclerosis (CS). It has been found that CS induced a significant decrease in VFT. This CS-induced decrease in VFT was significantly reversed by intravenous administration of DPDPE (0.1 mg/kg) 10 min before VFT measurement. On the contrary, intravenous injection of DSLET (0.5 mg/kg) exacerbated the CS-induced cardiac electrical instability. Pretreatment with the selective delta opioid receptor antagonist, ICI 174,864 (0.5 mg/kg), completely abolished the changes in VFT produced by both DPDPE and DSLET. Previous administration of a nonselective peripherally acting opioid receptor antagonist, naloxone methiodide (5 mg/kg) also completely reversed the antifibrillatory action of DPDPE. Naloxone methiodide and ICI 174,864 alone had no effect on VFT. Pretreatment with the nonselective K(ATP) channel blocker, glibenclamide (0.3 mg/kg), or with the mitochondrial selective K(ATP) channel blocker, 5-hydroxydecanoic acid (5-HD, 5 mg/kg), completely abolished the DPDPE-induced increase in cardiac electrical stability. Glibenclamide and 5-HD alone had no effect on VFT. These results demonstrate that the delta opioid receptor plays an important role in the regulation of electrical stability in rats with post-infarction cardiosclerosis. We propose that peripheral delta(1) opioid receptor stimulation reverses CS-induced electrical instability via mitochondrial K(ATP) channels. On the contrary, delta(2) opioid receptor stimulation may exacerbate the CS-induced decrease in VFT. Further studies are necessary to determine the delta opioid receptor subtype which mediates the antifibrillatory effect of DPDPE and pro-fibrillatory effect of DSLET. PMID:12798419

  13. Coincident signalling between the Gi/Go-coupled delta-opioid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells.

    PubMed

    Yeo, A; Samways, D S; Fowler, C E; Gunn-Moore, F; Henderson, G

    2001-03-01

    In SH-SY5Y cells, activation of delta-opioid receptors with [D-Pen(2,5)]-enkephalin (DPDPE; 1 microM) did not alter the intracellular free Ca(2+) concentration [Ca(2+)](i). However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine-M, it produced an elevation of [Ca(2+)](i). The DPDPE-evoked increase in [Ca(2+)](i) was abolished when the carbachol-sensitive intracellular Ca(2+) store was emptied. There was a marked difference between the concentration-response relationship for the elevation of [Ca(2+)](i) by carbachol (EC(50) 13 microM, Hill slope 1) and the concentration-response relationship for carbachol's permissive action in revealing the delta-opioid receptor-mediated elevation of [Ca(2+)] (EC(50) 0.7 mM; Hill slope 1.8). Sequestration of free G protein beta gamma dimers by transient transfection of cells with a beta gamma binding protein (residues 495-689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of delta opioid receptor activation to elevate [Ca(2+)](i). However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that delta-opioid receptor activation did not stimulate phospholipase C. Furthermore, delta-opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the delta-opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca(2+)](i) in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coincident signalling between the endogenously expressed delta-opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca(2+) release signalling pathway at a step after the activation of phospholipase C. PMID:11259487

  14. Autoradiographic localization of delta opioid receptors within the mesocorticolimbic dopamine system using radioiodinated (2-D-penicillamine, 5-D-penicillamine)enkephalin ( sup 125 I-DPDPE)

    SciTech Connect

    Dilts, R.P.; Kalivas, P.W. )

    1990-01-01

    The enkephalin analog (2-D-penicillamine, 5-D-penicillamine)enkephalin was radioiodinated (125I-DPDPE) and shown to retain a pharmacological selectivity characteristic of the delta opioid receptor in in vitro binding studies. The distributions of 125I-DPDPE binding, using in vitro autoradiographic techniques, were similar to those previously reported for the delta opioid receptor. The nucleus accumbens, striatum, and medial prefrontal cortex contain dense gradients of 125I-DPDPE binding in regions known to receive dopaminergic afferents emanating from the mesencephalic tegmentum. Selective chemical lesions of the ventral tegmental area and substantia nigra were employed to deduce the location of the 125I-DPDPE binding within particular regions of the mesocorticolimbic dopamine system. Unilateral lesions of dopamine perikarya (A9 and A10) within the ventral tegmental area and substantia nigra produced by mesencephalic injection of 6-hydroxydopamine resulted in significant (20-30%) increases in 125I-DPDPE binding contralateral to the lesion within the striatum and nucleus accumbens. Lesions of the perikarya (dopaminergic and nondopaminergic) of the ventral tegmental area, induced by quinolinic acid injections, caused increases of less magnitude within these same nuclei. No significant alterations in 125I-DPDPE binding were observed within the mesencephalon as a result of either treatment. The specificity of the lesions was confirmed by immunocytochemistry for tyrosine hydroxylase. These results suggest that the enkephalins and opioid agonists acting through delta opioid receptors do not directly modulate dopaminergic afferents but do regulate postsynaptic targets of the mesocorticolimbic dopamine system.

  15. Opioid Peptidomimetics: Leads for the Design of Bioavailable Mixed Efficacy Mu Opioid Receptor (MOR) Agonist/Delta Opioid Receptor (DOR) Antagonist Ligands

    PubMed Central

    Mosberg, Henry I.; Yeomans, Larisa; Harland, Aubrie A.; Bender, Aaron M.; Sobczyk-Kojiro, Katarzyna; Anand, Jessica P.; Clark, Mary J.; Jutkiewicz, Emily M.; Traynor, John R.

    2013-01-01

    We have previously described opioid peptidomimetic, 1, employing a tetrahydroquinoline scaffold and modeled on a series of cyclic tetrapeptide opioid agonists. We have recently described modifications to these peptides that confer a mu opioid receptor (MOR) agonist, delta opioid receptor (DOR) antagonist profile, which has been shown to reduce the development of tolerance to the analgesic actions of MOR agonists. Several such bifunctional ligands have been reported, but none has been demonstrated to cross the blood brain barrier. Here we describe the transfer of structural features that evoked MOR agonist/DOR antagonist behavior in the cyclic peptides to the tetrahydroquinoline scaffold and show that the resulting peptidomimetics maintain the desired pharmacological profile. Further, the 4R diastereomer of 1 was fully efficacious and approximately equipotent to morphine in the mouse warm water tail withdrawal assay following intraperitoneal administration and thus a promising lead for the development of opioid analgesics with reduced tolerance. PMID:23419026

  16. Quantification of delta-opioid receptors in human brain with N1'-([11C]methyl) naltrindole and positron emission tomography.

    PubMed

    Smith, J S; Zubieta, J K; Price, J C; Flesher, J E; Madar, I; Lever, J R; Kinter, C M; Dannals, R F; Frost, J J

    1999-09-01

    The regional binding of N1'-([11C]methyl)naltrindole (MeNTI), a selective delta-opioid antagonist, was studied in healthy human subjects with positron emission tomography (PET). After the bolus intravenous administration of high specific activity [11C]MeNTI, PET was performed over 90 minutes. Arterial plasma samples were obtained during the scanning period and assayed for the presence of radiolabeled metabolites. The data were analyzed with various kinetic (two- and three-compartment models, Patlak graphical analysis) and nonkinetic (apparent volume of distribution and activity at a late scanning time) approaches. This tracer showed irreversible binding characteristics during the scanning period used. The results of the analyses also were compared with the density and distribution of delta-opioid receptors in the human brain in vitro. Additionally, computer simulations were performed to assess the effects of changes in receptor binding and tracer transport changes on the perceived binding parameters obtained with the models. A constrained three-compartment kinetic model was demonstrated to be superior to other quantification models for the description of MeNTI kinetics and quantification of delta receptor binding in the human brain with 11C-labeled MeNTI. PMID:10478647

  17. Cellular localization and adaptive changes of the cardiac delta opioid receptor system in an experimental model of heart failure in rats.

    PubMed

    Treskatsch, Sascha; Feldheiser, Aarne; Shaqura, Mohammed; Dehe, Lukas; Habazettl, Helmut; Röpke, Torsten K; Shakibaei, Mehdi; Schäfer, Michael; Spies, Claudia D; Mousa, Shaaban A

    2016-02-01

    The role of the cardiac opioid system in congestive heart failure (CHF) is not fully understood. Therefore, this project investigated the cellular localization of delta opioid receptors (DOR) in left ventricle (LV) myocardium and adaptive changes in DOR and its endogenous ligand, the precursor peptide proenkephalin (PENK), during CHF. Following IRB approval, DOR localization was determined by radioligand binding using [H(3)]Naltrindole and by double immunofluorescence confocal analysis in the LV of male Wistar rats. Additionally, 28 days following an infrarenal aortocaval fistula (ACF) the extent of CHF and adaptions in left ventricular DOR and PENK expression were examined by hemodynamic measurements, RT-PCR, and Western blot. DOR specific membrane binding sites were identified in LV myocardium. DOR were colocalized with L-type Ca(2+)-channels (Cav1.2) as well as with intracellular ryanodine receptors (RyR) of the sarcoplasmatic reticulum. Following ACF severe congestive heart failure developed in all rats and was accompanied by up-regulation of DOR and PENK on mRNA as well as receptor proteins representing consecutive adaptations. These findings might suggest that the cardiac delta opioid system possesses the ability to play a regulatory role in the cardiomyocyte calcium homeostasis, especially in response to heart failure. PMID:25552382

  18. Discrete mapping of brain Mu and delta opioid receptors using selective peptides: Quantitative autoradiography, species differences and comparison with kappa receptors

    SciTech Connect

    Sharif, N.A.; Hughes, J. )

    1989-05-01

    The opioid peptides, (3H)DAGO and (3H)DPDPE, bound to rat and guinea pig brain homogenates with a high, nanomolar affinity and to a high density of mu and delta receptors, respectively. (3H)DAGO binding to mu receptors was competitively inhibited by unlabelled opioids with the following rank order of potency: DAGO greater than morphine greater than DADLE greater than naloxone greater than etorphine much greater than U50488 much greater than DPDPE. In contrast, (3H)DPDPE binding to delta receptors was inhibited by compounds with the following rank order of potency: DPDPE greater than DADLE greater than etorphine greater than dynorphin(1-8) greater than naloxone much greater than U50488 much greater than DAGO. These profiles were consistent with specific labelling of the mu and delta opioid receptors, respectively. In vitro autoradiographic techniques coupled with computer-assisted image analyses revealed a discrete but differential anatomical localization of mu and delta receptors in the rat and guinea pig brain. In general, mu and delta receptor density in the rat exceeded that in the guinea pig brain and differed markedly from that of kappa receptors in these species. However, while mu receptors were distributed throughout the brain with hotspots in the fore-, mid- and hindbrain of the two rodents, the delta sites were relatively diffusely distributed, and were mainly concentrated in the forebrain with particularly high levels within the olfactory bulb (OB), n. accumbens and striatum. Notable regions of high density of mu receptors in the rat and guinea pig brain were the accessory olfactory bulb, striatal patches and streaks, amygdaloid nuclei, ventral hippocampal subiculum and dentate gyrus, numerous thalamic nuclei, geniculate bodies, central grey, superior and inferior colliculi, solitary and pontine nuclei and s. nigra.

  19. Activation of delta-opioid receptors inhibits neuronal-like calcium channels and distal steps of Ca(2+)-dependent secretion in human small-cell lung carcinoma cells.

    PubMed

    Sher, E; Cesare, P; Codignola, A; Clementi, F; Tarroni, P; Pollo, A; Magnelli, V; Carbone, E

    1996-06-01

    Human small-cell lung carcinoma (SCLC) cells express neuronal-like voltage-operated calcium channels (VOCCs) and release mitogenic hormones such as serotonin (5-HT). Opioid peptides, on the other hand, have been shown to reduce SCLC cell proliferation by an effective autocrine pathway. Here we show that in GLC8 SCLC cells, only delta-opioid receptor subtype mRNA is expressed. Consistently, the selective delta-opioid agonist [D-Pen2-Pen5]-enkephalin (DPDPE), but not mu and kappa agonists, potently and dose-dependently inhibits high-threshold (HVA) VOCCs in these cells. As in peripheral neurons, this modulation is largely voltage-dependent, mediated by pertussis toxin (PTX)-sensitive G-proteins, cAMP-independent, and mainly affecting N-type VOCCs. With the same potency and selectivity, DPDPE also antagonizes the Ca(2+)-dependent release of [3H]serotonin ([3H]5-HT) from GLC8 cells. However, DPDPE inhibits not only the depolarization-induced release, but also the Ca(2+)-dependent secretion induced by thapsigargin or ionomycin. This suggests that besides inhibiting HVA VOCCs, opioids also exert a direct depressive action on the secretory apparatus in GLC8 cells. This latter effect also is mediated by a PTX-sensitive G-protein but, contrary to VOCC inhibition, it can be reversed by elevations of cAMP levels. These results show for the first time that opioids effectively depress both Ca2+ influx and Ca(2+)-dependent hormone release in SCLC cells by using multiple modulatory pathways. It can be speculated that the two mechanisms may contribute to the opioid antimitogenic action on lung neuroendocrine carcinoma cells. PMID:8642411

  20. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    SciTech Connect

    Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  1. Control of the efficiency of agonist-induced information transfer and stability of the ternary complex containing the delta opioid receptor and the alpha subunit of G(i1) by mutation of a receptor/G protein contact interface.

    PubMed

    Moon, H E; Bahia, D S; Cavalli, A; Hoffmann, M; Milligan, G

    2001-09-01

    Fusion proteins were constructed between the delta opioid receptor and forms of the alpha subunit of G(i1) in which cysteine(351) was mutated to a range of amino acids. GDP reduced the binding of the agonist [(3)H]DADLE but not the antagonist [(3)H]naltrindole to both the receptor alone and all the delta opioid receptor-Cys(351)XaaG(i1)alpha fusion proteins. For the fusion proteins the pEC(50) for GDP was strongly correlated with the n-octanol/H(2)O partition co-efficient of G protein residue(351). Fusion proteins in which this residue was either isoleucine or glycine had similar observed binding kinetics for [(3)H]DADLE. However, the rate of dissociation of [(3)H]DADLE was substantially greater for the glycine-containing fusion protein than that containing isoleucine, indicating that more hydrophobic residues imbued greater stability to the agonist-receptor-G protein ternary complex. This resulted in a higher affinity of binding of [(3)H]DADLE to the fusion protein containing isoleucine(351). In expectation with the binding data, maximal DADLE-stimulated GTP hydrolysis by the isoleucine(351)-containing fusion protein was two-fold greater and the potency of DADLE seven-fold higher than for the version containing glycine. These results demonstrate that the stability of the ternary complex between delta opioid receptor, G(i1)alpha and an agonist (but not antagonist) ligand is dependent upon the nature of residue(351) of the G protein and that this determines the effectiveness of information flow from the receptor to the G protein. PMID:11522323

  2. Preparation and Evaluation at the Delta Opioid Receptor of a Series of Linear Leu-Enkephalin Analogues Obtained by Systematic Replacement of the Amides

    PubMed Central

    2013-01-01

    Leu-enkephalin analogues, in which the amide bonds were sequentially and systematically replaced either by ester or N-methyl amide bonds, were prepared using classical organic chemistry as well as solid phase peptide synthesis (SPPS). The peptidomimetics were characterized using competition binding, ERK1/2 phosphorylation, receptor internalization, and contractility assays to evaluate their pharmacological profile over the delta opioid receptor (DOPr). The lipophilicity (LogD7.4) and plasma stability of the active analogues were also measured. Our results revealed that the last amide bond can be successfully replaced by either an ester or an N-methyl amide bond without significantly decreasing the biological activity of the corresponding analogues when compared to Leu-enkephalin. The peptidomimetics with an N-methyl amide function between residues Phe and Leu were found to be more lipophilic and more stable than Leu-enkephalin. Findings from the present study further revealed that the hydrogen-bond donor properties of the fourth amide of Leu-enkephalin are not important for its biological activity on DOPr. Our results show that the systematic replacement of amide bonds by isosteric functions represents an efficient way to design and synthesize novel peptide analogues with enhanced stability. Our findings further suggest that such a strategy can also be useful to study the biological roles of amide bonds. PMID:23650868

  3. (D-Pen2,4 prime -125I-Phe4,D-Pen5)enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity

    SciTech Connect

    Knapp, R.J.; Sharma, S.D.; Toth, G.; Duong, M.T.; Fang, L.; Bogert, C.L.; Weber, S.J.; Hunt, M.; Davis, T.P.; Wamsley, J.K. )

    1991-09-01

    (D-Pen2,4{prime}-125I-Phe4,D-Pen5)enkephalin ((125I)DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. (125I)DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 {plus minus} 67 pM and a receptor density (Bmax) value of 36.4 {plus minus} 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM (125I) DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 {plus minus} 0.88 {times} 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM (3H) DPDPE and 0.8 nM (3H) (D-Pen2,4{prime}-Cl-Phe4, D-Pen5)enkephalin, respectively. The dissociation rate of (125I)DPDPE (0.917 {plus minus} 0.117 {times} 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of (125I)DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes (125I)DPDPE a valuable new radioligand for studies of delta opioid receptors.

  4. Protective effect of delta opioid receptor agonist (D-Ala2, D-Leu5) enkephalin on permanent focal cerebral ischemia in rats.

    PubMed

    Fu, Danyun; Liu, Haitong; Zhu, Hui; Li, Shitong; Yao, Junyan

    2016-07-01

    To investigate the effect of delta opioid receptor agonist (D-Ala, D-Leu) enkephalin (DADLE) on the permanent focal cerebral ischemia in rats. Thirty four male Sprague-Dawley rats were assigned randomly into three groups: sham group (group Sham, n=10), artificial cerebrospinal fluid group (group ACSF, n=12), and DADLE group (group DADLE, n=12). Permanent middle cerebral artery occlusion was performed to induce permanent focal cerebral ischemia in rats. Then, the animals in group DADLE and group ACSF were treated with DADLE or ACSF by an intracerebroventricular injection at 45 min after ischemia. Neurologic deficit scores were assessed according to the Garcia criterion at 24 h after ischemia. Infarct volume was determined using the 2,3,5-triphenyltetrazolium chloride staining method. The histological analysis was used to evaluate the extent of cerebral injury. Compared with the control group, the Garcia scores were significantly higher (P=0.000) and the infarct volumes (P=0.018) were significantly smaller in the DADLE treatment group at 24 h after ischemia. These neurologic changes were closely correlated with the outcome of the infarct volumes. In addition, the histological examination showed more intact neurons in rats treated with DADLE than those treated with ACSF at 24 h after ischemia (P=0.000). DADLE by intracerebroventricular administration at 45 min after ischemia can improve neurologic outcome and mitigate cortical neuronal injury induced by permanent focal cerebral ischemia in rats. PMID:27232517

  5. Chromosomal localization of the [delta] opioid receptor gene to human 1p34. 3-36. 1 and mouse 4D bands by in situ hybridization

    SciTech Connect

    Befort, K.; Kieffer, B. ); Mattei, M.G.; Roeckel, N. )

    1994-03-01

    The aim of the present study is to determine the precise localization of this gene in the murine as well as human genome by in situ hybridization. Southern analysis, using the noncoding part of the cDNA as a probe (NotI-BamHI fragment, 1040 bp) under high-stringency conditions, shows the existence of a single-copy gene in the murine genome. In a similar analysis performed on human genomic DNA, the coding part of the cDNA (PstI-NotI fragment, 976 bp) clearly detected a single gene in the human genome (not shown). The authors therefore used these two probes for the chromosomal localization of the murine gene and its human counterpart, respectively. Chromosome spread preparations from concanavalin A (mouse) or phytohemagglutinin (human)-stimulated lymphocytes were hybridized with tritium-labeled cDNAs, exposed for 15 days, and developed, as described previously. For the murine assignment, a total of 100 metaphase cells were examined, and 149 silver grains were found. Forty-two grains were associated with chromosome 4, and 73.8% of them mapped to the D1-D3 region of the chromosome. In the 120 metaphase human cells analyzed, 197 silver grains were found, 25.8% of which were located on chromosome 1. The distribution of grains allowed mapping of the human [delta] opioid receptor gene to the p34.3-p36.1 region of the short arm with a maximum in the p35 band.

  6. Delta-Opioid Receptor (δOR) Targeted Near-Infrared Fluorescent Agent for Imaging of Lung Cancer: Synthesis and Evaluation In Vitro and In Vivo.

    PubMed

    Cohen, Allison S; Patek, Renata; Enkemann, Steven A; Johnson, Joseph O; Chen, Tingan; Toloza, Eric; Vagner, Josef; Morse, David L

    2016-02-17

    In the United States, lung cancer is the leading cause of cancer death and ranks second in the number of new cases annually among all types of cancers. Better methods or tools for diagnosing and treating this disease are needed to improve patient outcomes. The delta-opioid receptor (δOR) is reported to be overexpressed in lung cancers and not expressed in normal lung. Thus, we decided to develop a lung cancer-specific imaging agent targeting this receptor. We have previously developed a δOR-targeted fluorescent imaging agent based on a synthetic peptide antagonist (Dmt-Tic) conjugated to a Cy5 fluorescent dye. In this work, we describe the synthesis of Dmt-Tic conjugated to a longer wavelength near-infrared fluorescent (NIRF) dye, Li-cor IR800CW. Binding affinity of Dmt-Tic-IR800 for the δOR was studied using lanthanide time-resolved fluorescence (LTRF) competitive binding assays in cells engineered to overexpress the δOR. In addition, we identified lung cancer cell lines with high and low endogenous expression of the δOR. We confirmed protein expression in these cell lines using confocal fluorescence microscopy imaging and used this technique to estimate the cell-surface receptor number in the endogenously expressing lung cancer cell lines. The selectivity of Dmt-Tic-IR800 for imaging of the δOR in vivo was shown using both engineered cell lines and endogenously expressing lung cancer cells in subcutaneous xenograft models in mice. In conclusion, the δOR-specific fluorescent probe developed in this study displays excellent potential for imaging of lung cancer. PMID:26488422

  7. Opposite role of delta 1- and delta 2-opioid receptors activated by endogenous or exogenous opioid agonists on the endogenous cholecystokinin system: further evidence for delta-opioid receptor heterogeneity.

    PubMed

    Noble, F; Fournie-Zaluski, M C; Roques, B P

    1996-12-01

    Using the mouse caudate-putamen, where delta-opioid receptor subtypes have been shown to regulate adenylyl cyclase activity, we show in this study that endogenous enkephalins inhibit enzyme activity through activation of delta 1- and delta 2-opioid receptors. Thus, naltriben or 7-benzylidenenaltrexone as well as the delta-selective antagonist naltrindole (mixed delta 1 and delta 2 antagonist) antagonized inhibition of adenylyl cyclase activity induced by methionine- or leucine-enkephalin, while the micro-antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) was without effect. Furthermore, we have previously shown that activation of delta-opioid receptors increases cholecystokinin release in the central nervous system, resulting in a potentiation of micro-opioid antinociceptive responses, and the respective role of delta 1- and delta 2-opioid receptors in this facilitatory effect has now been evaluated. Activation of delta 2-opioid receptors, either by endogenous enkephalins protected from catabolism by the complete enkephalin-degrading enzyme inhibitor N-((R,S)-2-benzyl-3((S)(2-amino-4-methyl-thio) butyldithio)-1-oxopropyl)-L-phenyl-alanine benzyl ester (RB 101), or by the delta 2-selective agonist Tyr-D-Ser(O-tert-butyl)-Gly-Phe-Leu-Thr(O-tert-butyl) (BUBU), potentiated micro-opioid antinociceptive responses in the hot-plate test in mice. This effect was antagonized by a selective cholecystokinin-A antagonist. Activation of delta 1-opioid receptors by endogenous opioid peptides decreased the micro-opioid responses. These results suggest that stimulation of delta 2-opioid receptors potentiates micro-opioid analgesia in the hot-plate test in mice through an increase in endogenous cholecystokinin release, while activation of delta 1-opioid receptors could decrease it. Thus, the pre-existing physiological balance between opioid and cholecystokinin systems seems to be modulated in opposite directions depending on whether delta 1- or delta 2-opioid receptors are

  8. Peripheral G protein-coupled inwardly rectifying potassium (GIRK) channels are involved in delta opioid receptor-mediated anti-hyperalgesia in rat masseter muscle

    PubMed Central

    Chung, Man-Kyo; Cho, Yi Sul; Bae, Young Chul; Lee, Jongseok; Zhang, Xia; Ro, Jin Y.

    2014-01-01

    Background Although the efficacy of peripherally administered opioid has been demonstrated in preclinical and clinical studies, the underlying mechanisms of its anti-hyperalgesic effects are poorly understood. G protein-coupled inwardly rectifying potassium (GIRK) channels are linked to opioid receptors in the brain. However, the role of peripheral GIRK channels in analgesia induced by peripherally administered opioid, especially in trigeminal system, is not clear. Methods Expression of GIRK subunits in rat trigeminal ganglia (TG) was examined with RT-PCR, western blot and immunohistochemistry. Chemical profiles of GIRK expressing neurons in TG were further characterized. Behavioral and Fos experiments were performed to examine the functional involvement of GIRK channels in delta opioid receptor (DOR)-mediated anti-hyperalgesia under an acute myositis condition. Results TG expressed mRNA and proteins for GIRK1 and GIRK2 subunits. Majority of GIRK1- and GIRK2-expressing neurons were non-peptidergic afferents. Inhibition of peripheral GIRK using Tertiapin-Q (TPQ) attenuated anti-nociceptive effects of peripherally administered DOR agonist, DPDPE, on mechanical hypersensitivity in masseter muscle. Furthermore, TPQ attenuated the suppressive effects of peripheral DPDPE on neuronal activation in the subnucleus caudalis of the trigeminal nucleus (Vc) following masseteric injection of capsaicin. Conclusions Our data indicate that peripheral DOR agonist-induced suppression of mechanical hypersensitivity in the masseter muscle involves the activity of peripheral GIRK channels. These results could provide a rationale for developing a novel therapeutic approach using peripheral GIRK channel openers to mimic or supplement the effects of peripheral opioid agonist. PMID:23740773

  9. Quantitative conformationally sampled pharmacophore for delta opioid ligands: reevaluation of hydrophobic moieties essential for biological activity.

    PubMed

    Bernard, Denzil; Coop, Andrew; MacKerell, Alexander D

    2007-04-19

    Recent studies have indicated several therapeutic applications for delta opioid agonists and antagonists. To exploit the therapeutic potential of delta opioids developing a structural basis for the activity of ligands at the delta opioid receptor is essential. The conformationally sampled pharmacophore (CSP) method (Bernard et al. J. Am. Chem. Soc. 2003, 125, 3103-3107) is extended here to obtain quantitative models of delta opioid ligand efficacy and affinity. Quantification is performed via overlap integrals of the conformational space sampled by ligands with respect to a reference compound. Iterative refinement of the CSP model identified hydrophobic groups other than the traditional phenylalanine residues as important for efficacy and affinity in DSLET and ICI 174 864. The obtained models for a structurally diverse set of peptidic and nonpeptidic delta opioid ligands offer good predictions with R2 values>0.9, and the predicted efficacy for a set of test compounds was consistent with the experimental values. PMID:17367120

  10. Delta-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2.

    PubMed Central

    McKenzie, F R; Milligan, G

    1990-01-01

    Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID

  11. Cloned delta-opioid receptors in GH(3) cells inhibit spontaneous Ca(2+) oscillations and prolactin release through K(IR) channel activation.

    PubMed

    Piros, E T; Charles, R C; Song, L; Evans, C J; Hales, T G

    2000-05-01

    Opioid receptors can couple to K(+) and Ca(2+) channels, adenylyl cyclase, and phosphatidyl inositol turnover. Any of these actions may be important in the regulation of neurotransmitter and hormone release from excitable cells. GH(3) cells exhibit spontaneous oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) and prolactin release. Activation of cloned delta-opioid receptors stably expressed in GH(3) cells inhibits both spontaneous Ca(2+) signaling and basal prolactin release. The objective of this study was to examine a possible role for K(+) channels in these processes using the patch-clamp technique, fluorescence imaging, and a sensitive ELISA for prolactin. The selective delta receptor agonist [D-Pen(2), D-Pen(2)]enkephalin (DPDPE) inhibited [Ca(2+)](i) oscillations in GH(3) cells expressing both mu and delta receptors (GH(3)MORDOR cells) but had no effect on control GH(3) cells or cells expressing mu receptors alone (GH(3)MOR cells). The inhibition of [Ca(2+)](i) oscillations by DPDPE was unaffected by thapsigargin pretreatment, suggesting that this effect is independent of inositol 1,4,5-triphosphate-sensitive Ca(2+) stores. DPDPE caused a concentration-dependent inhibition of prolactin release from GH(3)MORDOR cells with an IC(50) of 4 nM. DPDPE increased inward K(+) current recorded from GH(3)MORDOR cells but had no significant effect on K(+) currents recorded from control GH(3) cells or GH(3)MOR cells. The mu receptor agonist morphine also had no effect on currents recorded from control cells but activated inward K(+) currents recorded from GH(3)MOR and GH(3)MORDOR cells. Somatostatin activated inward currents recorded from all three cell lines. The DPDPE-sensitive K(+) current was inwardly rectifying and was inhibited by Ba(2+) but not TEA. DPDPE had no effect on delayed rectifier-, Ca(2+)-, and voltage-activated or A-type K(+) currents, recorded from GH(3)MORDOR cells. Ba(2+) attenuated the inhibition of [Ca(2+)](i) and prolactin release

  12. Plasticity of α2-adrenergic spinal antinociception following nerve injury: selective, bidirectional interaction with the delta opioid receptor.

    PubMed

    Aira, Zigor; Barrenetxea, Teresa; Buesa, Itsaso; Azkue, Jon Jatsu

    2015-01-12

    Interactions of opioid receptors with other receptor families can be made use of to improve analgesia and reduce adverse effects of opioid analgesics. We investigated interactions of the α2-adrenergic receptor (α2AR) with opioid receptors of the mu (MOR) and delta (DOR) types in the spinal dorsal horn in an animal model of neuropathic pain induced by spinal nerve ligation. Nine days after nerve injury, immunoreactivity for the α2AR subtype A (α2AAR) was increased both in tissue homogenates and at pre- and post-synaptic sites in transverse sections. The efficacy of spinally administered α2AAR agonist guanfacine at reducing C-fiber-evoked field potentials was increased in nerve-ligated rats. This reducing effect was impaired by simultaneous administration of DOR antagonist naltrindole, but not MOR antagonist CTOP, suggesting that concurrent DOR activation was required for α2AAR-mediated inhibition. While DOR agonist deltorphin II and MOR agonist DAMGO both effectively depressed C-fiber-evoked spinal field potentials, DOR- but not MOR-mediated depression was enhanced by subclinical guanfacine. In conscious, nerve-ligated rats, chronically administered deltorphin II produced stable thermal and mechanical antinociception over the 9 following days after nerve injury without apparent signs of habituation. Such an effect was dramatically enhanced by co-administration of a low dose of guanfacine, which reversed thermal and mechanical thresholds to levels near those prior to injury. The results suggest that spinal, α2AAR-mediated antinociception is increased after nerve injury and based on DOR co-activation. We demonstrate in vivo that α2AAR/DOR interaction can be exploited to provide effective behavioral antinociception during neuropathic pain. PMID:25446445

  13. Proglumide exhibits delta opioid agonist properties.

    PubMed

    Rezvani, A; Stokes, K B; Rhoads, D L; Way, E L

    1987-01-01

    Recently, it was reported that proglumide, a cholecystokinin (CCK) antagonist, potentiates the analgetic effects of morphine and endogenous opioid peptides and reverses morphine tolerance by antagonizing the CCK system in the central nervous system of the rat. In order to provide additional insight into the mode of action of this agent, we assessed the effect of proglumide in the isolated guinea pig ileum and the mouse, rat and rabbit vas deferens. Furthermore, we studied the in vitro binding affinity of this substance to mouse brain synaptosomes. Our results show that proglumide inhibits, dose dependently, the electrically stimulated twitches in the mouse vas deferens and guinea pig ileum, but not in the rat or rabbit vas deferens. The inhibitory action of proglumide on the mouse vas deferens, but not on the guinea pig ileum, is antagonized by naloxone and by the selective delta-antagonist, ICI 174,864, in a competitive fashion. Other CCK antagonists were found to be devoid of such activity on the mouse vas deferens. In vitro binding studies showed that proglumide displaces D-ala-D-[leucine]5-enkephalin (DADLE), a delta agonist, but not ethylketocyclazocine (EKC), a preferentially selective kappa agonist. The effect of proglumide appeared to be elicited presynaptically since it did not alter the norepinephrine-induced contractions of the mouse vas deferens. Our results suggest that proglumide might exert its opiate-like effects by activation of delta-opioid receptors. PMID:3030338

  14. ( sup 3 H)(D-PEN sup 2 , D-PEN sup 5 ) enkephalin binding to delta opioid receptors on intact neuroblastoma-glioma (NG 108-15) hybrid cells

    SciTech Connect

    Knapp, R.J.; Yamamura, H.I. )

    1990-01-01

    ({sup 3}H)(D-Pen{sup 2}, D-Pen{sup 5})enkephalin binding to intact NG 108-15 cells has been measured under physiological conditions of temperature and medium. The dissociation constant, receptor density, and Hill slope values measured under these conditions are consistent with values obtained by others using membranes prepared from these cells. Kinetic analysis of the radioligand binding to these cells show biphasic association and monophasic dissociation processes suggesting the presence of different receptor affinity states for the agonist. The data show that the binding affinity of ({sup 3}H)(D-Pen{sup 2}, D-Pen{sup 5})enkephalin under physiological conditions is not substantially different to that measured in 50 mM Tris buffer using cell membrane fractions. Unlike DPDPE, the {mu} opioid agonists morphine, normorphine, PL-17, and DAMGO, have much lower affinity for the {delta} receptor measured under these conditions than is observed by studies using 50 mM Tris buffer. The results described here suggest that this assay may serve as a useful model of {delta} opioid receptor binding in vivo.

  15. Modulation of MDMA-induced behavioral and transcriptional effects by the delta opioid antagonist naltrindole in mice

    PubMed Central

    Belkaï, Emilie; Scherrmann, Jean-Michel; Noble, Florence; Marie-Claire, Cynthia

    2009-01-01

    The delta opioid system is involved in the behavioral effects of various drugs of abuse. However, only few studies have focused on the possible interactions between the opioid system and the effects of MDMA. In order to examine the possible role of the delta opioid system in MDMA-induced behaviors in mice, locomotor activity and conditioned place preference were investigated in the presence of naltrindole, a selective delta opioid antagonist. Moreover, the consequences of acute and chronic MDMA administration on Penk (pro-enkephalin) and Pomc (pro-opioimelanocortin) gene expression were assessed by quantitative real-time PCR. The results showed that, after acute MDMA administration (9mg/kg; i.p.), naltrindole (5mg/kg, s.c.) was able to totally block MDMA-induced hyperlocomotion. Penk expression gene was not modulated by acute MDMA but a decrease of Pomc gene expression was observed that was not antagonized by naltrindole. Administration of the antagonist prevented the acquisition of MDMA-induced conditioned place preference, suggesting an implication of the delta opioid receptors in this behavior. Following chronic MDMA treatment only the level of Pomc was modulated. The observed increase was totally blocked by naltrindole pretreatment. All these results confirm the interactions between the delta opioid system (receptors and peptides) and the effects of MDMA. PMID:19523041

  16. Modulation of MDMA-induced behavioral and transcriptional effects by the delta opioid antagonist naltrindole in mice.

    PubMed

    Belkaï, Emilie; Scherrmann, Jean-Michel; Noble, Florence; Marie-Claire, Cynthia

    2009-07-01

    The delta opioid system is involved in the behavioral effects of various drugs of abuse. However, only a few studies have focused on the possible interactions between the opioid system and the effects of 3,4-methylenedioxymethamphetamine (MDMA). In order to examine the possible role of the delta opioid system in MDMA-induced behaviors in mice, locomotor activity and conditioned place preference (CPP) were investigated in the presence of naltrindole (NTI), a selective delta opioid antagonist. Moreover, the consequences of acute and chronic MDMA administration on pro-enkephalin (Penk) and pro-opiomelanocortin (Pomc) gene expression were assessed by real-time quantitative polymerase chain reaction (QPCR). The results showed that, after acute MDMA administration (9 mg/kg; i.p.), NTI (5 mg/kg, s.c.) was able to totally block MDMA-induced hyperlocomotion. Penk gene expression was not modulated by acute MDMA, but a decrease of Pomc gene expression was observed, which was not antagonized by NTI. Administration of the antagonist prevented the acquisition of MDMA-induced CPP, suggesting an implication of the delta opioid receptors in this behavior. Following chronic MDMA treatment, only the level of Pomc was modulated. The observed increase was totally blocked by NTI pre-treatment. All these results confirm the interactions between the delta opioid system (receptors and peptides) and the effects of MDMA. PMID:19523041

  17. Case-control association analysis of polymorphisms in the delta-opioid receptor, OPRD1, with cocaine and opioid addicted populations*

    PubMed Central

    Crist, R.C.; Ambrose-Lanci, L.M.; Vaswani, M.; Clarke, T.K.; Zeng, A.; Yuan, C.; Ferraro, T.N.; Hakonarson, H.; Kampman, K.M.; Dackis, C.A.; Pettinati, H.M.; O’Brien, C.P.; Oslin, D.W.; Doyle, G.A.; Lohoff, F.W.; Berrettini, W.H.

    2012-01-01

    BACKGROUND Addiction susceptibility and treatment responsiveness are greatly influenced by genetic factors. Sequence variation in genes involved in the mechanisms of drug action have the potential to influence addiction risk and treatment outcome. The opioid receptor system is involved in mediating the rewarding effects of cocaine and opioids. The µ-opioid receptor (MOR) has traditionally been considered the primary target for opioid addiction. The MOR, however, interacts with and is regulated by many known MOR interacting proteins (MORIPs), including the δ-opioid receptor (DOR). METHODS The present study evaluated the contribution of OPRD1, the gene encoding the DOR, to the risk of addiction to opioids and cocaine. The association of OPRD1 polymorphisms with both opioid addiction (OA) and cocaine addiction (CA) was analyzed in African American (OA n=336, CA n=503) and European American (OA n=1007, CA n=336) populations. RESULTS The primary finding of this study is an association of rs678849 with cocaine addiction in African Americans (allelic p=0.0086). For replication purposes, this SNP was analyzed in a larger independent population of cocaine addicted African Americans and controls and the association was confirmed (allelic p=4.53×10−5; n=993). By performing a meta-analysis on the expanded populations, the statistical evidence for an association was substantially increased (allelic p=8.5 × 10 −7) (p-values non-FDR corrected). CONCLUSION The present study suggests that polymorphisms in OPRD1 are relevant for cocaine addiction in the African American population and provides additional support for a broad role for OPRD1 variants in drug dependence. PMID:22795689

  18. Opposite effects of mu and delta opioid receptor agonists on excitatory propagation induced in rat somatosensory and insular cortices by dental pulp stimulation.

    PubMed

    Yokota, Eiko; Koyanagi, Yuko; Nakamura, Hiroko; Horinuki, Eri; Oi, Yoshiyuki; Kobayashi, Masayuki

    2016-08-15

    The insular cortex (IC) contributes to nociceptive information processing. IC neurons express opioid receptors, including the mu (MOR), kappa (KOR), and delta (DOR) subtypes. Opioidergic agonists suppress excitatory synaptic transmission in the cerebral cortex. In addition, morphine injection into the IC reduces responses to noxious thermal stimuli. However, the mechanisms of the opioid-dependent modulation of cortical excitation at the macroscopic level, which bridge the cellular and behavioral findings, have remained unknown. The present in vivo optical imaging study aimed to examine the effects of the agonists of each subtype on cortical excitatory propagation in the IC and the neighboring cortices, the primary (S1) and secondary somatosensory (S2) areas. To assess the opioidergic effects on the cortical circuits, we applied electrical stimulation to the maxillary 1st molar pulp, which induced excitation in the ventral part of S1 and the S2/insular oral region (IOR). The initial excitatory response was observed 10-14ms after stimulation, and then excitation propagated concentrically. DAMGO (10-100μM), an MOR agonist, suppressed the amplitude of cortical excitation and shrank the maximum excitation areas in S1 and S2/IOR. In contrast, 10-100μM DPDPE, a DOR agonist, increased the amplitude of excitation and expanded the area of maximum excitation. U50488 (10-100μM), a KOR agonist, had little effect on cortical excitation. These results suggest that MOR-induced suppression of excitatory propagation in the IC is an underlying mechanism of the powerful analgesic effects of MOR agonists. In contrast, DOR may play a minor role in suppressing acute pain. PMID:27246300

  19. Endocytosis and Intracellular Trafficking of Human Natural Killer Cell Receptors

    PubMed Central

    Masilamani, Madhan; Peruzzi, Giovanna; Borrego, Francisco; Coligan, John E.

    2009-01-01

    Natural killer (NK) cells play a vital role in the defense against viral infections and tumor development. NK cell function is primarily regulated by the sum of signals from a broad array of activation and inhibitory receptors. Key to generating the input level of either activating or inhibitory signals is the maintenance of receptor expression levels on the cell surface. Although the mechanisms of endocytosis and trafficking for some cell surface receptors, such as transferrin receptor, and certain immune receptors, are very well known, that is not the situation for receptors expressed by NK cells. Recent studies have uncovered that endocytosis and trafficking routes characteristic for specific activation and inhibitory receptors can regulate the functional responses of NK cells. In this review, we summarize the current knowledge of receptor endocytosis and trafficking, and integrate this with our current understanding of NK cell receptor trafficking. PMID:19719476

  20. Death-receptor activation halts clathrin-dependent endocytosis

    PubMed Central

    Austin, Cary D.; Lawrence, David A.; Peden, Andrew A.; Varfolomeev, Eugene E.; Totpal, Klara; De Mazière, Ann M.; Klumperman, Judith; Arnott, David; Pham, Victoria; Scheller, Richard H.; Ashkenazi, Avi

    2006-01-01

    Endocytosis is crucial for various aspects of cell homeostasis. Here, we show that proapoptotic death receptors (DRs) trigger selective destruction of the clathrin-dependent endocytosis machinery. DR stimulation induced rapid, caspase-mediated cleavage of key clathrin-pathway components, halting cellular uptake of the classic cargo protein transferrin. DR-proximal initiator caspases cleaved the clathrin adaptor subunit AP2α between functionally distinct domains, whereas effector caspases processed clathrin’s heavy chain. DR5 underwent ligand-induced, clathrin-mediated endocytosis, suggesting that internalization of DR signaling complexes facilitates clathrin-pathway targeting by caspases. An endocytosis-blocking, temperature-sensitive dynamin-1 mutant attenuated DR internalization, enhanced caspase stimulation downstream of DRs, and increased apoptosis. Thus, DR-triggered caspase activity disrupts clathrin-dependent endocytosis, leading to amplification of programmed cell death. PMID:16801533

  1. Multiscale Modeling of Virus Entry via Receptor-Mediated Endocytosis

    NASA Astrophysics Data System (ADS)

    Liu, Jin

    2012-11-01

    Virus infections are ubiquitous and remain major threats to human health worldwide. Viruses are intracellular parasites and must enter host cells to initiate infection. Receptor-mediated endocytosis is the most common entry pathway taken by viruses, the whole process is highly complex and dictated by various events, such as virus motions, membrane deformations, receptor diffusion and ligand-receptor reactions, occurring at multiple length and time scales. We develop a multiscale model for virus entry through receptor-mediated endocytosis. The binding of virus to cell surface is based on a mesoscale three dimensional stochastic adhesion model, the internalization (endocytosis) of virus and cellular membrane deformation is based on the discretization of Helfrich Hamiltonian in a curvilinear space using Monte Carlo method. The multiscale model is based on the combination of these two models. We will implement this model to study the herpes simplex virus entry into B78 cells and compare the model predictions with experimental measurements.

  2. Pyrrolo- and Pyridomorphinans: Non-selective opioid antagonists and delta opioid agonists/mu opioid partial agonists

    PubMed Central

    Kumar, V.; Clark, M.J.; Traynor, J.R.; Lewis, J.W.; Husbands, S.M.

    2014-01-01

    Opioid ligands have found use in a number of therapeutic areas, including for the treatment of pain and opiate addiction (using agonists) and alcohol addiction (using antagonists such as naltrexone and nalmefene). The reaction of imines, derived from the opioid ligands oxymorphone and naltrexone, with Michael acceptors leads to pyridomorphinans with structures similar to known pyrrolo- and indolomorphinans. One of the synthesized compounds, 5e, derived from oxymorphone had substantial agonist activity at delta opioid receptors but not at mu and/or kappa opioid receptors and in that sense profiled as a selective delta opioid receptor agonist. The pyridomorphinans derived from naltrexone and naloxone were all found to be non-selective potent antagonists and as such could have utility as treatments for alcohol abuse. PMID:24973818

  3. Pyrrolo- and pyridomorphinans: non-selective opioid antagonists and delta opioid agonists/mu opioid partial agonists.

    PubMed

    Kumar, V; Clark, M J; Traynor, J R; Lewis, J W; Husbands, S M

    2014-08-01

    Opioid ligands have found use in a number of therapeutic areas, including for the treatment of pain and opiate addiction (using agonists) and alcohol addiction (using antagonists such as naltrexone and nalmefene). The reaction of imines, derived from the opioid ligands oxymorphone and naltrexone, with Michael acceptors leads to pyridomorphinans with structures similar to known pyrrolo- and indolomorphinans. One of the synthesized compounds, 5e, derived from oxymorphone had substantial agonist activity at delta opioid receptors but not at mu and/or kappa opioid receptors and in that sense profiled as a selective delta opioid receptor agonist. The pyridomorphinans derived from naltrexone and naloxone were all found to be non-selective potent antagonists and as such could have utility as treatments for alcohol abuse. PMID:24973818

  4. Crosslinking-Induced Endocytosis of Acetylcholine Receptors by Quantum Dots

    PubMed Central

    Geng, Lin; Peng, H. Benjamin

    2014-01-01

    In a majority of patients with myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies target postsynaptic AChR clusters and thus compromise the membrane integrity of neuromuscular junctions (NMJs) and lead to muscle weakness. Antibody-induced endocytosis of AChRs in the postsynaptic membrane represents the initial step in the pathogenesis of MG; however, the molecular mechanisms underlying AChR endocytosis remain largely unknown. Here, we developed an approach to mimic the pathogenic antibodies for inducing the crosslinking and internalization of AChRs from the postsynaptic membrane. Using biotin-α-bungarotoxin and quantum dot (QD)-streptavidin, cell-surface and internalized AChRs could be readily distinguished by comparing the size, fluorescence intensity, trajectory, and subcellular localization of the QD signals. QD-induced AChR endocytosis was mediated by clathrin-dependent and caveolin-independent mechanisms, and the trafficking of internalized AChRs in the early endosomes required the integrity of microtubule structures. Furthermore, activation of the agrin/MuSK (muscle-specific kinase) signaling pathway strongly suppressed QD-induced internalization of AChRs. Lastly, QD-induced AChR crosslinking potentiated the dispersal of aneural AChR clusters upon synaptic induction. Taken together, our results identify a novel approach to study the mechanisms of AChR trafficking upon receptor crosslinking and endocytosis, and demonstrate that agrin-MuSK signaling pathways protect against crosslinking-induced endocytosis of AChRs. PMID:24587270

  5. Delta opioid modulation of hypothalamic serotonergic neurotransmission in the ovariectomized and steroid-primed rat.

    PubMed

    Yilmaz, Bayram; Gilmore, Desmond P.

    2000-01-01

    OBJECTIVES: We have investigated the modulating effects of DPDPE (a delta opioid agonist) and opioid receptor antagonists on both LH release and indoleamine concentrations in specific hypothalamic regions of the ovx and steroid-primed rat. METHODS: DPDPE was intracerebroventricularly infused alone or with either ICI 154129 (a delta opioid antagonist) or naloxone under ketamine anesthesia. Blood samples were collected at hourly intervals on the afternoon of the anticipated LH surge. The rats were then decapitated and the medial preoptic area (MPOA), suprachiasmatic nucleus (SCN), median eminence (ME) and arcuate nucleus (ARN) surgically isolated by micropunch. Concentrations of 5-HT and its metabolite (5-HIAA) in these samples were determined by HPLC with ECD. Plasma LH levels were measured by RIA. RESULTS: The delta-agonist significantly reduced 5-HT concentrations in the SCN, ME and ARN, but not in the MPOA. 5-HIAA levels were decreased, but these changes were significant in only the MPOA and ARN compared to the control group. ICI 154129 had no significant effects on 5-HT release and turnover in any of the hypothalamic regions examined. However, co-administration of DPDPE with naloxone resulted in significant increases in 5-HT and 5-HIAA concentrations in the MPOA, SCN, ME and ARN compared to the DPDPE-treated group. Plasma LH levels were either low or undetectable in all groups. CONCLUSIONS: The present results suggest that delta-opioid receptors are involved in the opioid inhibition of the serotonergic neurotransmission in the hypothalamus. It is thought that the ketamine anesthesia interfered with LH secretory systems. PMID:11455339

  6. Constitutive Endocytosis of VEGFR2 Protects the Receptor against Shedding.

    PubMed

    Basagiannis, Dimitris; Christoforidis, Savvas

    2016-08-01

    VEGFR2 plays a fundamental role in blood vessel formation and in life threatening diseases, such as cancer angiogenesis and cardiovascular disorders. Although inactive growth factor receptors are mainly localized at the plasma membrane, VEGFR2 undergoes constitutive endocytosis (in the absence of ligand) and recycling. Intriguingly, the significance of these futile transport cycles of VEGFR2 remains unclear. Here we found that, unexpectedly, the function of constitutive endocytosis of VEGFR2 is to protect the receptor against plasma membrane cleavage (shedding), thereby preserving the functional state of the receptor until the time of activation by VEGF. Inhibition of constitutive endocytosis of VEGFR2, by interference with the function of clathrin, dynamin, or Rab5, increases dramatically the cleavage/shedding of VEGFR2. Shedding of VEGFR2 produces an N-terminal soluble fragment (100 kDa, s100), which is released in the extracellular space, and a residual C-terminal part (130 kDa, p130) that remains integrated at the plasma membrane. The released soluble fragment (s100) co-immunoprecipitates with VEGF, in line with the topology of the VEGF-binding domain at the N terminus of VEGFR2. Increased shedding of VEGFR2 (via inhibition of constitutive endocytosis) results in reduced response to VEGF, consistently with the loss of the VEGF-binding domain from the membrane remnant of VEGFR2. These data suggest that constitutive internalization of VEGFR2 protects the receptor against shedding and provides evidence for an unprecedented mechanism via which endocytosis can regulate the fate and activity of growth factor receptors. PMID:27298320

  7. Revisiting the Endocytosis of the M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Ockenga, Wymke; Tikkanen, Ritva

    2015-01-01

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles. PMID:25985102

  8. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

    PubMed

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

    2016-01-15

    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis. PMID:26711579

  9. Targeted gene delivery via N-acetylglucosamine receptor mediated endocytosis.

    PubMed

    Singh, Bijay; Maharjan, Sushila; Kim, You-Kyoung; Jiang, Tai; Islam, Mohammad Ariful; Kang, Sang-Kee; Cho, Myung-Haing; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-01

    Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational

  10. Receptor-mediated endocytosis and brain delivery of therapeutic biologics.

    PubMed

    Xiao, Guangqing; Gan, Liang-Shang

    2013-01-01

    Transport of macromolecules across the blood-brain-barrier (BBB) requires both specific and nonspecific interactions between macromolecules and proteins/receptors expressed on the luminal and/or the abluminal surfaces of the brain capillary endothelial cells. Endocytosis and transcytosis play important roles in the distribution of macromolecules. Due to the tight junction of BBB, brain delivery of traditional therapeutic proteins with large molecular weight is generally not possible. There are multiple pathways through which macromolecules can be taken up into cells through both specific and nonspecific interactions with proteins/receptors on the cell surface. This review is focused on the current knowledge of receptor-mediated endocytosis/transcytosis and brain delivery using the Angiopep-2-conjugated system and the molecular Trojan horses. In addition, the role of neonatal Fc receptor (FcRn) in regulating the efflux of Immunoglobulin G (IgG) from brain to blood, and approaches to improve the pharmacokinetics of therapeutic biologics by generating Fc fusion proteins, and increasing the pH dependent binding affinity between Fc and FcRn, are discussed. PMID:23840214

  11. Receptor-Mediated Endocytosis and Brain Delivery of Therapeutic Biologics

    PubMed Central

    Xiao, Guangqing

    2013-01-01

    Transport of macromolecules across the blood-brain-barrier (BBB) requires both specific and nonspecific interactions between macromolecules and proteins/receptors expressed on the luminal and/or the abluminal surfaces of the brain capillary endothelial cells. Endocytosis and transcytosis play important roles in the distribution of macromolecules. Due to the tight junction of BBB, brain delivery of traditional therapeutic proteins with large molecular weight is generally not possible. There are multiple pathways through which macromolecules can be taken up into cells through both specific and nonspecific interactions with proteins/receptors on the cell surface. This review is focused on the current knowledge of receptor-mediated endocytosis/transcytosis and brain delivery using the Angiopep-2-conjugated system and the molecular Trojan horses. In addition, the role of neonatal Fc receptor (FcRn) in regulating the efflux of Immunoglobulin G (IgG) from brain to blood, and approaches to improve the pharmacokinetics of therapeutic biologics by generating Fc fusion proteins, and increasing the pH dependent binding affinity between Fc and FcRn, are discussed. PMID:23840214

  12. USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

    PubMed Central

    Jaworski, Jakub; de la Vega, Michelle; Fletcher, Sarah J.; McFarlane, Cheryl; Greene, Michelle K.; Smyth, Andrew W.; Van Schaeybroeck, Sandra; Johnston, James A.; Scott, Christopher J.; Rappoport, Joshua Z.; Burrows, James F.

    2014-01-01

    Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of ‘CaaX’ motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis. PMID:25026282

  13. The selective non-peptidic delta opioid agonist SNC80 does not facilitate intracranial self-stimulation in rats

    PubMed Central

    Carmo, Gail Pereira Do; Folk, John E.; Rice, Kenner C.; Chartoff, Elena; Carlezon, William A.; Negus, S. Stevens

    2009-01-01

    Delta opioid receptor agonists are under development for a variety of clinical applications, and some findings in rats raise the possibility that agents with this mechanism have abuse liability. The present study assessed the effects of the non-peptidic delta opioid agonist SNC80 in an assay of intracranial self-stimulation (ICSS) in rats. ICSS was examined at multiple stimulation frequencies to permit generation of frequency-response rate curves and evaluation of curve shifts produced by experimental manipulations. Drug-induced leftward shifts in ICSS frequency-rate curves are often interpreted as evidence of abuse liability. However, SNC80 (1.0-10 mg/kg s.c.; 10-56 mg/kg i.p.) failed to alter ICSS frequency-rate curves at doses up to those that produced convulsions in the present study or other effects (e.g. antidepressant effects) in previous studies. For comparison, the monoamine releaser d-amphetamine (0.1-1.0 mg/kg, i.p.) and the kappa agonist U69,593 (0.1-0.56 mg/kg, i.p.) produced dose-dependent leftward and rightward shifts, respectively, in ICSS frequency-rate curves, confirming the sensitivity of the procedure to drug effects. ICSS frequency-rate curves were also shifted by two non-pharmacological manipulations (reductions in stimulus intensity and increases in response requirement). Thus, SNC80 failed to facilitate or attenuate ICSS-maintained responding under conditions in which other pharmacological and non-pharmacological manipulations were effective. These results suggest that non-peptidic delta opioid receptor agonists have negligible abuse-related effects in rats. PMID:19133255

  14. The selective delta opioid agonist SNC80 enhances amphetamine-mediated efflux of dopamine from rat striatum.

    PubMed

    Bosse, Kelly E; Jutkiewicz, Emily M; Gnegy, Margaret E; Traynor, John R

    2008-10-01

    The highly selective delta opioid agonist, SNC80, elicits dopamine-related behaviors including locomotor stimulation and conditioned place-preference. In contrast, it has been reported that SNC80 fails to promote dopamine efflux from the striatum of freely moving rats. However, SNC80 does enhance behavioral responses to the stimulants, amphetamine and cocaine, suggesting an interaction between delta opioids and psychostimulants. Since the increase in locomotor activity elicited by amphetamine and related stimulants acting at the dopamine transporter is associated with increases in extracellular concentrations of dopamine within the striatum, we hypothesized that SNC80 enhances this activity by potentiating the overflow of dopamine through the transporter. To test this hypothesis, striatal preparations from Sprague Dawley rats were assayed for dopamine efflux in response to amphetamine challenge. SNC80 was given either in vivo or in vitro directly to rat striatal tissue, prior to in vitro amphetamine challenge. Both in vivo and in vitro administration of SNC80 enhanced amphetamine-mediated dopamine efflux in a concentration- and time-dependent manner. However, SNC80 in either treatment paradigm produced no stimulation of dopamine efflux in the absence of amphetamine. The effect of SNC80 on amphetamine-mediated dopamine overflow, but not the effect of amphetamine alone, was blocked by the delta selective antagonist, naltrindole and was also observed with other delta agonists. The results of this study demonstrate that even though SNC80 does not stimulate dopamine efflux alone, it is able to augment amphetamine-mediated dopamine efflux through a delta opioid receptor mediated action locally in the striatum. PMID:18602932

  15. A luminescent assay for real-time measurements of receptor endocytosis in living cells.

    PubMed

    Robers, Matthew B; Binkowski, Brock F; Cong, Mei; Zimprich, Chad; Corona, Cesear; McDougall, Mark; Otto, George; Eggers, Christopher T; Hartnett, Jim; Machleidt, Thomas; Fan, Frank; Wood, Keith V

    2015-11-15

    Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs. PMID:26278171

  16. Kainate receptor activation induces glycine receptor endocytosis through PKC deSUMOylation.

    PubMed

    Sun, Hao; Lu, Li; Zuo, Yong; Wang, Yan; Jiao, Yingfu; Zeng, Wei-Zheng; Huang, Chao; Zhu, Michael X; Zamponi, Gerald W; Zhou, Tong; Xu, Tian-Le; Cheng, Jinke; Li, Yong

    2014-01-01

    Surface expression and regulated endocytosis of glycine receptors (GlyRs) play a critical function in balancing neuronal excitability. SUMOylation (SUMO modification) is of critical importance for maintaining neuronal function in the central nervous system. Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices. This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC. SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels. Altogether, we have identified a SUMOylation-dependent regulatory pathway for GlyR endocytosis, which may have important physiological implications for proper neuronal excitability. PMID:25236484

  17. [Molecular physiology of receptor mediated endocytosis and its role in overcoming multidrug resistance].

    PubMed

    Severin, E S; Posypanova, G A

    2011-06-01

    Receptor-mediated endocytosis plays important role in the selective uptake of proteins at the plasma membrane of eukaryotic cells. Endocytosis regulates many processes of cell signalling by controlling the number of functional receptors on the cell surface. The article reviews the mechanism of clathrin-dependent endocytosis and the possibility of using this phenomenon for the targeted delivery of drugs. Use of certain proteins as targeting component of drug delivery systems can significantly improve the selectivity of this drug, as well as to overcome the multidrug resistance of cells resulting from the activity of the ABC-transporters. PMID:21874867

  18. Size and shape effects on receptor-mediated endocytosis of nanoparticles

    NASA Astrophysics Data System (ADS)

    Li, Xinlei

    2012-01-01

    We present a thermodynamic approach to elucidate the effects of the size and shape of nanoparticles (NPs) on endocytosis. It is found that endocytosis needs to surmount a thermodynamic energy barrier and has a minimum radius of NPs for endocytosis. Through referring to the concept of "diffusion length of receptors," we obtain a simple and analytical expression for the optimal size of NPs. Furthermore, a phase diagram has been constructed, which can clarify the interrelated effects of the radius and the aspect ratio of NPs. We can identify from the phase the relation between the geometry of NP and its endocytosis rate. The theoretical results are in good agreement with the experimental observations and reveal physical mechanisms involved in the effects of the size and shape of NPs on endocytosis, which implies that these studies may provide useful guidance to the conscious design of NPs for diagnostic agents and drug delivery applications.

  19. Endocytosis as a Biological Response in Receptor Pharmacology: Evaluation by Fluorescence Microscopy

    PubMed Central

    Varela, María J.; de la Rocha, Arlet M. Acanda; Fernandez-Troyano, Juan C.; Barreiro, R. Belén; Lopez-Gimenez, Juan F.

    2015-01-01

    The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method –the Q-Endosomes algorithm– that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution. PMID:25849355

  20. The Ubiquitin-like Protein PLIC-2 Is a Negative Regulator of G Protein-coupled Receptor Endocytosis

    PubMed Central

    N'Diaye, Elsa-Noah; Hanyaloglu, Aylin C.; Kajihara, Kimberly K.; Puthenveedu, Manojkumar A.; Wu, Ping; von Zastrow, Mark

    2008-01-01

    The activity of many signaling receptors is regulated by their endocytosis via clathrin-coated pits (CCPs). For G protein-coupled receptors (GPCRs), recruitment of the adaptor protein arrestin to activated receptors is thought to be sufficient to drive GPCR clustering in CCPs and subsequent endocytosis. We have identified an unprecedented role for the ubiquitin-like protein PLIC-2 as a negative regulator of GPCR endocytosis. Protein Linking IAP to Cytoskeleton (PLIC)-2 overexpression delayed ligand-induced endocytosis of two GPCRs: the V2 vasopressin receptor and β-2 adrenergic receptor, without affecting endocytosis of the transferrin or epidermal growth factor receptor. The closely related isoform PLIC-1 did not affect receptor endocytosis. PLIC-2 specifically inhibited GPCR concentration in CCPs, without affecting membrane recruitment of arrestin-3 to activated receptors or its cellular levels. Depletion of cellular PLIC-2 accelerated GPCR endocytosis, confirming its regulatory function at endogenous levels. The ubiquitin-like domain of PLIC-2, a ligand for ubiquitin-interacting motifs (UIMs), was required for endocytic inhibition. Interestingly, the UIM-containing endocytic adaptors epidermal growth factor receptor protein substrate 15 and Epsin exhibited preferential binding to PLIC-2 over PLIC-1. This differential interaction may underlie PLIC-2 specific effect on GPCR endocytosis. Identification of a negative regulator of GPCR clustering reveals a new function of ubiquitin-like proteins and highlights a cellular requirement for exquisite regulation of receptor dynamics. PMID:18199683

  1. Electroencephalographic and convulsant effects of the delta opioid agonist SNC80 in rhesus monkeys

    PubMed Central

    Danielsson, Ingela; Gasior, Maciej; Stevenson, Glenn W.; Folk, John E.; Rice, Kenner C.; Negus, S. Stevens

    2007-01-01

    Non-peptidic delta opioid receptor agonists are being evaluated for a wide range of clinical applications; however, the clinical utility of piperazinyl benzamide delta agonists such as SNC80 may be limited by convulsant activity. The purpose of the present study was to evaluate the electroencephalographic and convulsant activity produced by a high dose of 10 mg/kg SNC80 IM in rhesus monkeys. EEG and behavioral activity were examined in four adult male rhesus monkeys after IM administration of SNC80. Monkeys were seated in a standard primate restraint chair, and EEG activity was recorded using an array of 16 needle electrodes implanted subcutaneously in the scalp in a bipolar (scalp-to scalp) montage in a longitudinal direction, with bilateral frontal, central, temporal, and occipital leads. Behavior was recorded using video monitoring equipment. Initially, all monkeys were tested with 10 mg/kg SNC80, which is a relatively high dose 3–10 fold greater than doses necessary to produce a variety of other behavioral effects. Behavioral convulsions and EEG seizures were observed in one of the four monkeys. In this monkey, neither behavioral convulsions nor EEG seizures were observed when a lower dose of 3.2 mg/kg was administered nine weeks later or when the same dose of 10 mg/kg SNC80 was administered one year later. These results suggest that IM administration of SNC80 is less potent in producing convulsant effects than in producing other, potentially useful behavioral effects (e.g. antinociception) in rhesus monkeys. PMID:17112570

  2. Scavenger receptor-mediated endocytosis by sinusoidal cells in rat bone marrow

    SciTech Connect

    Geoffroy, J.S.

    1987-01-01

    Endocytosis of serum albumin by sinusoidal endothelial cells in rat bone marrow was investigated initially at the ultrastructural level with subsequent biochemical investigation of the specificity mediating this event. Bovine serum albumin adsorbed to 20nm colloidal gold particles (AuBSA) was chosen as the electron microscopic probe. Morphological data strongly suggested that a receptor was involved in uptake of AuBSA. Confirmation of receptor involvement in the uptake of AuBSA by marrow sinusoidal endothelial cells was achieved utilizing an in situ isolated hind limb perfusion protocol in conjunction with unlabeled, radiolabeled, and radio-/colloidal gold labeled probes. The major findings of competition and saturation experiments were: (1) endocytosis of AuBSA was mediated by a receptor for modified/treated serum albumin; (2) endocytosis of formaldehyde-treated serum albumin was mediated by a binding site which may be the same or closely related to the site responsible for the uptake of AuBSA; and (3) endocytosis of native untreated albumin was not mediated by receptor and probably represents fluid-phase pinocitosis.

  3. AMPA Receptor Endocytosis in Rat Perirhinal Cortex Underlies Retrieval of Object Memory

    ERIC Educational Resources Information Center

    Cazakoff, Brittany N.; Howland, John G.

    2011-01-01

    Mechanisms consistent with long-term depression in the perirhinal cortex (PRh) play a fundamental role in object recognition memory; however, whether AMPA receptor endocytosis is involved in distinct phases of recognition memory is not known. To address this question, we used local PRh infusions of the cell membrane-permeable Tat-GluA2[subscript…

  4. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    PubMed Central

    Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

    2016-01-01

    The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis. PMID:27463710

  5. Effects of particle size and ligand density on the kinetics of receptor-mediated endocytosis of nanoparticles

    NASA Astrophysics Data System (ADS)

    Yuan, Hongyan; Zhang, Sulin

    2010-01-01

    We elucidate, from thermodynamic arguments, the governing factors of receptor-mediated endocytosis of nanoparticles (NPs). We show that the endocytic energetics specifies a minimal particle size and a minimal ligand density below which endocytosis is not possible. Due to the entropic penalty involved in ligand-receptor binding, endocytosis may occur with a large fraction of ligands unbound with receptors. Our analyses suggest that the endocytic time depends interrelatedly on the particle size and ligand density. There exists an optimal condition at which the endocytic time minimizes. These findings may provide valuable guidance to the rational designs of NP-based biomarkers and anticancer bioagents.

  6. FUNCTION OF NON-VISUAL ARRESTINS IN SIGNALING AND ENDOCYTOSIS OF THE GASTRIN-RELEASING PEPTIDE RECEPTOR (GRP RECEPTOR)

    PubMed Central

    Schumann, Michael; Nakagawa, Tomoo; Mantey, Samuel A.; Howell, Brian; Jensen, Robert T.

    2008-01-01

    Little is known about the role of arrestins in gastrointestinal hormone/neurotransmitter receptor endocytosis. With other G protein-coupled receptors, arrestins induce G protein-uncoupling and receptor endocytosis. In this study, we used arrestin wild-type and dominant-negative mutant constructs to analyze the arrestin dependence of endocytosis and desensitization of the gastrin- releasing peptide receptor (GRP-R). Co-expression of the GRP-R with wild-type arrestin-2 and arrestin-3 increased not only GRP-R endocytosis but also GRP-R desensitization in arrestin-overexpressing cells. Co-expression of the dominant-negative mutants V53D-arrestin-2 or V54D-arrestin-3 reduced GRP-R endocytosis. Notably, different trafficking routes for agonist-activated GRPR-arrestin-2 and GRPR-arrestin-3 complexes were found. Arrestin-3 internalizes with GRP-R to intracellular vesicles, arrestin-2 splits from the GRP-R and localizes to the cell membrane. Also, the recycling pathway of the GRP-R was different if co-expressed with arrestin-2 or arrestin-3. Using different GRP-R mutants, the C-terminus and the 2nd intracellular loop of the GRP-R were found to be important for the GRPR-arrestin interaction and for the difference in GRP receptor trafficking with the two arrestin subtypes. Our results show that both non-visual arrestins play an important role in GRP-R internalization and desensitization. PMID:18199425

  7. Endocytosis and Trafficking of Natriuretic Peptide Receptor-A: Potential Role of Short Sequence Motifs

    PubMed Central

    Pandey, Kailash N.

    2015-01-01

    The targeted endocytosis and redistribution of transmembrane receptors among membrane-bound subcellular organelles are vital for their correct signaling and physiological functions. Membrane receptors committed for internalization and trafficking pathways are sorted into coated vesicles. Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) and elicit the generation of intracellular second messenger cyclic guanosine 3',5'-monophosphate (cGMP), which lowers blood pressure and incidence of heart failure. After ligand binding, the receptor is rapidly internalized, sequestrated, and redistributed into intracellular locations. Thus, NPRA is considered a dynamic cellular macromolecule that traverses different subcellular locations through its lifetime. The utilization of pharmacologic and molecular perturbants has helped in delineating the pathways of endocytosis, trafficking, down-regulation, and degradation of membrane receptors in intact cells. This review describes the investigation of the mechanisms of internalization, trafficking, and redistribution of NPRA compared with other cell surface receptors from the plasma membrane into the cell interior. The roles of different short-signal peptide sequence motifs in the internalization and trafficking of other membrane receptors have been briefly reviewed and their potential significance in the internalization and trafficking of NPRA is discussed. PMID:26151885

  8. Inhibition of clathrin-mediated endocytosis selectively attenuates specific insulin receptor signal transduction pathways.

    PubMed

    Ceresa, B P; Kao, A W; Santeler, S R; Pessin, J E

    1998-07-01

    To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor. PMID:9632770

  9. Inhibition of Clathrin-Mediated Endocytosis Selectively Attenuates Specific Insulin Receptor Signal Transduction Pathways

    PubMed Central

    Ceresa, Brian P.; Kao, Aimee W.; Santeler, Scott R.; Pessin, Jeffrey E.

    1998-01-01

    To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor. PMID:9632770

  10. Effects of mu and delta opioid agonists and antagonists on affective vocal and reflexive pain responses during social stress in rats.

    PubMed

    Vivian, J A; Miczek, K A

    1998-10-01

    The present experiments evaluated the influence of intraventricular mu and delta opioid receptors on affective vocal and reflexive responses to aversive stimuli in socially inexperienced, as well as defensive and submissive responses in defeated, adult male Long-Evans rats. Defeat stress consisted of: (1) an aggressive confrontation in which the experimental intruder rat exhibited escape, defensive and submissive behaviors [i.e., upright, supine postures and ultrasonic vocalizations (USV)], and subsequently, (2) protection from the resident stimulus rat with a wire mesh screen for 10-20 min. Defeat stress was immediately followed by an experimental session with tactile startle (20 psi). The mu opioid receptor agonists morphine (0.1-0.6 microg i.c.v.) and [D-Ala2-N-Me-Phe4-Gly5-ol]-enkephalin (DAMGO; 0.01-0.3 microg i.c.v.), and the delta opioid receptor agonist [D-Pen2,5]-enkephalin (DPDPE; 10-100 microg i.c.v.) dose-dependently decreased startle-induced USV and increased tail-flick latencies in socially inexperienced and defeated rats. Of greater interest, morphine, DAMGO and DPDPE increased the occurrence of the submissive crouch posture, and defeated rats were more sensitive than socially inexperienced rats to the startle-induced USV-suppressive and antinociceptive effects of morphine and DPDPE. The antinociceptive effects of DAMGO were likewise obtained at lower doses in defeated rats. Finally, the USV-suppressive effects of morphine and DAMGO were reversed with the mu receptor antagonist naltrexone (0.1 mg/kg i.p.), but the USV-suppressive effects produced by DPDPE were not reversed with the delta receptor antagonist naltrindole (1 mg/kg i.p.). These results confirm mu, but not delta opioid receptor activation as significant in affective vocal, passive-submissive behavior, as well as reflexive antinociception. Furthermore, similar to previous studies with restraint and electric shock stress, the facilitation of mu opioid effects on vocal responses and

  11. Expression of folate receptors in nasopharyngeal and laryngeal carcinoma and folate receptor-mediated endocytosis by molecular targeted nanomedicine

    PubMed Central

    Xie, M; Zhang, H; Xu, Y; Liu, T; Chen, S; Wang, J; Zhang, T

    2013-01-01

    Immunohistochemistry and an immunofluorescence technique was used to detect folate receptor expression in tissue samples and cell lines of head and neck squamous carcinoma, including 20 tissue samples of nasopharyngeal carcinoma, 16 tissue samples of laryngeal carcinoma, and HNE-1, HNE-2, CNE-1, CNE-2, SUNE-1, 5–8F, and Hep-2 cell lines. Iron staining, electron microscopy, and magnetic resonance imaging were used to observe endocytosis of folate-conjugated cisplatin-loaded magnetic nanoparticles (CDDP-FA-ASA-MNP) in cultured cells and transplanted tumors. As shown by immunohistochemistry, 83.3% (30/36) of the head and neck squamous carcinomas expressed the folate receptor versus none in the control group (0/24). Only the HNE-1 and Hep-2 cell lines expressed the folate receptor, and the other five cell lines did not. Endocytosis of CDDP-FA-ASA-MNP was seen in HNE-1 and Hep-2 cells by iron staining and electron microscopy. A similar result was seen in transplanted tumors in nude mice. Magnetic resonance imaging showed low signal intensity of HNE-1 cells and HNE-1 transplanted tumors on T2-weighted images after uptake of CDDP-FA-ASA-MNP, and this was not seen in CNE-2 transplanted tumors. In conclusion, head and neck squamous carcinoma cell strongly expressed the folate receptor, while normal tissue did not. The folate receptor can mediate endocytosis of folate-conjugated anticancer nanomedicines, and lays the foundation for molecular targeted treatment of cancer. PMID:23874095

  12. Endocytosis of a Functionally Enhanced GFP-Tagged Transferrin Receptor in CHO Cells

    PubMed Central

    Chu, Chong; Jiang, Qing; Zhu, Huifen; He, Yong; Yue, Tingting; Wang, Ruibo; Lei, Ping; Shen, Guanxin

    2015-01-01

    The endocytosis of transferrin receptor (TfR) has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP)-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81±3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs. PMID:25803700

  13. Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis

    PubMed Central

    Richards, David M.; Endres, Robert G.

    2016-01-01

    Phagocytosis and receptor-mediated endocytosis are vitally important particle uptake mechanisms in many cell types, ranging from single-cell organisms to immune cells. In both processes, engulfment by the cell depends critically on both particle shape and orientation. However, most previous theoretical work has focused only on spherical particles and hence disregards the wide-ranging particle shapes occurring in nature, such as those of bacteria. Here, by implementing a simple model in one and two dimensions, we compare and contrast receptor-mediated endocytosis and phagocytosis for a range of biologically relevant shapes, including spheres, ellipsoids, capped cylinders, and hourglasses. We find a whole range of different engulfment behaviors with some ellipsoids engulfing faster than spheres, and that phagocytosis is able to engulf a greater range of target shapes than other types of endocytosis. Further, the 2D model can explain why some nonspherical particles engulf fastest (not at all) when presented to the membrane tip-first (lying flat). Our work reveals how some bacteria may avoid being internalized simply because of their shape, and suggests shapes for optimal drug delivery. PMID:27185939

  14. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis

    PubMed Central

    Ferrandon, Sébastien; Feinstein, Timothy N; Castro, Marian; Wang, Bin; Bouley, Richard; Potts, John T; Gardella, Thomas J; Vilardaga, Jean-Pierre

    2011-01-01

    Cell signaling mediated by the G protein-coupled parathyroid hormone receptor type 1 (PTHR) is fundamental to bone and kidney physiology. It has been unclear how the two ligand systems—PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine—can effectively operate with only one receptor and trigger different durations of the cAMP responses. Here we analyze the ligand response by measuring the kinetics of activation and deactivation for each individual reaction step along the PTHR signaling cascade. We found that during the time frame of G protein coupling and cAMP production, PTHrP1–36 action was restricted to the cell surface, whereas PTH1–34 had moved to internalized compartments where it remained associated with the PTHR and Gαs, potentially as a persistent and active ternary complex. Such marked differences suggest a mechanism by which PTH and PTHrP induce differential responses, and these results indicate that the central tenet that cAMP production originates exclusively at the cell membrane must be revised. PMID:19701185

  15. Endocytosis of Ligand-Activated Sphingosine 1-Phosphate Receptor 1 Mediated by the Clathrin-Pathway.

    PubMed

    Reeves, Patrick M; Kang, Yuan-Lin; Kirchhausen, Tom

    2016-01-01

    The sphingosine 1-phosphate receptor 1 (S1PR1) is one of five G protein-coupled receptors activated by the lipid sphingosine 1-phosphate (S1P). Stimulation of S1PR1 by binding S1P or the synthetic agonist FTY720P results in rapid desensitization, associated in part with depletion of receptor from the cell surface. We report here combining spinning disc confocal fluorescence microscopy and flow cytometry to show that rapid internalization of activated S1PR1 relies on a functional clathrin-mediated endocytic pathway. Uptake of activated S1PR1 was strongly inhibited in cells disrupted in their clathrin-mediated endocytosis by depleting clathrin or AP-2 or by treating cells with dynasore-OH. The uptake of activated S1P1R was strongly inhibited in cells lacking both β-arrestin 1 and β-arrestin 2, indicating that activated S1PR1 follows the canonical route of endocytosis for G-protein coupled receptor's (GPCR)'s. PMID:26481905

  16. Endocytosis and the Src family of non-receptor tyrosine kinases

    PubMed Central

    Reinecke, James; Caplan, Steve

    2014-01-01

    The regulated intracellular transport of nutrient, adhesion, and growth factor receptors is crucial for maintaining cell and tissue homeostasis. Endocytosis, or endocytic membrane trafficking, involves the steps of intracellular transport that include, but are not limited to: internalization from the plasma membrane, sorting in early endosomes, transport to late endosomes/lysosomes followed by degradation, and/or recycling back to the plasma membrane via tubular recycling endosomes. In addition to regulating the localization of transmembrane receptor proteins, the endocytic pathway also controls the localization of non-receptor molecules. The non-receptor tyrosine kinase c-Src (Src), and its closely related family members Yes and Fyn, represent three proteins whose localization and signaling activities are tightly regulated by endocytic trafficking. Here, we provide a brief overview of endocytosis, Src function and its biochemical regulation. We will then concentrate on recent advances in understanding how Src intracellular localization is regulated and how its subcellular localization ultimately dictates downstream functioning. Since Src kinases are hyperactive in many cancers, it is essential to decipher the spatiotemporal regulation of this important family of tyrosine kinases. PMID:25372749

  17. The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells

    SciTech Connect

    Kindberg, G.M.; Gudmundsen, O.; Berg, T. )

    1990-06-05

    Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions. Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C; vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content.

  18. CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice

    PubMed Central

    Shimokawa, Noriaki; Haglund, Kaisa; Hölter, Sabine M; Grabbe, Caroline; Kirkin, Vladimir; Koibuchi, Noriyuki; Schultz, Christian; Rozman, Jan; Hoeller, Daniela; Qiu, Chun-Hong; Londoño, Marina B; Ikezawa, Jun; Jedlicka, Peter; Stein, Birgit; Schwarzacher, Stephan W; Wolfer, David P; Ehrhardt, Nicole; Heuchel, Rainer; Nezis, Ioannis; Brech, Andreas; Schmidt, Mirko H H; Fuchs, Helmut; Gailus-Durner, Valerie; Klingenspor, Martin; Bogler, Oliver; Wurst, Wolfgang; Deller, Thomas; de Angelis, Martin Hrabé; Dikic, Ivan

    2010-01-01

    Despite extensive investigations of Cbl-interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85Δex2) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85Δex2 animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post-synaptic compartment of striatal neurons in which it co-clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85Δex2 mice. PMID:20551902

  19. Role of Spinophilin in Group I Metabotropic Glutamate Receptor Endocytosis, Signaling, and Synaptic Plasticity.

    PubMed

    Di Sebastiano, Andrea R; Fahim, Sandra; Dunn, Henry A; Walther, Cornelia; Ribeiro, Fabiola M; Cregan, Sean P; Angers, Stephane; Schmid, Susanne; Ferguson, Stephen S G

    2016-08-19

    Activation of Group I metabotropic glutamate receptors (mGluRs) activates signaling cascades, resulting in calcium release from intracellular stores, ERK1/2 activation, and long term changes in synaptic activity that are implicated in learning, memory, and neurodegenerative diseases. As such, elucidating the molecular mechanisms underlying Group I mGluR signaling is important for understanding physiological responses initiated by the activation of these receptors. In the current study, we identify the multifunctional scaffolding protein spinophilin as a novel Group I mGluR-interacting protein. We demonstrate that spinophilin interacts with the C-terminal tail and second intracellular loop of Group I mGluRs. Furthermore, we show that interaction of spinophilin with Group I mGluRs attenuates receptor endocytosis and phosphorylation of ERK1/2, an effect that is dependent upon the interaction of spinophilin with the C-terminal PDZ binding motif encoded by Group I mGluRs. Spinophilin knock-out results in enhanced mGluR5 endocytosis as well as increased ERK1/2, AKT, and Ca(2+) signaling in primary cortical neurons. In addition, the loss of spinophilin expression results in impaired mGluR5-stimulated LTD. Our results indicate that spinophilin plays an important role in regulating the activity of Group I mGluRs as well as their influence on synaptic activity. PMID:27358397

  20. Macrophage Receptor with Collagenous Structure (MARCO) Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway

    PubMed Central

    Hirano, Seishiro; Kanno, Sanae

    2015-01-01

    The Macrophage Receptor with COllagenous structure (MARCO) protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome) generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2–3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways. PMID:26545255

  1. Endocytosis via coated pits mediated by glycoprotein receptor in which the cytoplasmic tail is replaced by unrelated sequences.

    PubMed Central

    Verrey, F; Gilbert, T; Mellow, T; Proulx, G; Drickamer, K

    1990-01-01

    Rat 6 fibroblast cell lines expressing wild-type chicken liver glycoprotein receptor (CHL) or chimeric receptors with alternate cytoplasmic tails were produced to study the role of the cytoplasmic tail in mediating receptor localization in coated pits and endocytosis of ligand. Cells expressing CHL or cells expressing a hybrid receptor that contains the cytoplasmic tail of the asialoglycoprotein receptor display high-efficiency endocytosis of N-acetylglucosamine-conjugated bovine serum albumin in experiments designed to measure an initial internalization step, as well as in studies of continuous uptake and degradation. Substitution of the cytoplasmic tail by the equivalent domain of rat Na,K-ATPase beta subunit or by a stretch of Xenopus laevis globin beta chain does not abolish endocytosis but decreases the endocytosis rate constant from 15%-16%/min to 2.4% and 6.5%/min, respectively. Electron microscopy was used to visualize the glycoprotein binding sites at the surface of Rat 6 cells transfected with the various receptors. The percentage of receptors found in coated areas ranged from 32% for CHL to 9% for the Na,K-ATPase hybrid, indicating that clustering in coated pits correlates with efficiency of endocytosis. We concluded that replacement of the CHL cytoplasmic tail with unrelated sequences does not prevent, but decreases to varying extents, coated-pit localization and endocytosis efficiency. The construct with NH2-terminal globin tail lacks a signal for high-efficiency localization in coated pits but nevertheless is directed to the pits by an alternative mechanism. Images PMID:1963794

  2. Understanding magnetic nanoparticle osteoblast receptor-mediated endocytosis using experiments and modeling

    NASA Astrophysics Data System (ADS)

    Tran, Nhiem; Webster, Thomas J.

    2013-05-01

    Iron oxide nanoparticles are promising candidates for controlling drug delivery through an external magnetic force to treat a wide range of diseases, including osteoporosis. Previous studies have demonstrated that in the presence of hydroxyapatite coated magnetite (Fe3O4) nanoparticles, osteoblast (or bone forming cell) proliferation and long-term functions (such as calcium deposition) were significantly enhanced. Hydroxyapatite is the major inorganic component of bone. As a further attempt to understand why, in the current study, the uptake of such nanoparticles into osteoblasts was experimentally investigated and mathematically modeled. Magnetite nanoparticles were synthesized using a co-precipitation method and were coated with hydroxyapatite. A cellular uptake experiment at low temperatures indicated that receptor-mediated endocytosis contributed to the internalization of the magnetic nanoparticles into osteoblasts. A model was further developed to explain the uptake of magnetic nanoparticles into osteoblasts using receptor-mediated endocytosis. This model may explain the internalization of hydroxyapatite into osteoblasts to elevate intracellular calcium levels necessary to promote osteoblast functions to treat a wide range of orthopedic problems, including osteoporosis.

  3. Endocytosis of lutropin by Leydig cells through a pathway distinct from the high-affinity receptor.

    PubMed

    Bozon, V; Pajot-Augy, E; Vignon, X; Salesse, R

    1998-08-25

    In porcine Leydig cells in primary culture, 95% of the internalization of [125I]porcine lutropin ([125I]pLH, which bears sulfated GalNAc) could not be ascribed to the high-affinity LH receptor (LHR). In contrast, >40% of [125I]human choriogonadotropin (hCG, with sialylated sugar chains) uptake was performed by the LHR itself. When the LHR was down-regulated by excess unlabeled hormone, the LHR-independent incorporation of [125I]pLH could be inhibited in a dose-dependent fashion by sulfated polysaccharides such as fucoidan or chondroitin-(4 or 6)-sulfate, but not by other polyanionic compounds, nor by sulfated chondroitin disaccharides. Endocytosis occurred through a clathrin-dependent pathway and was inhibited by low temperature, endocytosis inhibitors, increased ionic strength, or by EDTA and dithiothreitol. Taken together, these results suggest that a Leydig cell membrane protein (possibly a lectin, or a glycosaminoglycan receptor) could perform specific LH clearance in the testis via recognition of its sulfated sugars. PMID:9806348

  4. Administration of pyrene lipids by receptor-mediated endocytosis and their degradation in skin fibroblasts

    SciTech Connect

    Agmon, V.; Dinur, T.; Cherbu, S.; Dagan, A.; Gatt, S. )

    1991-10-01

    Sphingomyelin and seven glycosphingolipids were labeled with the fluorescent probe pyrene and administered into cultured fibroblasts by receptor-mediated endocytosis. For this purpose pyrene sphingomyelin or mixtures of pyrene glycolipid and unlabeled sphingomyelin were dispersed as small, unilamellar liposomes. Apolipoprotein E was then added and the receptor for this ligand on the cell surface was utilized for uptake of the liposomes and their transport to the lysosomes, where the respective pyrene lipids were degraded. Following incubation with each of the respective pyrene lipids, only the administered compound and the pyrene ceramide were present; intermediate hydrolysis products were not detected. This indicated that, in skin fibroblasts, the lysosomal ceramidase was limiting and controlled the rate of total degradation of the pyrene sphingolipids.

  5. Functional roles of short sequence motifs in the endocytosis of membrane receptors

    PubMed Central

    Pandey, Kailash N.

    2009-01-01

    Internalization and trafficking of cell-surface membrane receptors and proteins into subcellular compartments is mediated by specific short-sequence signal motifs, which are usually located within the cytoplasmic domains of these receptor and protein molecules. The signals usually consist of short linear amino acid sequences, which are recognized by adaptor coat proteins along the endocytic and sorting pathways. The complex arrays of signals and recognition proteins ensure the dynamic movement, accurate trafficking, and designated distribution of transmembrane receptors and ligands into intracellular compartments, particularly to the endosomal-lysosomal system. This review summarizes the new information and concepts, integrating them with the current and established views of endocytosis, intracellular trafficking, and sorting of membrane receptors and proteins. Particular emphasis has been given to the functional roles of short-sequence signal motifs responsible for the itinerary and destination of membrane receptors and proteins moving into the subcellular compartments. The specific characteristics and functions of short-sequence motifs, including various tyrosine-based, dileucine-type, and other short-sequence signals in the trafficking and sorting of membrane receptors and membrane proteins are presented and discussed. PMID:19482617

  6. Distinct CCR7 glycosylation pattern shapes receptor signaling and endocytosis to modulate chemotactic responses.

    PubMed

    Hauser, Mark A; Kindinger, Ilona; Laufer, Julia M; Späte, Anne-Katrin; Bucher, Delia; Vanes, Sarah L; Krueger, Wolfgang A; Wittmann, Valentin; Legler, Daniel F

    2016-06-01

    The homeostatic chemokines CCL19 and CCL21 and their common cognate chemokine receptor CCR7 orchestrate immune cell trafficking by eliciting distinct signaling pathways. Here, we demonstrate that human CCR7 is N-glycosylated on 2 specific residues in the N terminus and the third extracellular loop. Conceptually, CCR7 glycosylation adds steric hindrance to the receptor N terminus and extracellular loop 3, acting as a "swinging door" to regulate receptor sensitivity and cell migration. We found that freshly isolated human B cells, as well as expanded T cells, but not naïve T cells, express highly sialylated CCR7. Moreover, we identified that human dendritic cells imprint T cell migration toward CCR7 ligands by secreting enzymes that deglycosylate CCR7, thereby boosting CCR7 signaling on T cells, permitting enhanced T cell locomotion, while simultaneously decreasing receptor endocytosis. In addition, dendritic cells proteolytically convert immobilized CCL21 to a soluble form that is more potent in triggering chemotactic movement and does not desensitize the receptor. Furthermore, we demonstrate that soluble CCL21 functionally resembles neither the CCL19 nor the CCL21 phenotype but acts as a chemokine with unique features. Thus, we advance the concept of dendritic cell-dependent generation of micromilieus and lymph node conditioning by demonstrating a novel layer of CCR7 regulation through CCR7 sialylation. In summary, we demonstrate that leukocyte subsets express distinct patterns of CCR7 sialylation that contribute to receptor signaling and fine-tuning chemotactic responses. PMID:26819318

  7. Effect of the anti-receptor ligand-blocking 225 monoclonal antibody on EGF receptor endocytosis and sorting

    SciTech Connect

    Jaramillo, Maria L. . E-mail: maria.jaramillo@nrc.ca; Leon, Zully; Grothe, Suzanne; Paul-Roc, Beatrice; Abulrob, Abedelnasser; O'Connor McCourt, Maureen

    2006-09-10

    The anti-receptor antibody, 225 mAb, is known to block binding of ligand to the epidermal growth factor receptor (EGFR). However, the effect of this neutralizing antibody on EGFR endocytosis, trafficking and degradation remains unclear. Here, we demonstrate that endocytosis of {sup 125}I-225 mAb occurs, albeit with a slower rate than that of EGF. Using pulse chase assays, we show that internalized {sup 125}I-225 mAb is recycled to the surface much more efficiently than internalized {sup 125}I-EGF. Also, we found that internalization of {sup 125}I-225 mAb, in contrast to that of EGF, is independent of receptor tyrosine kinase activity, as evidenced by its insensitivity to AG1478, a specific EGFR tyrosine kinase inhibitor. Analysis of the levels of cell surface and total EGFR showed that treatment with 225 mAb results in a 30-40% decrease in surface EGFR and a relatively slow downregulation of total EGFR. Taken together, these data indicate that 225 mAb induces internalization and downregulation of EGFR via a mechanism distinct from that underlying EGF-induced EGFR internalization and downregulation.

  8. Separation of the convulsions and antidepressant-like effects produced by the delta-opioid agonist SNC80 in rats

    PubMed Central

    Jutkiewicz, E. M.; Traynor, J. R.; Woods, J. H.; Rice, K. C.

    2005-01-01

    Rationale Delta-opioid agonists produce a number of behavioral effects, including convulsions, anti-nociception, locomotor stimulation, and antidepressant-like effects. The development of these compounds as treatments for depression is limited by their convulsive effects. Therefore, determining how to separate the convulsive and antidepressant-like characteristics of these compounds is essential for their potential clinical use. Objective The present study tests the hypothesis that the rate of delta-opioid agonist administration greatly contributes to the convulsive properties, but not the anti-depressant-like effects, of delta-opioid agonists. Materials and methods The delta-opioid agonist SNC80 (1, 3.2, and 10 mg kg−1 or vehicle) was administered to Sprague–Dawley rats by intravenous infusion over different durations of time (20 s, 20, or 60 min). Convulsions were measured by observation prior to determining antidepressant-like effects in the forced swim test. Results Slowing the rate of SNC80 administration minimized delta agonist-induced convulsions without altering the effects of SNC80 in the forced swim test. Conclusions These data suggest that delta agonist-induced antidepressant properties are independent of convulsive effects, and that it may be possible to eliminate the convulsions produced by delta agonists, further promoting their potential clinical utility. PMID:16163520

  9. Delivery of liposomes into cultured KB cells via folate receptor-mediated endocytosis.

    PubMed

    Lee, R J; Low, P S

    1994-02-01

    Folic acid was covalently conjugated to 66-nm liposomes via spacers of various lengths in an attempt to target the liposomes to KB cells expressing folate receptors. Spacers of short and intermediate lengths were unable to mediate association of folate-conjugated liposomes with receptor-bearing cells, however, use of a 250 A polyethyleneglycol spacer (PEG, M(r) approximately 3350) permitted avid uptake of the liposomes at approximately 2.5 x 10(5) sites/cell. The binding of folate-PEG liposomes to KB cells could be competitively inhibited by excess free folate or by antiserum against the folate receptor, demonstrating the interaction is mediated by the cell surface folate-binding protein. Following binding, cell-associated folate-PEG liposomes were internalized by folate-receptor-mediated endocytosis at 37 degrees C but not at 4 degrees C. These folate-PEG liposomes show potential for delivering large quantities of low molecular weight compounds nondestructively into folate receptor-bearing cells. PMID:8106354

  10. Regulation of Epidermal Growth Factor Receptor Signaling by Endocytosis and Intracellular Trafficking

    SciTech Connect

    Burke, Patrick; Schooler, Kevin; Wiley, H S.

    2001-06-01

    Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated following endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules using reversibly-biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. Using a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that following internalization, EGFR remained active in the early endosomes. However, receptors were inactivated prior to degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, while others, such as Eps8, were only found with intracellular receptors. During the inactivation phase, c-Cbl became EGFR-associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment-specific . In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.

  11. Neurokinin1 receptors regulate morphine-induced endocytosis and desensitization of mu opioid receptors in CNS neurons

    PubMed Central

    Yu, Y. Joy; Arttamangkul, Seksiri; Evans, Christopher J.; Williams, John T.; von Zastrow, Mark

    2009-01-01

    Mu opioid receptors (MORs) are G protein-coupled receptors (GPCRs) that mediate the physiological effects of endogenous opioid neuropeptides and opiate drugs such as morphine. MORs are co-expressed with neurokinin 1 receptors (NK1Rs) in several regions of the central nervous system (CNS) that control opioid dependence and reward. NK1R activation affects opioid reward specifically, however, and the cellular basis for this specificity is unknown. We found that ligand-induced activation of NK1Rs produces a cell autonomous and non-reciprocal inhibition of MOR endocytosis induced by diverse opioids. Studies using epitope-tagged receptors expressed in cultured striatal neurons and a neuroblastoma cell model indicated that this heterologous regulation is mediated by NK1R-dependent sequestration of arrestins on endosome membranes. First, endocytic inhibition mediated by wild type NK1Rs was overcome in cells over-expressing β-arrestin2, a major arrestin isoform expressed in striatum. Second, NK1R activation promoted sequestration of β-arrestin2 on endosomes, whereas MOR activation did not. Third, heterologous inhibition of MOR endocytosis was prevented by mutational disruption of β-arrestin2 sequestration by NK1Rs. NK1R-mediated regulation of MOR trafficking was associated with reduced opioid-induced desensitization of adenylyl cyclase signaling in striatal neurons. Further, heterologous regulation of MOR trafficking was observed in both amygdala and locus coeruleus neurons that naturally co-express these receptors. These results identify a cell autonomous mechanism that may underlie the highly specific effects of NK1R on opioid signaling and suggest, more generally, that receptor-specific trafficking of arrestins may represent a fundamental mechanism for coordinating distinct GPCR-mediated signals at the level of individual CNS neurons. PMID:19129399

  12. Effects of chronic ethanol administration on receptor mediated endocytosis of asialoorosomucoid (ASOR) in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J.

    1986-05-01

    The authors have previously shown that acute and chronic ethanol administration decreases hepatic glycoprotein secretion and membrane biogenesis. The present study was undertaken to determine the effects of chronic ethanol feeding on receptor-mediated endocytosis using the endocytosis of ASOR as a model system. Rats were fed either rat chow ad lib or pair-fed with Lieber-DeCarli diet (ethanol or isocaloric glucose as 36% of total calories) for 5 to 7 weeks. Binding of /sup 125/I ASOR to isolated hepatocytes was studied at 0-4/sup 0/C. Internalization (cell-associated acid precipitable radioactivity) and degradation (acid soluble radioactivity) were determined at 37/sup 0/C for periods up to 240 min. Results were expressed as pmoles ASOR bound, degraded or internalized/10/sup 6/ cells. In ethanol-fed rats the number of pmoles ASOR bound/10/sup 6/ cells was decreased by 40-50% (p< 0.01) as compared to pair-fed and chow-fed animals. Rates of degradation and internalization of the ligand were also 50-70% lower (p< 0.01) in chronic ethanol-treated animals. No significant differences were observed for either binding or internalization of ASOR between chow-fed and pair-fed animals. These results indicate that chronic ethanol feeding decreases internalization and degradation of ASOR in rat hepatocytes.

  13. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-05-15

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

  14. Endocytosis and trafficking of BMP receptors: Regulatory mechanisms for fine-tuning the signaling response in different cellular contexts.

    PubMed

    Ehrlich, Marcelo

    2016-02-01

    Signaling by bone morphogenetic protein (BMP) receptors is regulated at multiple levels in order to ensure proper interpretation of BMP stimuli in different cellular settings. As with other signaling receptors, regulation of the amount of exposed and signaling-competent BMP receptors at the plasma-membrane is predicted to be a key mechanism in governing their signaling output. Currently, the endocytosis of BMP receptors is thought to resemble that of the structurally related transforming growth factor-β (TGF-β) receptors, as BMP receptors are constitutively internalized (independently of ligand binding), with moderate kinetics, and mostly via clathrin-mediated endocytosis. Also similar to TGF-β receptors, BMP receptors are able to signal from the plasma membrane, while internalization to endosomes may have a signal modulating effect. When at the plasma membrane, BMP receptors localize to different membrane domains including cholesterol rich domains and caveolae, suggesting a complex interplay between membrane distribution and internalization. An additional layer of complexity stems from the putative regulatory influence on the signaling and trafficking of BMP receptors exerted by ligand traps and/or co-receptors. Furthermore, the trafficking and signaling of BMP receptors are subject to alterations in cellular context. For example, genetic diseases involving changes in the expression of auxiliary factors of endocytic pathways hamper retrograde BMP signals in neurons, and perturb the regulation of synapse formation. This review summarizes current understanding of the trafficking of BMP receptors and discusses the role of trafficking in regulation of BMP signals. PMID:26776724

  15. A simple method to evaluate the optimal size of nanoparticles for endocytosis based on kinetic diffusion of receptors

    NASA Astrophysics Data System (ADS)

    Li, Xinlei; Xing, Da

    2010-10-01

    We have presented an analytic thermodynamic model to elucidate the mechanism of receptor-mediated endocytosis of nanoparticles (NPs) and provided a simple method to evaluate the optimal size of NPs by minimizing the kinetic diffusion time of the free receptors around the bound region toward the contact surface with NPs. It is found that the average density of receptors and chemical energy release upon the binding of a ligand-receptor pair determine the optimal size of NPs if the bending modulus of membranes and the cross-sectional area of the receptor are constants. The optimal radius of NPs can be calculated based on our model.

  16. Endocytosis of pro-inflammatory cytokine receptors and its relevance for signal transduction.

    PubMed

    Hermanns, Heike M; Wohlfahrt, Julia; Mais, Christine; Hergovits, Sabine; Jahn, Daniel; Geier, Andreas

    2016-08-01

    The pro-inflammatory cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) are key players of the innate and adaptive immunity. Their activity needs to be tightly controlled to allow the initiation of an appropriate immune response as defense mechanism against pathogens or tissue injury. Excessive or sustained signaling of either of these cytokines leads to severe diseases, including rheumatoid arthritis, inflammatory bowel diseases (Crohn's disease, ulcerative colitis), steatohepatitis, periodic fevers and even cancer. Studies carried out in the last 30 years have emphasized that an elaborate control system for each of these cytokines exists. Here, we summarize what is currently known about the involvement of receptor endocytosis in the regulation of these pro-inflammatory cytokines' signaling cascades. Particularly in the last few years it was shown that this cellular process is far more than a mere feedback mechanism to clear cytokines from the circulation and to shut off their signal transduction. PMID:27071147

  17. The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

    SciTech Connect

    Watanabe, H.; Grubb, J.H.; Sly, W.S. )

    1990-10-01

    The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.

  18. Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase reduce receptor-mediated endocytosis in opossum kidney cells.

    PubMed

    Sidaway, James E; Davidson, Robert G; McTaggart, Fergus; Orton, Terry C; Scott, Robert C; Smith, Graham J; Brunskill, Nigel J

    2004-09-01

    Renal proximal tubule cells are responsible for the reabsorption of proteins that are present in the tubular lumen. This occurs by receptor-mediated endocytosis, a process that has a requirement for some GTP-binding proteins. Statins are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase used for the therapeutic reduction of cholesterol-containing plasma lipoproteins. However, they can also reduce intracellular levels of isoprenoid pyrophosphates that are derived from the product of the enzyme, mevalonate, and are required for the prenylation and normal function of GTP-binding proteins. The hypothesis that inhibition of HMG-CoA reductase in renal proximal tubule cells could reduce receptor mediated-endocytosis was therefore tested. Five different statins inhibited the uptake of FITC-labeled albumin by the proximal tubule-derived opossum kidney cell line in a dose-dependent manner and in the absence of cytotoxicity. The reduction in albumin uptake was related to the degree of inhibition of HMG-CoA reductase. Simvastatin (e.g., statin) inhibited receptor-mediated endocytosis of both FITC-albumin and FITC-beta(2)-microglobulin to similar extents but without altering the binding of albumin to the cell surface. The effect on albumin endocytosis was prevented by mevalonate and by the isoprenoid geranylgeranyl pyrophosphate but not by cholesterol. Finally, evidence that the inhibitory effect of statins on endocytosis of proteins may be caused by reduced prenylation and thereby decreased function of one or more GTP-binding proteins is provided. These data establish the possibility in principle that inhibition of HMG-CoA reductase by statins in proximal tubule cells may reduce tubular protein reabsorption. PMID:15339975

  19. Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria.

    PubMed

    Seliverstova, E V; Prutskova, N P

    2015-01-01

    The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals. PMID:26150156

  20. Receptor-Mediated Endocytosis of Lysozyme in Renal Proximal Tubules of the Frog Rana Temporaria

    PubMed Central

    Seliverstova, E.V.

    2015-01-01

    The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endo-somes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intra-cellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals. PMID:26150156

  1. Endocytosis and degradative sorting of NMDA receptors by conserved membrane-proximal signals.

    PubMed

    Scott, Derek B; Michailidis, Ioannis; Mu, Yuanyue; Logothetis, Diomedes; Ehlers, Michael D

    2004-08-11

    Regulation of the abundance of NMDA receptors (NMDARs) at excitatory synapses is critical during changes in synaptic efficacy underlying learning and memory as well as during synapse formation throughout neural development. However, the molecular signals that govern NMDAR delivery, maintenance, and internalization remain unclear. In this study, we identify a conserved family of membrane-proximal endocytic signals, two within the NMDAR type 1 (NR1) subunit and one within the NR2A and NR2B subunits, necessary and sufficient to drive the internalization of NMDARs. These endocytic motifs reside in the region of NMDAR subunits immediately after the fourth membrane segment, a region implicated in use-dependent rundown and NMDA channel inactivation. Although endocytosis driven by the distal C-terminal domain of NR2B is followed by rapid recycling, internalization mediated by membrane-proximal motifs selectively targets receptors to late endosomes and accelerates degradation. These results define a novel conserved signature of NMDARs regulating internalization and postendocytic trafficking. PMID:15306643

  2. Nuclear-cytoplasmic transport of EGFR involves receptor endocytosis, importin beta1 and CRM1.

    PubMed

    Lo, Hui-Wen; Ali-Seyed, Mohamed; Wu, Yadi; Bartholomeusz, Geoffrey; Hsu, Sheng-Chieh; Hung, Mien-Chie

    2006-08-15

    Many receptor tyrosine kinases (RTKs) can be detected in the cell nucleus, such as EGFR, HER-2, HER-3, HER-4, and fibroblast growth factor receptor. EGFR, HER-2 and HER-4 contain transactivational activity and function as transcription co-factors to activate gene promoters. High EGFR in tumor nuclei correlates with increased tumor proliferation and poor survival in cancer patients. However, the mechanism by which cell-surface EGFR translocates into the cell nucleus remains largely unknown. Here, we found that EGFR co-localizes and interacts with importins alpha1/beta1, carriers that are critical for macromolecules nuclear import. EGFR variant mutated at the nuclear localization signal (NLS) is defective in associating with importins and in entering the nuclei indicating that EGFR's NLS is critical for EGFR/importins interaction and EGFR nuclear import. Moreover, disruption of receptor internalization process using chemicals and forced expression of dominant-negative Dynamin II mutant suppressed nuclear entry of EGFR. Additional evidences suggest an involvement of endosomal sorting machinery in EGFR nuclear translocalization. Finally, we found that nuclear export of EGFR may involve CRM1 exportin as we detected EGFR/CRM1 interaction and markedly increased nuclear EGFR following exposure to leptomycin B, a CRM1 inhibitor. Collectively, these data suggest the importance of receptor endocytosis, endosomal sorting machinery, interaction with importins alpha1/beta1, and exportin CRM1 in EGFR nuclear-cytoplasmic trafficking. Together, our work sheds light into the nature and regulation of the nuclear EGFR pathway and provides a plausible mechanism by which cells shuttle cell-surface EGFR and potentially other RTKs through the nuclear pore complex and into the nuclear compartment. PMID:16552725

  3. Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively

    PubMed Central

    Shojima, Kensaku; Sato, Akira; Hanaki, Hideaki; Tsujimoto, Ikuko; Nakamura, Masahiro; Hattori, Kazunari; Sato, Yuji; Dohi, Keiji; Hirata, Michinari; Yamamoto, Hideki; Kikuchi, Akira

    2015-01-01

    Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity. For this action, Wnt5a-induced receptor endocytosis with clathrin is required. Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. Recently, it was reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. In this study, we generated a neutralizing anti-Wnt5a monoclonal antibody (mAb5A16) to investigate the mechanism by which Wnt5a regulates cancer cell proliferation. Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively. PMID:25622531

  4. Initial hepatic removal of chylomicron remnants is unaffected but endocytosis is delayed in mice lacking the low density lipoprotein receptor.

    PubMed Central

    Herz, J; Qiu, S Q; Oesterle, A; DeSilva, H V; Shafi, S; Havel, R J

    1995-01-01

    Two endocytic receptors, the low density lipoprotein (LDL) receptor (LDLR) and the LDLR-related protein (LRP), are thought to act in concert in the hepatic uptake of partially metabolized dietary lipoproteins, the chylomicron remnants. We have evaluated the role of these two receptors in the hepatic metabolism of chylomicron remnants in normal mice and in LDLR-deficient [LDLR (-/-)] mice. The rate of chylomicron remnant removal by the liver was normal up to 30 min after intravenous injection of chylomicrons into LDLR (-/-) mice and was unaffected by receptor-associated protein (RAP), a potent inhibitor of ligand binding to LRP. In contrast, endocytosis of the remnants by the hepatocytes, measured by their accumulation in the endosomal fraction and by the rate of hydrolysis of component cholesteryl esters, was dramatically reduced in the absence of the LDLR. Coadministration of RAP prevented the continuing hepatic removal of chylomicron remnants in LDL (-/-) mice after 30 min, consistent with blockade of the slow endocytosis by a RAP-sensitive process. Taken together with previous studies, our results are consistent with a model in which the initial hepatic removal of chylomicron remnants is primarily mediated by mechanisms that do not include LDLR or LRP, possibly involving glycosaminoglycan-bound hepatic lipase and apolipoprotein E. After the remnants bind to these alternative sites on the hepatocyte surface, endocytosis is predominantly mediated by the LDLR and also by a slower and less efficient backup process that is RAP sensitive and therefore most likely involves LRP. PMID:7753850

  5. Complex Formation Facilitates Endocytosis of the Varicella-Zoster Virus gE:gI Fc Receptor

    PubMed Central

    Olson, Julie K.; Grose, Charles

    1998-01-01

    Open reading frames within the unique short segment of alphaherpesvirus genomes participate in egress and cell-to-cell spread. The case of varicella-zoster virus (VZV) is of particular interest not only because the virus is highly cell associated but also because its most prominent cell surface protein, gE, bears semblance to the mammalian Fc receptor FcγRII. A previous study demonstrated that when expressed alone in cells, VZV gE was endocytosed from the cell surface through a tyrosine localization motif in its cytoplasmic tail (J. K. Olson and C. Grose, J. Virol. 71:4042–4054, 1997). Since VZV gE is normally found in association with gI in the infected cell, the present study was directed at defining the trafficking of the VZV gE:gI protein complex. First, VZV gI underwent endocytosis and recycling when it was expressed alone in cells, and interestingly, VZV gI contained a methionine-leucine internalization motif in its cytoplasmic tail. Second, VZV gI was found by confocal microscopy to colocalize with VZV gE during endocytosis and recycling in cells. Third, by a quantitative internalization assay, VZV gE:gI was shown to undergo endocytosis more efficiently (steady state, 55 to 60%) than either gE alone (steady state, ∼32%) or gI alone (steady state, ∼45%). Further, examination of endocytosis-deficient mutant proteins demonstrated that VZV gI exerted a more pronounced effect than gE on internalization of the complex. Most importantly, therefore, these studies suggest that VZV gI behaves as an accessory component by facilitating the endocytosis of the major constituent gE and thereby modulating the trafficking of the entire cell surface gE:gI Fc receptor complex. PMID:9445058

  6. Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes.

    PubMed

    Pinilla-Macua, Itziar; Watkins, Simon C; Sorkin, Alexander

    2016-02-23

    Signaling from epidermal growth factor receptor (EGFR) to extracellular-stimuli-regulated protein kinase 1/2 (ERK1/2) is proposed to be transduced not only from the cell surface but also from endosomes, although the role of endocytosis in this signaling pathway is controversial. Ras is the only membrane-anchored component in the EGFR-ERK signaling axis, and therefore, its location determines intracellular sites of downstream signaling. Hence, we labeled endogenous H-Ras (HRas) with mVenus fluorescent protein using gene editing in HeLa cells. mVenus-HRas was primarily located at the plasma membrane, and in small amounts in tubular recycling endosomes and associated vesicles. EGF stimulation resulted in fast but transient activation of mVenus-HRas. Although EGF:EGFR complexes were rapidly accumulated in endosomes together with the Grb2 adaptor, very little, if any, mVenus-HRas was detected in these endosomes. Interestingly, the activities of MEK1/2 and ERK1/2 remained high beyond the point of the physical separation of HRas from EGF:EGFR complexes and down-regulation of Ras activity. Paradoxically, this sustained MEK1/2 and ERK1/2 activation was dependent on the active EGFR kinase. Cell surface biotinylation and selective inactivation of surface EGFRs suggested that a small fraction of active EGFRs remaining in the plasma membrane is responsible for continuous signaling to MEK1/2 and ERK1/2. We propose that, under physiological conditions of cell stimulation, EGFR endocytosis serves to spatially separate EGFR-Grb2 complexes and Ras, thus terminating Ras-mediated signaling. However, sustained minimal activation of Ras by a small pool of active EGFRs in the plasma membrane is sufficient for extending MEK1/2 and ERK1/2 activities. PMID:26858456

  7. Avr4 promotes Cf-4 receptor-like protein association with the BAK1/SERK3 receptor-like kinase to initiate receptor endocytosis and plant immunity.

    PubMed

    Postma, Jelle; Liebrand, Thomas W H; Bi, Guozhi; Evrard, Alexandre; Bye, Ruby R; Mbengue, Malick; Kuhn, Hannah; Joosten, Matthieu H A J; Robatzek, Silke

    2016-04-01

    The first layer of plant immunity is activated by cell surface receptor-like kinases (RLKs) and proteins (RLPs) that detect infectious pathogens. Constitutive interaction with the SUPPRESSOR OF BIR1 (SOBIR1) RLK contributes to RLP stability and kinase activity. As RLK activation requires transphosphorylation with a second associated RLK, it remains elusive how RLPs initiate downstream signaling. We employed live-cell imaging, gene silencing and coimmunoprecipitation to investigate the requirement of associated kinases for functioning and ligand-induced subcellular trafficking of Cf RLPs that mediate immunity of tomato against Cladosporium fulvum. Our research shows that after elicitation with matching effector ligands Avr4 and Avr9, BRI1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) associates with Cf-4 and Cf-9. BAK1/SERK3 is required for the effector-triggered hypersensitive response and resistance of tomato against C. fulvum. Furthermore, Cf-4 interacts with SOBIR1 at the plasma membrane and is recruited to late endosomes upon Avr4 trigger, also depending on BAK1/SERK3. These observations indicate that RLP-mediated resistance and endocytosis require ligand-induced recruitment of BAK1/SERK3, reminiscent of BAK1/SERK3 interaction and subcellular fate of the FLAGELLIN SENSING 2 (FLS2) RLK. This reveals that diverse classes of cell surface immune receptors share common requirements for initiation of resistance and endocytosis. PMID:26765243

  8. Mechanism-Based Tumor-Targeting Drug Delivery System. Validation of Efficient Vitamin Receptor-Mediated Endocytosis and Drug Release

    SciTech Connect

    Chen, S.; Wong, S.; Zhao, X.; Chen, J.; Chen, J.; Kuznetsova, L.; Ojima, I.

    2010-05-01

    An efficient mechanism-based tumor-targeting drug delivery system, based on tumor-specific vitamin-receptor mediated endocytosis, has been developed. The tumor-targeting drug delivery system is a conjugate of a tumor-targeting molecule (biotin: vitamin H or vitamin B-7), a mechanism-based self-immolative linker and a second-generation taxoid (SB-T-1214) as the cytotoxic agent. This conjugate (1) is designed to be (i) specific to the vitamin receptors overexpressed on tumor cell surface and (ii) internalized efficiently through receptor-mediated endocytosis, followed by smooth drug release via glutathione-triggered self-immolation of the linker. In order to monitor and validate the sequence of events hypothesized, i.e., receptor-mediated endocytosis of the conjugate, drug release, and drug-binding to the target protein (microtubules), three fluorescent/fluorogenic molecular probes (2, 3, and 4) were designed and synthesized. The actual occurrence of these processes was unambiguously confirmed by means of confocal fluorescence microscopy (CFM) and flow cytometry using L1210FR leukemia cells, overexpressing biotin receptors. The molecular probe 4, bearing the taxoid linked to fluorescein, was also used to examine the cell specificity (i.e., efficacy of receptor-based cell targeting) for three cell lines, L1210FR (biotin receptors overexpressed), L1210 (biotin receptors not overexpressed), and WI38 (normal human lung fibroblast, biotin receptor negative). As anticipated, the molecular probe 4 exhibited high specificity only to L1210FR. To confirm the direct correlation between the cell-specific drug delivery and anticancer activity of the probe 4, its cytotoxicity against these three cell lines was also examined. The results clearly showed a good correlation between the two methods. In the same manner, excellent cell-specific cytotoxicity of the conjugate 1 (without fluorescein attachment to the taxoid) against the same three cell lines was confirmed. This mechanism

  9. Dynamin 2-dependent endocytosis sustains T-cell receptor signaling and drives metabolic reprogramming in T lymphocytes

    PubMed Central

    Willinger, Tim; Staron, Matthew; Ferguson, Shawn M.; De Camilli, Pietro; Flavell, Richard A.

    2015-01-01

    Prolonged T-cell receptor (TCR) signaling is required for the proliferation of T lymphocytes. Ligation of the TCR activates signaling, but also causes internalization of the TCR from the cell surface. How TCR signaling is sustained for many hours despite lower surface expression is unknown. Using genetic inhibition of endocytosis, we show here that TCR internalization promotes continued TCR signaling and T-lymphocyte proliferation. T-cell–specific deletion of dynamin 2, an essential component of endocytosis, resulted in reduced TCR signaling strength, impaired homeostatic proliferation, and the inability to undergo clonal expansion in vivo. Blocking endocytosis resulted in a failure to maintain mammalian target of rapamycin (mTOR) activity and to stably induce the transcription factor myelocytomatosis oncogene (c-Myc), which led to metabolic stress and a defect in cell growth. Our results support the concept that the TCR can continue to signal after it is internalized from the cell surface, thereby enabling sustained signaling and cell proliferation. PMID:25831514

  10. Syndecan-4 Is a Receptor for Clathrin-Mediated Endocytosis of Arginine-Rich Cell-Penetrating Peptides.

    PubMed

    Kawaguchi, Yoshimasa; Takeuchi, Toshihide; Kuwata, Keiko; Chiba, Junya; Hatanaka, Yasumaru; Nakase, Ikuhiko; Futaki, Shiroh

    2016-04-20

    Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photo-cross-linking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins nonspecifically bound to the affinity beads. To address this issue, here we developed a photo-cross-linking probe in which a cleavable linker of a diazobenzene moiety was employed to allow selective elution of cross-linked proteins by reducing agent-mediated cleavage. We demonstrated that introduction of the diazobenzene moiety into the photoaffinity probe enables efficient purification of cross-linked proteins with significant reduction of nonspecific binding proteins, leading to successful identification of 17 membrane-associated proteins that would interact with R8 peptide. RNAi-mediated knockdown experiments in combination with the pharmacological inhibitors revealed that, among the proteins identified, syndecan-4, one of the heparan sulfate proteoglycans, is an endogenous membrane-associated receptor for the cellular uptake of R8 peptide via clathrin-mediated endocytosis. This syndecan-4-dependent pathway was also involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. These results reveal that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis. PMID:27019270

  11. Zonal differences in ethanol-induced impairments in receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J. )

    1991-02-01

    We have shown previously that ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid occurred as early as 1 wk after ethanol feeding. This study was undertaken as an initial attempt to establish a possible role of defective receptor-mediated endocytosis in liver injury by investigating whether differences exist in the effects of ethanol on receptor-mediated endocytosis in hepatocytes isolated from different regions of the liver. Perivenule cells, present in the distal half of the liver, are thought to be more susceptible to ethanol-induced liver injury than are the periportal cells located in the proximal half of the liver acini. For these studies, we fed male Sprague-Dawley rats for 7 days with liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate. Perivenule and periportal hepatocytes were then isolated using a digitonin-collagenase perfusion method. In control animals, cells isolated from the perivenule region bound significantly more ligand than did cells from the periportal region. Amounts of ligand internalized and degraded were also greater in perivenule than in periportal cells in these animals. After ethanol feeding, cells isolated from both the perivenule and periportal regions bound significantly less ligand than their respective controls. This impairment in surface and total binding was more pronounced in perivenule than in periportal cells. Internalization and degradation of the ligand were also more adversely affected in the centrilobular region as shown by decreases of greater than 60% in perivenule cells and by only 20% to 30% in periportal cells of ethanol-fed animals compared with controls.

  12. Adaptor Protein 2 Regulates Receptor-Mediated Endocytosis and Cyst Formation in Giardia lamblia

    PubMed Central

    Rivero, Maria R.; Vranych, Cecilia V.; Bisbal, Mariano; Maletto, Belkys A.; Ropolo, Andrea S.; Touz, Maria C.

    2010-01-01

    Synopsis The parasite Giardia lamblia possesses peripheral vacuoles (PVs) that function as both endosomes and lysosomes and are implicated in the adaptation, differentiation, and survival of the parasite in different environments. The mechanisms by which Giardia traffics essential proteins to these organelles and regulates their secretion have important implications in the control of parasite dissemination. In this study, we describe the participation of the heterotetrameric clathrin-adaptor protein gAP2 complex in lysosomal protein trafficking. A specific monoclonal antibody against the medium subunit (gμ2) of gAP2 showed localization of this complex to the PVs, cytoplasm, and plasma membrane in the growing trophozoites. gAP2 also colocalized with clathrin in the PVs, suggesting its involvement in endocytosis. Uptake experiments using standard molecules for the study of endocytosis revealed that gAP2 specifically participated in the endocytosis of LDL. Targeted downregulation of the gene encoding gμ2 in growing and encysting trophozoites resulted in a large decrease in the amount of cell growth and cyst wall formation, suggesting a distinct mechanism in which gAP2 is directly involved in both endocytosis and vesicular trafficking. PMID:20199400

  13. Delta opioid receptors: reflexive, defensive and vocal affective responses in female rats.

    PubMed

    Haney, M; Miczek, K A

    1995-09-01

    Ultrasonic vocalizations may be an expression of the affective pain response in laboratory animals. The present experiment compares the effects of morphine to the delta agonist, DPDPE (D-Pen2,D-Pen5 enkephalin) on a range of reflexive, behavioral and affective responses during an aggressive interaction. In experiment 1, naive female Long-Evans rats received morphine (0, 1, 3, 6, 10 micrograms ICV), or DPDPE (0, 30, 60, 100 micrograms ICV). In experiment 2, female rats were treated with naltrindole (1.0 mg/kg IP) 20 min before DPDPE (0, 60, 100 micrograms ICV). The following endpoints were measured: (1) latency to tail flick in response to heat stimuli; (2) high (33-65 kHz) and low (20-32 kHz) frequency ultrasonic and audible vocalizations; (3) defensive behavior; and (4) motoric activity. Following a brief exposure to attack, rats were threatened by the aggressor but protected from further attack by a large, wire mesh cage, thereby allowing for continued behavioral and vocal measurement without the risk of physical injury; video and audio recordings were made during the attack and then during a portion of the protected encounter (2 min). Morphine suppressed pain reactions varying in complexity from a spinal reflex, to an organized escape reaction, to an affective vocal response. The delta agonist, DPDPE, attenuated high frequency ultrasonic calling and tail flick responding. Defensive behaviors were also modulated by DPDPE at doses that had no effect on walking or rearing, indicating behavioral specificity. By contrast, doses of morphine that decreased defensive upright and escape also decreased motor activity. In female rats, morphine and DPDPE share a common profile of effects on a range of functional end-points, but DPDPE appears to modulate more selectively the reactions related to aversiveness without exerting sedative effects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8545526

  14. Corticosterone suppresses vasotocin-enhanced clasping behavior in male rough-skinned newts by novel mechanisms interfering with V1a receptor availability and receptor-mediated endocytosis.

    PubMed

    Davis, Audrey; Abraham, Emily; McEvoy, Erin; Sonnenfeld, Sarah; Lewis, Christine; Hubbard, Catherine S; Dolence, E Kurt; Rose, James D; Coddington, Emma

    2015-03-01

    In rough-skinned newts, Taricha granulosa, exposure to an acute stressor results in the rapid release of corticosterone (CORT), which suppresses the ability of vasotocin (VT) to enhance clasping behavior. CORT also suppresses VT-induced spontaneous activity and sensory responsiveness of clasp-controlling neurons in the rostromedial reticular formation (Rf). The cellular mechanisms underlying this interaction remain unclear. We hypothesized that CORT blocks VT-enhanced clasping by interfering with V1a receptor availability and/or VT-induced endocytosis. We administered a physiologically active fluorescent VT conjugated to Oregon Green (VT-OG) to the fourth ventricle 9 min after an intraperitoneal injection of CORT (0, 10, 40 μg/0.1mL amphibian Ringers). The brains were collected 30 min post-VT-OG, fixed, and imaged with confocal microscopy. CORT diminished the number of endocytosed vesicles, percent area containing VT-OG, sum intensity of VT-OG, and the amount of VT-V1a within each vesicle; indicating that CORT was interfering with V1a receptor availability and VT-V1a receptor-mediated endocytosis. CORT actions were brain location-specific and season-dependent in a manner that is consistent with the natural and context-dependent expression of clasping behavior. Furthermore, the sensitivity of the Rf to CORT was much higher in animals during the breeding season, arguing for ethologically appropriate seasonal variation in CORT's ability to prevent VT-induced endocytosis. Our data are consistent with the time course and interaction effects of CORT and VT on clasping behavior and neurophysiology. CORT interference with VT-induced endocytosis may be a common mechanism employed by hormones across taxa for mediating rapid context- and season-specific behavioral responses. PMID:25528549

  15. CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors.

    PubMed

    Loebrich, Sven; Benoit, Marc Robert; Konopka, Jaclyn Aleksandra; Cottrell, Jeffrey Richard; Gibson, Joanne; Nedivi, Elly

    2016-02-01

    Internalization of glutamate receptors at the postsynaptic membrane via clathrin-mediated endocytosis (CME) is a key mechanism for regulating synaptic strength. A role for the F-actin cytoskeleton in CME is well established, and recently, PKA-dependent association of candidate plasticity gene 2 (CPG2) with the spine-cytoskeleton has been shown to mediate synaptic glutamate receptor internalization. Yet, how the endocytic machinery is physically coupled to the actin cytoskeleton to facilitate glutamate receptor internalization has not been demonstrated. Moreover, there has been no distinction of endocytic-machinery components that are specific to activity-dependent versus constitutive glutamate receptor internalization. Here, we show that CPG2, through a direct physical interaction, recruits endophilin B2 (EndoB2) to F-actin, thus anchoring the endocytic machinery to the spine cytoskeleton and facilitating glutamate receptor internalization. Regulation of CPG2 binding to the actin cytoskeleton by protein kinase A directly impacts recruitment of EndoB2 and clathrin. Specific disruption of EndoB2 or the CPG2-EndoB2 interaction impairs activity-dependent, but not constitutive, internalization of both NMDA- and AMPA-type glutamate receptors. These results demonstrate that, through direct interactions with F-actin and EndoB2, CPG2 physically bridges the spine cytoskeleton and the endocytic machinery, and this tripartite association is critical specifically for activity-dependent CME of synaptic glutamate receptors. PMID:26776730

  16. Kainate Receptor Post-translational Modifications Differentially Regulate Association with 4.1N to Control Activity-dependent Receptor Endocytosis*

    PubMed Central

    Copits, Bryan A.; Swanson, Geoffrey T.

    2013-01-01

    Kainate receptors exhibit a highly compartmentalized distribution within the brain; however, the molecular and cellular mechanisms that coordinate their expression at neuronal sites of action are poorly characterized. Here we report that the GluK1 and GluK2 kainate receptor subunits interact with the spectrin-actin binding scaffolding protein 4.1N through a membrane-proximal domain in the C-terminal tail. We found that this interaction is important for the forward trafficking of GluK2a receptors, their distribution in the neuronal plasma membrane, and regulation of receptor endocytosis. The association between GluK2a receptors and 4.1N was regulated by both palmitoylation and protein kinase C (PKC) phosphorylation of the receptor subunit. Palmitoylation of the GluK2a subunit promoted 4.1N association, and palmitoylation-deficient receptors exhibited reduced neuronal surface expression and compromised endocytosis. Conversely, PKC activation decreased 4.1N interaction with GluK2/3-containing kainate receptors in acute brain slices, an effect that was reversed after inhibition of PKC. Our data and previous studies therefore demonstrate that these two post-translational modifications have opposing effects on 4.1N association with GluK2 kainate and GluA1 AMPA receptors. The convergence of the signaling pathways regulating 4.1N protein association could thus result in the selective removal of AMPA receptors from the plasma membrane while simultaneously promoting the insertion and stabilization of kainate receptors, which may be important for tuning neuronal excitability and synaptic plasticity. PMID:23400781

  17. Kainate receptor post-translational modifications differentially regulate association with 4.1N to control activity-dependent receptor endocytosis.

    PubMed

    Copits, Bryan A; Swanson, Geoffrey T

    2013-03-29

    Kainate receptors exhibit a highly compartmentalized distribution within the brain; however, the molecular and cellular mechanisms that coordinate their expression at neuronal sites of action are poorly characterized. Here we report that the GluK1 and GluK2 kainate receptor subunits interact with the spectrin-actin binding scaffolding protein 4.1N through a membrane-proximal domain in the C-terminal tail. We found that this interaction is important for the forward trafficking of GluK2a receptors, their distribution in the neuronal plasma membrane, and regulation of receptor endocytosis. The association between GluK2a receptors and 4.1N was regulated by both palmitoylation and protein kinase C (PKC) phosphorylation of the receptor subunit. Palmitoylation of the GluK2a subunit promoted 4.1N association, and palmitoylation-deficient receptors exhibited reduced neuronal surface expression and compromised endocytosis. Conversely, PKC activation decreased 4.1N interaction with GluK2/3-containing kainate receptors in acute brain slices, an effect that was reversed after inhibition of PKC. Our data and previous studies therefore demonstrate that these two post-translational modifications have opposing effects on 4.1N association with GluK2 kainate and GluA1 AMPA receptors. The convergence of the signaling pathways regulating 4.1N protein association could thus result in the selective removal of AMPA receptors from the plasma membrane while simultaneously promoting the insertion and stabilization of kainate receptors, which may be important for tuning neuronal excitability and synaptic plasticity. PMID:23400781

  18. Towards predicting the lung fibrogenic activity of MWCNT: Key role of endocytosis, kinase receptors and ERK 1/2 signaling.

    PubMed

    Vietti, Giulia; Ibouraadaten, Saloua; Palmai-Pallag, Mihaly; Yakoub, Yousof; Piret, Jean-Pascal; Marbaix, Etienne; Lison, Dominique; van den Brule, Sybille

    2016-05-01

    Carbon nanotubes (CNT) have been reported to induce lung inflammation and fibrosis in rodents. We investigated the direct and indirect cellular mechanisms mediating the fibrogenic activity of multi-wall (MW) CNT on fibroblasts. We showed that MWCNT indirectly stimulate lung fibroblast (MLg) differentiation, via epithelial cells and macrophages, whereas no direct effect of MWCNT on fibroblast differentiation or collagen production was detected. MWCNT directly stimulated the proliferation of fibroblasts primed with low concentrations of growth factors, such as PDGF, TGF-β or EGF. MWCNT prolonged ERK 1/2 phosphorylation induced by low concentrations of PDGF or TGF-β in fibroblasts. This phenomenon and the proliferative activity of MWCNT on fibroblasts was abrogated by the inhibitors of ERK 1/2, PDGF-, TGF-β- and EGF-receptors. This activity was also reduced by amiloride, an endocytosis inhibitor. Finally, the lung fibrotic response to several MWCNT samples (different in length and diameter) correlated with their in vitro capacity to stimulate the proliferation of fibroblasts and to prolong ERK 1/2 signaling in these cells. Our findings point to a crosstalk between MWCNT, kinase receptors, ERK 1/2 signaling and endocytosis which stimulates the proliferation of fibroblasts. The mechanisms of action identified in this study contribute to predict the fibrogenic potential of MWCNT. PMID:26444902

  19. Restoration of Physiologically Responsive Low-Density Lipoprotein Receptor-Mediated Endocytosis in Genetically Deficient Induced Pluripotent Stem Cells.

    PubMed

    Ramakrishnan, Venkat M; Yang, Jeong-Yeh; Tien, Kevin T; McKinley, Thomas R; Bocard, Braden R; Maijub, John G; Burchell, Patrick O; Williams, Stuart K; Morris, Marvin E; Hoying, James B; Wade-Martins, Richard; West, Franklin D; Boyd, Nolan L

    2015-01-01

    Acquiring sufficient amounts of high-quality cells remains an impediment to cell-based therapies. Induced pluripotent stem cells (iPSC) may be an unparalleled source, but autologous iPSC likely retain deficiencies requiring correction. We present a strategy for restoring physiological function in genetically deficient iPSC utilizing the low-density lipoprotein receptor (LDLR) deficiency Familial Hypercholesterolemia (FH) as our model. FH fibroblasts were reprogrammed into iPSC using synthetic modified mRNA. FH-iPSC exhibited pluripotency and differentiated toward a hepatic lineage. To restore LDLR endocytosis, FH-iPSC were transfected with a 31 kb plasmid (pEHZ-LDLR-LDLR) containing a wild-type LDLR (FH-iPSC-LDLR) controlled by 10 kb of upstream genomic DNA as well as Epstein-Barr sequences (EBNA1 and oriP) for episomal retention and replication. After six months of selective culture, pEHZ-LDLR-LDLR was recovered from FH-iPSC-LDLR and transfected into Ldlr-deficient CHO-a7 cells, which then exhibited feedback-controlled LDLR-mediated endocytosis. To quantify endocytosis, FH-iPSC ± LDLR were differentiated into mesenchymal cells (MC), pretreated with excess free sterols, Lovastatin, or ethanol (control), and exposed to DiI-LDL. FH-MC-LDLR demonstrated a physiological response, with virtually no DiI-LDL internalization with excess sterols and an ~2-fold increase in DiI-LDL internalization by Lovastatin compared to FH-MC. These findings demonstrate the feasibility of functionalizing genetically deficient iPSC using episomal plasmids to deliver physiologically responsive transgenes. PMID:26307169

  20. Restoration of Physiologically Responsive Low-Density Lipoprotein Receptor-Mediated Endocytosis in Genetically Deficient Induced Pluripotent Stem Cells

    PubMed Central

    Ramakrishnan, Venkat M.; Yang, Jeong-Yeh; Tien, Kevin T.; McKinley, Thomas R.; Bocard, Braden R.; Maijub, John G.; Burchell, Patrick O.; Williams, Stuart K.; Morris, Marvin E.; Hoying, James B.; Wade-Martins, Richard; West, Franklin D.; Boyd, Nolan L.

    2015-01-01

    Acquiring sufficient amounts of high-quality cells remains an impediment to cell-based therapies. Induced pluripotent stem cells (iPSC) may be an unparalleled source, but autologous iPSC likely retain deficiencies requiring correction. We present a strategy for restoring physiological function in genetically deficient iPSC utilizing the low-density lipoprotein receptor (LDLR) deficiency Familial Hypercholesterolemia (FH) as our model. FH fibroblasts were reprogrammed into iPSC using synthetic modified mRNA. FH-iPSC exhibited pluripotency and differentiated toward a hepatic lineage. To restore LDLR endocytosis, FH-iPSC were transfected with a 31 kb plasmid (pEHZ-LDLR-LDLR) containing a wild-type LDLR (FH-iPSC-LDLR) controlled by 10 kb of upstream genomic DNA as well as Epstein-Barr sequences (EBNA1 and oriP) for episomal retention and replication. After six months of selective culture, pEHZ-LDLR-LDLR was recovered from FH-iPSC-LDLR and transfected into Ldlr-deficient CHO-a7 cells, which then exhibited feedback-controlled LDLR-mediated endocytosis. To quantify endocytosis, FH-iPSC ± LDLR were differentiated into mesenchymal cells (MC), pretreated with excess free sterols, Lovastatin, or ethanol (control), and exposed to DiI-LDL. FH-MC-LDLR demonstrated a physiological response, with virtually no DiI-LDL internalization with excess sterols and an ~2-fold increase in DiI-LDL internalization by Lovastatin compared to FH-MC. These findings demonstrate the feasibility of functionalizing genetically deficient iPSC using episomal plasmids to deliver physiologically responsive transgenes. PMID:26307169

  1. Adenosine A1 Receptor-Mediated Endocytosis of AMPA Receptors Contributes to Impairments in Long-Term Potentiation (LTP) in the Middle-Aged Rat Hippocampus.

    PubMed

    Chen, Zhicheng; Stockwell, Jocelyn; Cayabyab, Francisco S

    2016-05-01

    Aging causes multiple changes in the mammalian brain, including changes in synaptic signaling. Previous reports have shown increased extracellular adenosine in the aging brain, and we recently reported that activation of adenosine A1 receptors (A1Rs) induces AMPA receptor (AMPAR) internalization in rat hippocampus. This study investigated whether aging-related changes in the rat hippocampus include altered surface expression of adenosine A1 and A2A receptors, and whether these changes correspond to changes in AMPAR surface expression and altered synaptic plasticity. We found reduced A1R surface expression in middle-aged rat hippocampus, and also reduced GluA1 and GluA2 AMPAR subunit surface expression. Using a chemically-induced LTP (cLTP) experimental protocol, we recorded fEPSPs in young (1 month old) and middle-aged (7-12 month old) rat hippocampal slices. There were significant impairments in cLTP in middle-aged slices, suggesting impaired synaptic plasticity. Since we previously showed that the A1R agonist N(6)-cyclopentyladenosine (CPA) reduced both A1Rs and GluA2/GluA1 AMPARs, we hypothesized that the observed impaired synaptic plasticity in middle-aged brains is regulated by A1R-mediated AMPAR internalization by clathrin-mediated endocytosis. Following cLTP, we found a significant increase in GluA1 and GluA2 surface expression in young rats, which was blunted in middle-aged brains or in young brains pretreated with CPA. Blocking A1Rs with 8-cyclopentyl-1,3-dipropylxanthine or AMPAR endocytosis with either Tat-GluA2-3Y peptide or dynasore (dynamin inhibitor) similarly enhanced AMPAR surface expression following cLTP. These data suggest that age-dependent alteration in adenosine receptor expression contributes to increased AMPAR endocytosis and impaired synaptic plasticity in aged brains. PMID:26700433

  2. Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2.

    PubMed

    Grassart, Alexandre; Dujeancourt, Annick; Lazarow, Paul B; Dautry-Varsat, Alice; Sauvonnet, Nathalie

    2008-04-01

    There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism-clathrin- and caveolae-independent endocytosis-used by the interleukin-2 receptor beta (IL-2R beta). We address the question of its regulation in comparison with the clathrin-dependent pathway. First, we show that Ras-related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL-2R beta entry, and we identify p21-activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL-2R beta uptake, in contrast to the clathrin-dependent route. We observe that cortactin, a partner of actin and dynamin-two essential endocytic factors-is required for IL-2R beta uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL-2R beta internalization. This study indicates Paks as the first specific regulators of the clathrin-independent endocytosis pathway. PMID:18344974

  3. Synergistic antidepressant-like effects between a kappa opioid antagonist (LY2444296) and a delta opioid agonist (ADL5859) in the mouse forced swim test.

    PubMed

    Huang, Peng; Tunis, Julia; Parry, Christopher; Tallarida, Ronald; Liu-Chen, Lee-Yuan

    2016-06-15

    Kappa opioid (KOP) receptor antagonists and delta opioid (DOP) receptor agonists have antidepressant-like effects in animal tests and may be useful for treatment-resistant depression in humans. In this study, we examined whether the combination of a KOP receptor antagonist and a DOP receptor agonist would produce a better than additive effect (i.e. synergy). LY2444296 is a short-acting selective nonpeptide KOP receptor antagonist. ADL5859 is a selective nonpeptide DOP receptor agonist which does not produce seizures and EEG disturbances. Each compound and combinations of the two were examined in the forced swim test (FST) one h post injection, a screening test for antidepressant-like effect, in male adult C57BL/6J mice (Jackson Lab). LY2444296 [subcutaneous (s.c.) injection] at 10 and 30mg/kg, but not 3mg/kg, significantly decreased immobility time in a dose-dependent manner. Intraperitoneal (i.p.) injections of ADL5859 also reduced immobility time dose-dependently at doses of 3 and 10mg/kg, but not at 1mg/kg. An analysis was conducted using the method of Tallarida and Raffa (2010), which employed dose equivalence. The relative potency of the drugs was determined to be LY2444296: ADL5859=1:0.28, which was the dose ratio for combination studies. Six combinations of the two compounds were tested in mice at a fixed dose ratio. We found that LY2444296 and ADL5859 yielded significant synergistic effects for the antidepressant-like effect at the combined dose ranging from 3.84mg/kg to 9.0mg/kg. ADL5859 (10mg/kg), LY2444296 (30mg/kg) and their combined dose (3.84mg/kg) had no effects on locomotor activities. Since the two drugs have distinct pharmacological profiles, such a synergism will allow use of lower doses of both drugs to achieve desired antidepressant effects with fewer side effects. PMID:27044434

  4. Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor

    PubMed Central

    Amsalem, Ayelet R.; Marom, Barak; Shapira, Keren E.; Hirschhorn, Tal; Preisler, Livia; Paarmann, Pia; Knaus, Petra; Henis, Yoav I.; Ehrlich, Marcelo

    2016-01-01

    The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and –SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3’ terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane. PMID:26739752

  5. Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor.

    PubMed

    Amsalem, Ayelet R; Marom, Barak; Shapira, Keren E; Hirschhorn, Tal; Preisler, Livia; Paarmann, Pia; Knaus, Petra; Henis, Yoav I; Ehrlich, Marcelo

    2016-02-15

    The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and -SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3' terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane. PMID:26739752

  6. Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

    PubMed Central

    Garay, Camilo; Judge, Gurjeet; Lucarelli, Stefanie; Bautista, Stephen; Pandey, Rohan; Singh, Tanveer; Antonescu, Costin N.

    2015-01-01

    Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation. PMID:26246598

  7. Altered Cortical GABAA Receptor Composition, Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome*

    PubMed Central

    Zhou, Chengwen; Huang, Zhiling; Ding, Li; Deel, M. Elizabeth; Arain, Fazal M.; Murray, Clark R.; Patel, Ronak S.; Flanagan, Christopher D.; Gallagher, Martin J.

    2013-01-01

    Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABAA receptors (GABAARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knock-out (Hetα1KO) of the human epilepsy gene, the GABAAR α1 subunit, produced absence epilepsy in mice. Here, we determined the effects of Hetα1KO on the expression and physiology of GABAARs in the mouse cortex. We found that Hetα1KO caused modest reductions in the total and surface expression of the β2 subunit but did not alter β1 or β3 subunit expression, results consistent with a small reduction of GABAARs. Cortices partially compensated for Hetα1KO by increasing the fraction of residual α1 subunit on the cell surface and by increasing total and surface expression of α3, but not α2, subunits. Co-immunoprecipitation experiments revealed that Hetα1KO increased the fraction of α1 subunits, and decreased the fraction of α3 subunits, that associated in hybrid α1α3βγ receptors. Patch clamp electrophysiology studies showed that Hetα1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Hetα1KO reduced base-line GABAAR endocytosis, an effect that probably contributes to the observed changes in GABAAR expression. These findings demonstrate that Hetα1KO exerts two principle disinhibitory effects on cortical GABAAR-mediated inhibitory neurotransmission: 1) a modest reduction of GABAAR number and 2) a partial compensation with GABAAR isoforms that possess physiological properties different from those of the otherwise predominant α1βγ GABAARs. PMID:23744069

  8. Mannose 6-phosphate receptor-dependent endocytosis of lysosomal enzymes is increased in sulfatide-storing kidney cells.

    PubMed

    Klein, Diana; Yaghootfam, Afshin; Matzner, Ullrich; Koch, Bettina; Braulke, Thomas; Gieselmann, Volkmar

    2009-01-01

    Metachromatic leukodystrophy is a lysosomal disorder caused by the deficiency of arylsulfatase A (ASA). This leads to the storage of the sphingolipid 3-O-sulfogalactosylceramide (sulfatide) in various cell types, such as renal tubular cells. Examination of mannose 6-phosphate receptor (MPR300)-dependent endocytosis revealed that uptake of lysosomal enzymes is more than two-fold increased in sulfatide-storing kidney cells. Expression of MPR300 and its internalization rate is increased in these cells, whereas the recycling rate is decreased. Similar alterations can be found for the transferrin receptor, indicating that sulfatide storage leads to a general alteration of the endocytotic pathway. These data allow calculating that the endosomal pool from which receptors can recycle is 1.4- to 2-fold increased in lipid-storing cells. Immunocytochemistry demonstrates that the MPR300 in lipid-storing cells does not co-localize with accumulated sulfatide, suggesting that the kinetics of internalization and recycling appear to be altered indirectly. PMID:19007310

  9. Receptor-Mediated Endocytosis of Two-Dimensional Nanomaterials Undergoes Flat Vesiculation and Occurs by Revolution and Self-Rotation.

    PubMed

    Mao, Jian; Chen, Pengyu; Liang, Junshi; Guo, Ruohai; Yan, Li-Tang

    2016-01-26

    Two-dimensional nanomaterials, such as graphene and transitional metal dichalcogenide nanosheets, are promising materials for the development of antimicrobial surfaces and the nanocarriers for intracellular therapy. Understanding cell interaction with these emerging materials is an urgently important issue to promoting their wide applications. Experimental studies suggest that two-dimensional nanomaterials enter cells mainly through receptor-mediated endocytosis. However, the detailed molecular mechanisms and kinetic pathways of such processes remain unknown. Here, we combine computer simulations and theoretical derivation of the energy within the system to show that the receptor-mediated transport of two-dimensional nanomaterials, such as graphene nanosheet across model lipid membrane, experiences a flat vesiculation event governed by the receptor density and membrane tension. The graphene nanosheet is found to undergo revolution relative to the membrane and, particularly, unique self-rotation around its normal during membrane wrapping. We derive explicit expressions for the formation of the flat vesiculation, which reveals that the flat vesiculation event can be fundamentally dominated by a dimensionless parameter and a defined relationship determined by complicated energy contributions. The mechanism offers an essential understanding on the cellular internalization and cytotoxicity of the emerging two-dimensional nanomaterials. PMID:26741298

  10. The endocytosis of epidermal growth factor in A431 cells: A pH of microenvironment and the dynamics of receptor complex dissociation

    SciTech Connect

    Sorkin, A.D.; Teslenko, L.V.; Nikolsky, N.N. )

    1988-03-01

    The endocytosis and intracellular fate of epidermal growth factor (EGF) were studied in A431 cells. After 15-20 min of internalization at 37{degree}C, rhodomaine-labeled ({sup 125}-I) EGF (EGR-Rh) accumulated into large juxtanuclear compartment consisting of closely related vesicles. This structure was shown to be localized in the para-Golgi region. Fluorescein-labeled transferrin (Tr-FITC) was observed in the same region when added to the cell simultaneously with EGF-Rh. Using microscopy spectrofluorometer, the authors determined that the Tr-FITC-containing para-Golgi structures have a pH of 6.1{plus minus}0.3 while lysosomes containing dextran-fluorescein have a pH of 5.0{plus minus}0.2. To study the dynamics of EGF-receptor dissociation during endocytosis a mild detergent treatment of living cells was used for extraction of an intracellular receptor-unbound EGF. These results suggest that EGF remains associated with receptors during endocytosis in A431 cells until it is transferred to lysosomes where the pH of the EGF microenvironment is dropped to 5. A prolonged presence of EGF-receptor complexes in the para-Golgi region might be of importance in mitotic signaling.

  11. Dynamics of Virus-Receptor Interactions in Virus Binding, Signaling, and Endocytosis

    PubMed Central

    Boulant, Steeve; Stanifer, Megan; Lozach, Pierre-Yves

    2015-01-01

    During viral infection the first challenge that viruses have to overcome is gaining access to the intracellular compartment. The infection process starts when the virus contacts the surface of the host cell. A complex series of events ensues, including diffusion at the host cell membrane surface, binding to receptors, signaling, internalization, and delivery of the genetic information. The focus of this review is on the very initial steps of virus entry, from receptor binding to particle uptake into the host cell. We will discuss how viruses find their receptor, move to sub-membranous regions permissive for entry, and how they hijack the receptor-mediated signaling pathway to promote their internalization. PMID:26043381

  12. Ligand-Mediated Endocytosis and Intracellular Sequestration of Guanylyl Cyclase/Natriuretic Peptide Receptors: Role of GDAY Motif

    PubMed Central

    Pandey, Kailash N.

    2015-01-01

    The guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), also referred to as GC-A, is a single polypeptide molecule having a critical function in blood pressure regulation and cardiovascular homeostasis. GC-A/NPRA, which resides in the plasma membrane, consists of an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular cytoplasmic region containing a protein kinase-like homology domain (KHD) and a guanylyl cyclase (GC) catalytic domain. After binding with atrial and brain natriuretic peptides (ANP and BNP), GC-A/NPRA is internalized and sequestered into intracellular compartments. Therefore, GC-A/NPRA is a dynamic cellular macromolecule that traverses different subcellular compartments through its lifetime. This review describes the roles of short-signal sequences in the internalization, trafficking, and intracellular redistribution of GC-A/NPRA from cell surface to cell interior. Evidence indicates that, after internalization, the ligand-receptor complexes dissociate inside the cell and a population of GC-A/NPRA recycles back to the plasma membrane. Subsequently, the disassociated ligands are degraded in the lysosomes. However a small percentage of the ligand escapes the lysosomal degradative pathway and is released intact into culture medium. By using pharmacologic and molecular perturbants, emphasis has been placed on the cellular regulation and processing of ligand-bound GC-A/NPRA in terms of receptor trafficking and down-regulation in intact cells. The discussion is concluded by examining the functions of short-signal sequence motifs in the cellular life-cycle of GC-A/NPRA, including endocytosis, trafficking, metabolic processing, inactivation, and/or down-regulation in model cell systems. PMID:19941037

  13. Second-messenger regulation of receptor association with clathrin-coated pits: a novel and selective mechanism in the control of CD4 endocytosis.

    PubMed Central

    Foti, M; Carpentier, J L; Aiken, C; Trono, D; Lew, D P; Krause, K H

    1997-01-01

    CD4, a member of the immunoglobulin superfamily, is not only expressed in T4 helper lymphocytes but also in myeloid cells. Receptor-mediated endocytosis plays a crucial role in the regulation of surface expression of adhesion molecules such as CD4. In T lymphocytes p56lck, a CD4-associated tyrosine kinase, prevents CD4 internalization, but in myeloid cells p56lck is not expressed and CD4 is constitutively internalized. In this study, we have investigated the role of cyclic AMP (cAMP) in the regulation of CD4 endocytosis in the myeloid cell line HL-60. Elevations of cellular cAMP were elicited by 1) cholera toxin, 2) pertussis toxin, 3) forskolin and IBMX, 4) NaF, or 5) the physiological receptor agonist prostaglandin E1. All five interventions led to an inhibition of CD4 internalization. Increased cAMP levels did not inhibit endocytosis per se, because internalization of insulin receptors and transferrin receptors and fluid phase endocytosis were either unchanged or slightly enhanced. The mechanism of cAMP inhibition was further analyzed at the ultrastructural level. CD4 internalization, followed either by quantitative electron microscopy autoradiography or by immunogold labeling, showed a rapid and temperature-dependent association of CD4 with clathrin-coated pits in control cells. This association was markedly inhibited in cells with elevated cAMP levels. Thus these findings suggest a second-messenger regulation of CD4 internalization through an inhibition of CD4 association with clathrin-coated pits in p56lck-negative cells. Images PMID:9243514

  14. Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen

    PubMed Central

    Sprott, G. Dennis; Sad, Subash; Fleming, L. Perry; DiCaire, Chantal J.; Patel, Girishchandra B.; Krishnan, Lakshmi

    2003-01-01

    Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis ofarchaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation. PMID:15803661

  15. Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2

    PubMed Central

    Grassart, Alexandre; Dujeancourt, Annick; Lazarow, Paul B; Dautry-Varsat, Alice; Sauvonnet, Nathalie

    2008-01-01

    There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism—clathrin- and caveolae-independent endocytosis—used by the interleukin-2 receptor β (IL-2Rβ). We address the question of its regulation in comparison with the clathrin-dependent pathway. First, we show that Ras-related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL-2Rβ entry, and we identify p21-activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL-2Rβ uptake, in contrast to the clathrin-dependent route. We observe that cortactin, a partner of actin and dynamin—two essential endocytic factors—is required for IL-2Rβ uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL-2Rβ internalization. This study indicates Paks as the first specific regulators of the clathrin-independent endocytosis pathway. PMID:18344974

  16. UBIQUITINATION OF β-ARRESTIN LINKS 7-TRANSMEMBRANE RECEPTOR ENDOCYTOSIS AND ERK ACTIVATION

    PubMed Central

    Shenoy, Sudha K.; Barak, Larry S.; Xiao, Kunhong; Ahn, Seungkirl; Berthouze, Magali; Shukla, Arun K.; Luttrell, Louis M.; Lefkowitz, Robert J.

    2007-01-01

    β-arrestin2 and its ubiquitination play crucial roles in both internalization and signaling of seven-transmembrane receptors (7TMRs). To understand the connection between ubiquitination and β-arrestin’s endocytic and signaling functions, we generated a β-arrestin2 mutant that is defective in ubiquitination (β-arrestin20K), by mutating all the ubiquitin acceptor lysines to arginines and compared its properties with the wild type and a stably ubiquitinated β-arrestin2-Ub chimera. In vitro translated β-arrestin2 and β-arrestin20K displayed equivalent binding to recombinant β2 adrenergic receptor (β2AR) reconstituted in vesicles, while β-arrestin2-Ub bound approximately four fold more. In cellular coimmunoprecipitation assays, β-arrestin20K bound non-receptor partners such as AP-2 and c-Raf and scaffolded pERK robustly, but displayed weak binding to clathrin. Moreover, β-arrestin20K was recruited only transiently to activated receptors at the membrane, did not enhance receptor internalization and decreased the amount of pERK assimilated into isolated β2AR complexes. While the wild type β-arrestin2 formed ERK signaling complexes with the β2AR at the membrane, a stably ubiquitinated β-arrestin2-Ub chimera, not only stabilized the ERK signalosomes but also led to their endosomal targeting. Interestingly, in cellular fractionation assays the ubiquitination state of β-arrestin2 favors its distribution in membrane fractions suggesting that ubiquitination increases β-arrestin’s propensity for membrane association. Our findings suggest that although β-arrestin ubiquitination is dispensable for β-arrestin’s cytosol to membrane translocation and its ‘constitutive’ interactions with some cytosolic proteins, it nevertheless is a prerequisite both for the formation of tight complexes with 7TMRs in vivo as well as for membrane compartment interactions that are crucial for downstream endocytic and signaling processes. PMID:17666399

  17. Calcium-Sensing Receptor: Trafficking, Endocytosis, Recycling, and Importance of Interacting Proteins.

    PubMed

    Ray, Kausik

    2015-01-01

    The cloning of the extracellular calcium-sensing receptor (CaSR) provided a new paradigm in G-protein-coupled receptor (GPCR) signaling in which principal physiological ligand is a cation, namely, extracellular calcium (Ca(o)(2+)). A wealth of information has accumulated in the past two decades about the CaSR's structure and function, its contribution to pathology in disorders of calcium in humans, and CaSR-based therapeutics. The CaSR unlike many other GPCRs must function in the presence of its ligand, thus understanding the mechanisms such as anterograde trafficking and endocytic pathways of this receptor are complex and fallen behind other classical GPCRs. Factors controlling CaSR signaling include various proteins affecting the expression of the CaSR as well as modulation of its trafficking to and from the cell surface. The dimeric cell-surface CaSR links to various heterotrimeric G-proteins (G(q/11), G(i/o), G(12/13), and G(s)) to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. This chapter describes key features of CaSR structure and function and discusses novel mechanisms by which the level of cell-surface receptor expression can be regulated including forward trafficking during biosynthesis, desensitization, internalization and recycling from the cell surface, and degradation. These processes are impacted by its interactions with several proteins in addition to signaling molecules per se (i.e., G-proteins, protein kinases, inositol phosphates, etc.) and include small molecular weight G-proteins (Sar1, Rabs, ARF, P24A, RAMPs, filamin A, 14-3-3 proteins, calmodulin, and caveolin-1). Moreover, CaSR signaling seems compartmentalized in cell-type-specific manner, and caveolin and filamin A likely act as scaffolds that bind signaling components and other key cellular

  18. Acute cross-tolerance to opioids in heroin delta-opioid-responding Swiss Webster mice.

    PubMed

    Rady, J J; Fujimoto, J M

    2000-01-01

    It is generally thought that the mu receptor actions of metabolites, 6-monoacetylmorphine (6MAM) and morphine, account for the pharmacological actions of heroin. However, upon intracerebroventricular (i.c.v.) administration in Swiss Webster mice, heroin and 6MAM act on delta receptors while morphine acts on mu receptors. Swiss Webster mice made tolerant to subcutaneous (s.c.) morphine by morphine pellet were not cross-tolerant to s.c. heroin (at 20 min in the tail flick test). Now, opioids were given in combination, s.c. (6.5 h) and i.c.v. (3 h) preceding testing the challenging agonist i.c.v. (at 10 min in the tail flick test). The combination (s.c. + i.c.v.) morphine pretreatment induced tolerance to the mu action of morphine but no cross-tolerance to the delta action of heroin, 6MAM and DPDPE and explained why morphine pelleting did not produce cross-tolerance to s.c. heroin above. Heroin plus heroin produced tolerance to delta agonists but not to mu agonists. Surprisingly, all combinations of morphine with the delta agonists produced tolerance to morphine which now acted through delta receptors (inhibited by i.c.v. naltrindole), an unusual change in receptor selectivity for morphine. PMID:10810246

  19. Knockdown of GnT-Va expression inhibits ligand-induced downregulation of the epidermal growth factor receptor and intracellular signaling by inhibiting receptor endocytosis

    PubMed Central

    Guo, Hua-Bei; Johnson, Heather; Randolph, Matthew; Lee, Intaek; Pierce, Michael

    2009-01-01

    Changes in the expression of N-glycan branching glycosyltransferases can alter cell surface receptor functions, involving their levels of cell surface retention, rates of internalization into the endosomal compartment, and subsequent intracellular signaling. To study in detail the regulation of signaling of the EGF receptor (EGFR) by GlcNAcβ(1,6)Man branching, we utilized specific siRNA to selectively knockdown GnT-Va expression in the highly invasive human breast carcinoma line MDA-MB231, which resulted in the attenuation of its invasiveness-related phenotypes. Compared to control cells, ligand-induced downregulation of EGFR was significantly inhibited in GnT-Va-suppressed cells. This effect could be reversed by re-expression of GnT-Va, indicating that changes in ligand-induced receptor downregulation were dependent on GnT-Va activity. Knockdown of GnT-Va had no significant effect on c-Cbl mediated receptor ubiquitination and degradation, but did cause the inhibition of receptor internalization, showing that altered signaling and delayed ligand-induced downregulation of EGFR expression resulted from decreased EGFR endocytosis. Similar results were obtained with HT1080 fibrosarcoma cells treated with GnT-Va siRNA. Inhibited receptor internalization caused by the expression of GnT-Va siRNA appeared to be independent of galectin binding since decreased EGFR internalization in the knockdown cells was not affected by the treatment of the cells with lactose, a galectin inhibitor. Our results show that decreased GnT-Va activity due to siRNA expression in human carcinoma cells inhibits ligand-induced EGFR internalization, consequently resulting in delayed downstream signal transduction and inhibition of the EGF-induced, invasiveness-related phenotypes. PMID:19225046

  20. Stat-mediated Signaling Induced by Type I and Type II Interferons (IFNs) Is Differentially Controlled through Lipid Microdomain Association and Clathrin-dependent Endocytosis of IFN Receptors

    PubMed Central

    Marchetti, Marta; Monier, Marie-Noelle; Fradagrada, Alexandre; Mitchell, Keith; Baychelier, Florence; Eid, Pierre; Johannes, Ludger

    2006-01-01

    Type I (α/β) and type II (γ) interferons (IFNs) bind to distinct receptors, although they activate the same signal transducer and activator of transcription, Stat1, raising the question of how signal specificity is maintained. Here, we have characterized the sorting of IFN receptors (IFN-Rs) at the plasma membrane and the role it plays in IFN-dependent signaling and biological activities. We show that both IFN-α and IFN-γ receptors are internalized by a classical clathrin- and dynamin-dependent endocytic pathway. Although inhibition of clathrin-dependent endocytosis blocked the uptake of IFN-α and IFN-γ receptors, this inhibition only affected IFN-α–induced Stat1 and Stat2 signaling. Furthermore, the antiviral and antiproliferative activities induced by IFN-α but not IFN-γ were also affected. Finally, we show that, unlike IFN-α receptors, activated IFN-γ receptors rapidly become enriched in plasma membrane lipid microdomains. We conclude that IFN-R compartmentalization at the plasma membrane, through clathrin-dependent endocytosis and lipid-based microdomains, plays a critical role in the signaling and biological responses induced by IFNs and contributes to establishing specificity within the Jak/Stat signaling pathway. PMID:16624862

  1. [Reabsorption of yellow fluorescent protein in the Rana temporaria kidney by receptor-mediated endocytosis].

    PubMed

    Seliverstova, E V; Prutskova, N P

    2014-01-01

    The absorption of yellow fluorescent protein (YFP) and the expression of the endocytic receptors, megalin and cubilin, were investigated in the renal proximal tubules (PT) in frogs Rana temporaria after parenteral YFP injections. The methods of confocal microscopy and immunohistochemistry were used. The dynamics of YFP absorption was analyzed 2 h after injection. The logarithmic time dependence of the accumulation of YFP-containing endocytic vesicles in PT cells and the completion of absorption process 90-120 min after injection were shown. Unlike substantial megalin and cubilin expression 15-30 min after YFP introduction, immunolabeled endocytic receptors were not detected in PT cells after 2 h. The re-injection of YFP led to the appearance of apical endocytic vesicles containing megalin or cubilin colocalized with YFP. At the same time, the decrease of YFP uptake associated with reduction in the number of receptor-containing vesicles was demonstrated, suggesting a failure of megalin and cubilin expression. The decrease of absorption capacity of PT cells after YFP re-injection was similar to that found previously under conditions of the competitive absorption of green fluorescent protein (GFP) and YFP injected in different sequences. The data are the further demonstration of the proposed mechanism limiting the tubular protein absorption in the frog kidney and suggest the involvement of megalin and cubilin in uptake and vesicular transport of YFP. PMID:25782287

  2. Insulin and insulin like growth factor II endocytosis and signaling via insulin receptor B

    PubMed Central

    2013-01-01

    Background Insulin and insulin-like growth factors (IGFs) act on tetrameric tyrosine kinase receptors controlling essential functions including growth, metabolism, reproduction and longevity. The insulin receptor (IR) binds insulin and IGFs with different affinities triggering different cell responses. Results We showed that IGF-II induces cell proliferation and gene transcription when IR-B is over-expressed. We combined biotinylated ligands with streptavidin conjugated quantum dots and visible fluorescent proteins to visualize the binding of IGF-II and insulin to IR-B and their ensuing internalization. By confocal microscopy and flow cytometry in living cells, we studied the internalization kinetic through the IR-B of both IGF-II, known to elicit proliferative responses, and insulin, a regulator of metabolism. Conclusions IGF-II promotes a faster internalization of IR-B than insulin. We propose that IGF-II differentially activates mitogenic responses through endosomes, while insulin-activated IR-B remains at the plasma membrane. This fact could facilitate the interaction with key effector molecules involved in metabolism regulation. PMID:23497114

  3. Expression and endocytosis of VEGF and its receptors in human colonic vascular endothelial cells.

    PubMed

    Wang, Dongfang; Lehman, Richard E; Donner, David B; Matli, Mary R; Warren, Robert S; Welton, Mark L

    2002-06-01

    Normal human colonic microvascular endothelial cells (HUCMEC) have been isolated from surgical specimens by their adherence to Ulex europaeus agglutinin bound to magnetic dynabeads that bind alpha-L-fucosyl residues on the endothelial cell membrane. Immunocytochemistry demonstrated the presence of a range of endothelial-specific markers on HUCMEC, including the von Willebrand factor, Ulex europaeus agglutinin, and platelet endothelial cell adhesion molecule-1. The growing cells form monolayers with the characteristic cobblestone morphology of endothelial cells and eventually form tube-like structures. HUCMEC produce vascular endothelial growth factor (VEGF) and express the receptors, kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase, through which VEGF mediates its actions in the endothelium. VEGF induces the tyrosine phosphorylation of KDR and a proliferative response from HUCMEC comparable to that elicited from human umbilical vein endothelial cells (HUVEC). On binding to HUCMEC or HUVEC, (125)I-labeled VEGF internalizes or dissociates to the medium. Once internalized, (125)I-labeled VEGF is degraded and no evidence of ligand recycling was observed. However, significantly less VEGF is internalized, and more is released to the medium from HUCMEC than HUVEC. Angiogenesis results from the proliferation and migration of microvascular, not large-vessel, endothelial cells. The demonstration that microvascular endothelial cells degrade less and release more VEGF to the medium than large-vessel endothelial cells identifies a mechanism permissive of the role of microvascular cells in angiogenesis. PMID:12016135

  4. Light microscopic autoradiographic localization of mu and delta opioid binding sites in the mouse central nervous system

    SciTech Connect

    Moskowitz, A.S.; Goodman, R.R.

    1984-05-01

    Much work has been done on opioid systems in the rat CNS. Although the mouse is widely used in pharmacological studies of opioid action, little has been done to characterize opioid systems in this species. In the present study the distribution of mu and delta opioid binding sites in the mouse CNS was examined using a quantitative in vitro autoradiography procedure. Tritiated dihydromorphine was used to visualize mu sites and (3H-d-Ala2-d-Leu5)enkephalin with a low concentration of morphine was used to visualize delta sites. Mu and delta site localizations in the mouse are very similar to those previously described in the rat (Goodman, R.R., S.H. Snyder, M.J. Kuhar, and W.S. Young, 3d (1980) Proc. Natl. Acad. Sci. U.S.A. 77:6239-6243), with certain exceptions and additions. Mu and delta sites were observed in sensory processing areas, limbic system, extrapyramidal motor system, and cranial parasympathetic system. Differential distributions of mu and delta sites were noted in many areas. Mu sites were prominent in laminae I, IV, and VI of the neocortex, in patches in the striatum, and in the ventral pallidum, nucleus accumbens, medial and midline thalamic nuclei, medial habenular nucleus, interpeduncular nucleus, and laminae I and II of the spinal cord. In contrast, delta sites were prominent in all laminae of the neocortex, olfactory tubercle, diffusely throughout the striatum, and in the basal, lateral, and cortical nuclei of the amygdala. The determination of the differential distributions of opioid binding sites should prove useful in suggesting anatomical substrates for the actions of opiates and opioids.

  5. Agonist-induced changes in RalA activities allows the prediction of the endocytosis of G protein-coupled receptors.

    PubMed

    Zheng, Mei; Zhang, Xiaohan; Guo, Shuohan; Zhang, Xiaowei; Min, Chengchun; Cheon, Seung Hoon; Oak, Min-Ho; Kim, Young Ran; Kim, Kyeong-Man

    2016-01-01

    GTP binding proteins are classified into two families: heterotrimeric large G proteins which are composed of three subunits, and one subunit of small G proteins. Roles of small G proteins in the intracellular trafficking of G protein-coupled receptors (GPCRs) were studied. Among various small G proteins tested, GTP-bound form (G23V) of RalA inhibited the internalization of dopamine D2 receptor independently of the previously reported downstream effectors of RalA, such as Ral-binding protein 1 and PLD. With high affinity for GRK2, active RalA inhibited the GPCR endocytosis by sequestering the GRK2 from receptors. When it was tested for several GPCRs including an endogenous GPCR, lysophosphatidic acid receptor 1, agonist-induced conversion of GTP-bound to GDP-bound RalA, which presumably releases the sequestered GRK2, was observed selectively with the GPCRs which have tendency to undergo endocytosis. Conversion of RalA from active to inactive state occurred by translocation of RGL, a guanine nucleotide exchange factor, from the plasma membrane to cytosol as a complex with Gβγ. These results suggest that agonist-induced Gβγ-mediated conversion of RalA from the GTP-bound form to the GDP-bound form could be a mechanism to facilitate agonist-induced internalization of GPCRs. PMID:26477566

  6. Dynamin-dependent endocytosis of Bone Morphogenetic Protein2 (BMP2) and its receptors is dispensable for the initiation of Smad signaling.

    PubMed

    Paarmann, Pia; Dörpholz, Gina; Fiebig, Juliane; Amsalem, Ayelet R; Ehrlich, Marcelo; Henis, Yoav I; Müller, Thomas; Knaus, Petra

    2016-07-01

    Bone Morphogenetic Protein (BMP) signal transduction via the canonical Smad158 pathway has previously been linked to dynamin-dependent endocytosis, since the application of chemical inhibitors of clathrin or dynamin in functional cell culture based assays negatively affects initiation and propagation of the Smad response. More recent studies, however, demonstrated efficient Smad signaling by non-internalizable BMP2. The role of endocytosis in BMP signal transduction thus remained controversial. In our study we aimed to refine cell biological assays and to apply novel tools, including a new site-directed fluorescently labeled BMP2 ligand, to revisit key steps in BMP Smad signaling. We found that dynamin2 function was required for BMP2 uptake but was dispensable for C-terminal phosphorylation, nuclear translocation and transcriptional activity of BMP-dependent Smads. Furthermore, we demonstrated a role of dynamin2 in the regulation of steady-state and surface BMP receptor levels, as well as an impact on Smad1 protein level. Thus, dynamin2 allows for modulation of basal and ligand-dependent Smad signaling capacity. High levels of functional dynamin2 enhanced the myogenic differentiation of precursor cells. From our study we conclude that dynamin-dependent endocytosis serves as a regulatory mechanism to fine-tune Smad signaling, but it is not a prerequisite for signal initiation and propagation. Our findings contribute to the understanding of fundamental mechanisms of BMP signaling and thus provide important information for future consideration in the context of therapeutic applications of BMPs. PMID:27113717

  7. Real-time observation of the effect of iron on receptor-mediated endocytosis of transferrin conjugated with quantum dots

    NASA Astrophysics Data System (ADS)

    Zhang, Hai-Li; Li, Yong-Qiang; Zhang, Ming-Zhen; Zhao, Yuan-Di

    2010-07-01

    The optical properties of antiphotobleaching and the advantage of long-term fluorescence observation of quantum dots are fully adopted to study the effects of iron on the endocytosis of transferrin. Quantum dots are labeled for transferrin and endocytosis of transferrin in HeLa cells is observed under the normal state, iron overloading, and an iron-deficient state. In these three states, the fluorescence undergoes a gradual process of first dark, then light, and finally dark, indicating the endocytosis of transferrin. The fluorescence intensity analysis shows that a platform emerges when fluorescence changes to a certain degree in the three states. Experienced a same period of time after platform, the fluorescence strength of cells in the normal state is 1.2 times the first value, and the iron-deficiency state is 1.4 times, but the iron overloading state was 0.85 times. We also find that the average fluorescence intensity in cells detected by the spectrophotometer in the iron-deficiency state is almost 7 times than that in a high iron state. All this proves that iron overloading would slow the process, but iron deficiency would accelerate endocytosis. We advance a direct observational method that may contribute to further study of the relationship of iron and transferrin.

  8. Phytophthora infestans RXLR-WY Effector AVR3a Associates with Dynamin-Related Protein 2 Required for Endocytosis of the Plant Pattern Recognition Receptor FLS2.

    PubMed

    Chaparro-Garcia, Angela; Schwizer, Simon; Sklenar, Jan; Yoshida, Kentaro; Petre, Benjamin; Bos, Jorunn I B; Schornack, Sebastian; Jones, Alexandra M E; Bozkurt, Tolga O; Kamoun, Sophien

    2015-01-01

    Pathogens utilize effectors to suppress basal plant defense known as PTI (Pathogen-associated molecular pattern-triggered immunity). However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that the co-receptor BAK1/SERK3 contributes to basal immunity against the potato pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated cell death by binding the plant E3 ligase CMPG1. In contrast, AVR3aKI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and does not suppress INF1-mediated cell death. Here, we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3-dependent PTI responses in N. benthamiana using the elicitor/receptor pair flg22/FLS2 as a model. We found that all tested variants of AVR3a suppress defense responses triggered by flg22 and reduce internalization of activated FLS2. Moreover, we discovered that AVR3a associates with the Dynamin-Related Protein 2 (DRP2), a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, silencing of DRP2 impaired ligand-induced FLS2 internalization but did not affect internalization of the growth receptor BRI1. Our results suggest that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis. We conclude that AVR3a is a multifunctional effector that can suppress BAK1/SERK3-mediated immunity through at least two different pathways. PMID:26348328

  9. Phytophthora infestans RXLR-WY Effector AVR3a Associates with Dynamin-Related Protein 2 Required for Endocytosis of the Plant Pattern Recognition Receptor FLS2

    PubMed Central

    Chaparro-Garcia, Angela; Schwizer, Simon; Sklenar, Jan; Yoshida, Kentaro; Petre, Benjamin; Bos, Jorunn I. B.; Schornack, Sebastian; Jones, Alexandra M. E.; Bozkurt, Tolga O.; Kamoun, Sophien

    2015-01-01

    Pathogens utilize effectors to suppress basal plant defense known as PTI (Pathogen-associated molecular pattern-triggered immunity). However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that the co-receptor BAK1/SERK3 contributes to basal immunity against the potato pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated cell death by binding the plant E3 ligase CMPG1. In contrast, AVR3aKI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and does not suppress INF1-mediated cell death. Here, we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3-dependent PTI responses in N. benthamiana using the elicitor/receptor pair flg22/FLS2 as a model. We found that all tested variants of AVR3a suppress defense responses triggered by flg22 and reduce internalization of activated FLS2. Moreover, we discovered that AVR3a associates with the Dynamin-Related Protein 2 (DRP2), a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, silencing of DRP2 impaired ligand-induced FLS2 internalization but did not affect internalization of the growth receptor BRI1. Our results suggest that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis. We conclude that AVR3a is a multifunctional effector that can suppress BAK1/SERK3-mediated immunity through at least two different pathways. PMID:26348328

  10. Ethanol-induced impairments in receptor-mediated endocytosis of asialoorosomucoid in isolated rat hepatocytes: Time course of impairments and recovery after ethanol withdrawal

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J.

    1989-04-01

    Chronic ethanol administration markedly impairs the process of receptor-mediated endocytosis (RME) of a representative asialoglycoprotein, asialoorosomucoid (ASOR), by the liver. In this study, we further characterized these impairments by identifying the time of onset for ethanol-induced changes in RME as well as establishing the time course for recovery to normal endocytotic values after ethanol withdrawal. Ethanol administration for 3 days did not alter any aspect of endocytosis examined in this study. After feeding ethanol to rats for 7 days, however, significant decreases in amounts of ligand bound, internalized, and degraded were apparent. These impairments persisted throughout the 5-week feeding study although the effects were somewhat attenuated with more prolonged ethanol feeding. In addition, an accumulation of intracellular receptors was observed in ethanol-fed animals relative to controls after 7 days of ethanol feeding. In all cases, recovery of endocytotic values to control levels was partially completed after 2 to 3 days of refeeding control diet and was fully completed after 7 days of refeeding. These results indicate that ethanol feeding for as little as 7 days profoundly impairs the process of RME by the liver. These impairments can be reversed after refeeding control diet for 7 days.

  11. Tritiation of delta opioid-receptor selective antagonist dipeptide ligands with extraordinary affinity containing 2', 6'dimethyltyrosine

    NASA Astrophysics Data System (ADS)

    Kertész, I.; Tóth, G.; Balboni, G.; Guerrini, R.; Salvadori, S.

    1999-01-01

    Recently a new class of δ opioid antagonists has been discovered by using Tyr-Tic sequence. The substitution of Tyr1 by Dmt resulted in a new analogue (H-Dmt-Tic-OH) with enhanced affinity and selectivity. Because of its excellent property we chose it for labelling with tritium. At the same time peptides containing Tic at position 2 undergo spontaneous diketopiperazine formation in some solvents, and they lose some of their binding ability. To avoid this unwanted side-reaction we synthetized the N-methylated analogue (N,N(Me)2-Dmt-Tic-OH), and it was more stable under storage condition, but δ affinity declined moderately. On the basis of this information we prepared diiodinated analogues of these dipeptides. Catalytic dehalotritiation of precursors resulted in tritiated peptides. High specific radioactivity, 44.67 Ci/mmol with [3H]Dmt-Tic-OH and 59.88 Ci/mmol with N,N(Me)2-[3H]Dmt-Tic-OH were achieved.

  12. Tritiation of delta opioid-receptor selective antagonist dipeptide ligands with extraordinary affinity containing 2‧, 6‧dimethyltyrosine

    NASA Astrophysics Data System (ADS)

    Kertész, I.; Tóth, G.; Balboni, G.; Guerrini, R.; Salvadori, S.

    1999-01-01

    Recently a new class of δ opioid antagonists has been discovered by using Tyr-Tic sequence. The substitution of Tyr1 by Dmt resulted in a new analogue (H-Dmt-Tic-OH) with enhanced affinity and selectivity. Because of its excellent property we chose it for labelling with tritium. At the same time peptides containing Tic at position 2 undergo spontaneous diketopiperazine formation in some solvents, and they lose some of their binding ability. To avoid this unwanted side-reaction we synthetized the N-methylated analogue (N,N(Me)2-Dmt-Tic-OH), and it was more stable under storage condition, but δ affinity declined moderately. On the basis of this information we prepared diiodinated analogues of these dipeptides. Catalytic dehalotritiation of precursors resulted in tritiated peptides. High specific radioactivity, 44.67 Ci/mmol with [3H]Dmt-Tic-OH and 59.88 Ci/mmol with N,N(Me)2-[3H]Dmt-Tic-OH were achieved.

  13. Role of {alpha}{sub v}{beta}{sub 5} integrin receptor in endocytosis of crocidolite and its effect on intracellular glutathione levels in human lung epithelial (A549) cells

    SciTech Connect

    Pande, Priyadarshini; Mosleh, Tariq A.; Aust, Ann E. . E-mail: aaust@cc.usu.edu

    2006-01-15

    Crocidolite, containing 27% iron by weight, is the most carcinogenic form of asbestos. Crocidolite fibers are endocytized by {alpha}{sub v}{beta}{sub 5} integrin receptors in rabbit pleural mesothelial cells. We show here that crocidolite fibers are endocytized in human lung epithelial (A549) cells and in primary small airway epithelial (SAEC) cells. Presence of the integrin {alpha}{sub v}{beta}{sub 5} blocking antibody, P1F6, significantly reduced the uptake of crocidolite fibers in A549 cells. Thus, the integrin {alpha}{sub v}{beta}{sub 5} receptor is involved in endocytosis of crocidolite fibers in A549 cells as well. Previously, it has been observed that asbestos fibers lead to changes in the intracellular redox environment, i.e. a marked decrease in intracellular glutathione concentrations and an increase in the extracellular glutathione in A549 cells. In addition, the decrease in intracellular glutathione was found to be largely independent of iron present on the surface of the fiber. A549 cells were treated with crocidolite in the presence of endocytosis inhibitor cytochalasin D. Our data indicate that, upon preventing endocytosis, we were able to reverse the decrease in total intracellular glutathione. The decrease in total intracellular glutathione could also be prevented in the presence of the monoclonal antibody P1F6. Thus, we observed that endocytosis of crocidolite fibers via integrin {alpha}{sub v}{beta}{sub 5} receptor is linked to the marked decrease in total intracellular glutathione in A549 cells.

  14. Effect of octreotide surface density on receptor-mediated endocytosis in vitro and anticancer efficacy of modified nanocarrier in vivo after optimization.

    PubMed

    Su, Zhigui; Shi, Yongping; Xiao, Yanyu; Sun, Minjie; Ping, Qineng; Zong, Li; Li, Sai; Niu, Jiangxiu; Huang, Aiwen; You, Weiliang; Chen, Yinan; Chen, Xi; Fei, Jia; Tian, Jia

    2013-04-15

    The objective of the present work was to investigate the optimum density of octreotide on the surface of nanostructured lipid carriers (NLC) loaded with hydroxycamptothencine (HCPT) to enhance receptor-mediated endocytosis and tumor targeting selectivity. Different amounts of octreotide-polyethylene glycol (100) monostearate (OPMS), a ligand for somatostatin receptors (SSTRs), were coupled into NLC. In vitro evaluation of OPMS modified NLCs (O-NLCs) was done by studying the physicochemical properties, drug release, cellular uptake and cytotoxicity. Whereas in vivo evaluation was done by studying the tissue distribution in S180 tumor-bearing mice through ex vivo fluorescence imaging and HCPT quantitative study. The results showed that O-NLCs with an average size of ∼100 nm possessed obvious sustained release. When OPMS was used in the amount of 5 μmol (O₅-NLC) highest cellular uptake, cytotoxicity in SMMC-7721 cell line and remarkable accumulation in S180 tumor were observed. The treatments of O₅-NLC brought about significant tumor inhibition and prolonged the median survival time as compared with HCPT, unmodified NLC and the pegylated NLC (P₅-NLC) groups. It appears that to achieve a more rational approach of receptor mediated tumor targeted drug delivery system the surface density of the targeting moiety on the surface of nanocarriers should be considered. PMID:23396258

  15. A Trypanosoma cruzi Phosphatidylinositol 3-Kinase (TcVps34) Is Involved in Osmoregulation and Receptor-mediated Endocytosis*S⃞

    PubMed Central

    Schoijet, Alejandra C.; Miranda, Kildare; Girard-Dias, Wendell; de Souza, Wanderley; Flawiá, Mirtha M.; Torres, Héctor N.; Docampo, Roberto; Alonso, Guillermo D.

    2008-01-01

    Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking. PMID:18801733

  16. The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling

    PubMed Central

    Cruse, Glenn; Beaven, Michael A.; Music, Stephen C.; Bradding, Peter; Gilfillan, Alasdair M.; Metcalfe, Dean D.

    2015-01-01

    MS4A family members differentially regulate the cell cycle, and aberrant, or loss of, expression of MS4A family proteins has been observed in colon and lung cancer. However, the precise functions of MS4A family proteins and their mechanistic interactions remain unsolved. Here we report that MS4A4 facilitates trafficking of the receptor tyrosine kinase KIT through endocytic recycling rather than degradation pathways by a mechanism that involves recruitment of KIT to caveolin-1–enriched microdomains. Silencing of MS4A4 in human mast cells altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes while reducing that in the plasma membrane, as exemplified by Akt and PLCγ1 phosphorylation, respectively. The altered endocytic trafficking of KIT also resulted in an increase in SCF-induced mast cell proliferation and migration, which may reflect altered signaling in these cells. Our data reveal a novel function for MS4A family proteins in regulating trafficking and signaling, which could have implications in both proliferative and immunological diseases. PMID:25717186

  17. 2,8-Diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine potent CCR4 antagonists capable of inducing receptor endocytosis.

    PubMed

    Shukla, Lena; Ajram, Laura A; Begg, Malcolm; Evans, Brian; Graves, Rebecca H; Hodgson, Simon T; Lynn, Sean M; Miah, Afjal H; Percy, Jonathan M; Procopiou, Panayiotis A; Richards, Stephen A; Slack, Robert J

    2016-06-10

    A number of potent 2,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine CCR4 antagonists binding to the extracellular allosteric site were synthesised. (R)-N-(2,4-Dichlorobenzyl)-2-(2-(pyrrolidin-2-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine (R)-(18a) has high affinity in both the [(125)I]-TARC binding assay with a pKi of 8.8, and the [(35)S]-GTPγS functional assay with a pIC50 of 8.1, and high activity in the human whole blood actin polymerisation assay (pA2 = 6.7). The most potent antagonists were also investigated for their ability to induce endocytosis of CCR4 and were found to internalise about 60% of the cell surface receptors, a property which is not commonly shared by small molecule antagonists of chemokine receptors. PMID:26991939

  18. The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling.

    PubMed

    Cruse, Glenn; Beaven, Michael A; Music, Stephen C; Bradding, Peter; Gilfillan, Alasdair M; Metcalfe, Dean D

    2015-05-01

    MS4A family members differentially regulate the cell cycle, and aberrant, or loss of, expression of MS4A family proteins has been observed in colon and lung cancer. However, the precise functions of MS4A family proteins and their mechanistic interactions remain unsolved. Here we report that MS4A4 facilitates trafficking of the receptor tyrosine kinase KIT through endocytic recycling rather than degradation pathways by a mechanism that involves recruitment of KIT to caveolin-1-enriched microdomains. Silencing of MS4A4 in human mast cells altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes while reducing that in the plasma membrane, as exemplified by Akt and PLCγ1 phosphorylation, respectively. The altered endocytic trafficking of KIT also resulted in an increase in SCF-induced mast cell proliferation and migration, which may reflect altered signaling in these cells. Our data reveal a novel function for MS4A family proteins in regulating trafficking and signaling, which could have implications in both proliferative and immunological diseases. PMID:25717186

  19. Quantum dots implementation as a label for analysis of early stages of EGF receptor endocytosis: a comparative study on cultured cells

    PubMed Central

    Salova, Anna V.; Belyaeva, Tatiana N.; Leontieva, Ekaterina A.; Zlobina, Maria V.; Kharchenko, Marianna V.; Kornilova, Elena S.

    2016-01-01

    EGF complexed to fluorescent photostable quantum dots by biotin-streptavidin system (bEGF-savQD) is attractive for both the basic research and therapeutic application such as targeted drug delivery in EGF-receptor (EGFR) expressing cancers. However, compared to native EGF, the large size of QD and its quasi-multivalency can have unpredictable effects on EGFR endocytosis changing the internalization portal and/or endosomal processing tightly bound to EGF signaling. We have found that bEGF-savQDs enter HeLa cells via the temperature-dependent clathrin-mediated EGF-receptor-specific pathway characteristic for native EGF. We also found that EGF-to-QD concentration ratios used for the complex preparation and the level of EGF receptor expression affect the number and integral densities of the formed endosomes. So, at EGF-to-QD ratio from 4:1 to 12:1 (at nanomolar bEGF concentrations) on average 100 bright endosomes per HeLa cell were formed 15 min after the complex addition, while 1:1 ratio resulted in formation of very few dim endosomes. However, in A431 cells overexpressing EGFR 1:1 ratio was effective. Using dynamin inhibition and Na-acidic washout we showed that bEGF-savQDs bind surface receptors and enter clathrin-coated pits slower than the same ligands without QD. Yet, the bEGF-savQD demonstrated similar to native EGF and bEGF-savCy3 co-localization dynamics with tethering protein EEA1 and HRS, the key component of sorting ESCRT0 complex. In conclusion, our comparative study reveals that in respect to entrapment into coated pits, endosomal recruitment, endosome fusions, and the initial steps of endosomal maturation, bEGF-savQD behaves like native EGF and QD implementation does not affect these important events. PMID:26716513

  20. Quantum dots implementation as a label for analysis of early stages of EGF receptor endocytosis: a comparative study on cultured cells.

    PubMed

    Salova, Anna V; Belyaeva, Tatiana N; Leontieva, Ekaterina A; Zlobina, Maria V; Kharchenko, Marianna V; Kornilova, Elena S

    2016-02-01

    EGF complexed to fluorescent photostable quantum dots by biotin-streptavidin system (bEGF-savQD) is attractive for both the basic research and therapeutic application such as targeted drug delivery in EGF-receptor (EGFR) expressing cancers. However, compared to native EGF, the large size of QD and its quasi-multivalency can have unpredictable effects on EGFR endocytosis changing the internalization portal and/or endosomal processing tightly bound to EGF signaling. We have found that bEGF-savQDs enter HeLa cells via the temperature-dependent clathrin-mediated EGF-receptor-specific pathway characteristic for native EGF. We also found that EGF-to-QD concentration ratios used for the complex preparation and the level of EGF receptor expression affect the number and integral densities of the formed endosomes. So, at EGF-to-QD ratio from 4:1 to 12:1 (at nanomolar bEGF concentrations) on average 100 bright endosomes per HeLa cell were formed 15 min after the complex addition, while 1:1 ratio resulted in formation of very few dim endosomes. However, in A431 cells overexpressing EGFR 1:1 ratio was effective. Using dynamin inhibition and Na-acidic washout we showed that bEGF-savQDs bind surface receptors and enter clathrin-coated pits slower than the same ligands without QD. Yet, the bEGF-savQD demonstrated similar to native EGF and bEGF-savCy3 co-localization dynamics with tethering protein EEA1 and HRS, the key component of sorting ESCRT0 complex. In conclusion, our comparative study reveals that in respect to entrapment into coated pits, endosomal recruitment, endosome fusions, and the initial steps of endosomal maturation, bEGF-savQD behaves like native EGF and QD implementation does not affect these important events. PMID:26716513

  1. Low concentrations of primaquine inhibit degradation but not receptor-mediated endocytosis of asialoorosomucoid by HepG2 cells

    SciTech Connect

    Reif, J.S.; Schwartz, A.L.; Fallon, R.J. )

    1991-02-01

    Asialoorosomucoid (ASOR) is internalized and degraded by HepG2 cells after binding to the asialoglycoprotein (ASGP) receptor, internalization through the coated pit/coated vesicle pathway, and trafficking to lysosomes. Primaquine, an 8-aminoquinoline antimalarial compound, inhibits ASOR degradation at concentrations greater than 0.2 mM by neutralizing intracellular acid compartments. This leads to alterations in surface receptor number, receptor-ligand dissociation, and receptor recycling. We have investigated the effects of primaquine on 125I-ASOR uptake and degradation as a function of primaquine concentration and duration of exposure. Concentrations below those required for neutralization of acidic compartments block 125I-ASOR degradation in HepG2 cells and lead to intracellular ligand accumulation. This effect is maximal at 80 microM primaquine. The intracellular 125I-ASOR is undegraded, dissociated from the ASGP receptor, and contained within vesicular compartments distinct from lysosomes, plasma membrane, or endosomes. In addition, the effect of 80 microM primaquine on 125I-ASOR degradation is very slowly reversible (greater than 6 h), in contrast to primaquine's rapidly reversible effect on receptor recycling and ligand uptake (10 min). Furthermore, the effect is ligand-specific. 125I-asialofetuin, another ASGP receptor ligand, is internalized and degraded in lysosomes at normal rates in HepG2 cells exposed to 80 microM primaquine. These findings indicate that primaquine has multiple effects on the uptake and degradation of ligand occurring in the endosome-lysosome pathway. These effects of primaquine differ in their concentration-dependence, site of action, reversibility, and ligand selectivity.

  2. Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells.

    PubMed

    Kosheverova, Vera V; Kamentseva, Rimma S; Gonchar, Ilya V; Kharchenko, Marianna V; Kornilova, Elena S

    2016-04-22

    Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. PMID:26993163

  3. Tyrosine phosphorylation regulates the endocytosis and surface expression of GluN3A-containing NMDA receptors.

    PubMed

    Chowdhury, Dhrubajyoti; Marco, Sonia; Brooks, Ian M; Zandueta, Aitor; Rao, Yijian; Haucke, Volker; Wesseling, John F; Tavalin, Steven J; Pérez-Otaño, Isabel

    2013-02-27

    Selective control of receptor trafficking provides a mechanism for remodeling the receptor composition of excitatory synapses, and thus supports synaptic transmission, plasticity, and development. GluN3A (formerly NR3A) is a nonconventional member of the NMDA receptor (NMDAR) subunit family, which endows NMDAR channels with low calcium permeability and reduced magnesium sensitivity compared with NMDARs comprising only GluN1 and GluN2 subunits. Because of these special properties, GluN3A subunits act as a molecular brake to limit the plasticity and maturation of excitatory synapses, pointing toward GluN3A removal as a critical step in the development of neuronal circuitry. However, the molecular signals mediating GluN3A endocytic removal remain unclear. Here we define a novel endocytic motif (YWL), which is located within the cytoplasmic C-terminal tail of GluN3A and mediates its binding to the clathrin adaptor AP2. Alanine mutations within the GluN3A endocytic motif inhibited clathrin-dependent internalization and led to accumulation of GluN3A-containing NMDARs at the cell surface, whereas mimicking phosphorylation of the tyrosine residue promoted internalization and reduced cell-surface expression as shown by immunocytochemical and electrophysiological approaches in recombinant systems and rat neurons in primary culture. We further demonstrate that the tyrosine residue is phosphorylated by Src family kinases, and that Src-activation limits surface GluN3A expression in neurons. Together, our results identify a new molecular signal for GluN3A internalization that couples the functional surface expression of GluN3A-containing receptors to the phosphorylation state of GluN3A subunits, and provides a molecular framework for the regulation of NMDAR subunit composition with implications for synaptic plasticity and neurodevelopment. PMID:23447623

  4. The Matricellular Receptor LRP1 Forms an Interface for Signaling and Endocytosis in Modulation of the Extracellular Tumor Environment.

    PubMed

    Van Gool, Bart; Dedieu, Stéphane; Emonard, Hervé; Roebroek, Anton J M

    2015-01-01

    The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1) has been attributed a role in cancer. However, its presumably often indirect involvement is far from understood. LRP1 has both endocytic and signaling activities. As a matricellular receptor it is involved in regulation, mostly by clearing, of various extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases, protease inhibitor complexes, and the endoglycosidase heparanase. Furthermore, by binding extracellular ligands including growth factors and subsequent intracellular interaction with scaffolding and adaptor proteins it is involved in regulation of various signaling cascades. LRP1 expression levels are often downregulated in cancer and some studies consider low LRP1 levels a poor prognostic factor. On the contrary, upregulation in brain cancers has been noted and clinical trials explore the use of LRP1 as cargo receptor to deliver cytotoxic agents. This mini-review focuses on LRP1's role in tumor growth and metastasis especially by modulation of the extracellular tumor environment. In relation to this role its diagnostic, prognostic and therapeutic potential will be discussed. PMID:26617523

  5. The Matricellular Receptor LRP1 Forms an Interface for Signaling and Endocytosis in Modulation of the Extracellular Tumor Environment

    PubMed Central

    Van Gool, Bart; Dedieu, Stéphane; Emonard, Hervé; Roebroek, Anton J. M.

    2015-01-01

    The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1) has been attributed a role in cancer. However, its presumably often indirect involvement is far from understood. LRP1 has both endocytic and signaling activities. As a matricellular receptor it is involved in regulation, mostly by clearing, of various extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases, protease inhibitor complexes, and the endoglycosidase heparanase. Furthermore, by binding extracellular ligands including growth factors and subsequent intracellular interaction with scaffolding and adaptor proteins it is involved in regulation of various signaling cascades. LRP1 expression levels are often downregulated in cancer and some studies consider low LRP1 levels a poor prognostic factor. On the contrary, upregulation in brain cancers has been noted and clinical trials explore the use of LRP1 as cargo receptor to deliver cytotoxic agents. This mini-review focuses on LRP1’s role in tumor growth and metastasis especially by modulation of the extracellular tumor environment. In relation to this role its diagnostic, prognostic and therapeutic potential will be discussed. PMID:26617523

  6. Impaired endocytosis in proximal tubule from subchronic exposure to cadmium involves angiotensin II type 1 and cubilin receptors

    PubMed Central

    2013-01-01

    Background Chronic exposure to low cadmium (Cd) levels produces urinary excretion of low molecular weight proteins, which is considered the critical effect of Cd exposure. However, the mechanisms involved in Cd-induced proteinuria are not entirely clear. Therefore, the present study was designed to evaluate the possible role of megalin and cubilin (important endocytic receptors in proximal tubule cells) and angiotensin II type 1 (AT1) receptor on Cd-induced microalbuminuria. Methods Four groups of female Wistar rats were studied. Control (CT) group, vehicle-treated rats; LOS group, rats treated with losartan (an AT1 antagonist) from weeks 5 to 8 (10 mg/kg/day by gavage); Cd group, rats subchronically exposed to Cd (3 mg/kg/day by gavage) during 8 weeks, and Cd + LOS group, rats treated with Cd for 8 weeks and LOS from weeks 5–8. Kidney Cd content, glomerular function (evaluated by creatinine clearance and plasma creatinine), kidney injury and tubular function (evaluated by Kim-1 expression, urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) and glucose, and microalbuminuria), oxidative stress (measured by lipid peroxidation and NAD(P)H oxidase activity), mRNA levels of megalin, expressions of megalin and cubilin (by confocal microscopy) and AT1 receptor (by Western blot), were measured in the different experimental groups. Data were analyzed by one-way ANOVA or Kruskal-Wallis test using GraphPad Prism 5 software (Version 5.00). P < 0.05 was considered statistically significant. Results Administration of Cd (Cd and Cd + LOS groups) increased renal Cd content. LOS-treatment decreased Cd-induced microalbuminuria without changes in: plasma creatinine, creatinine clearance, urinary NAG and glucose, oxidative stress, mRNA levels of megalin and cubilin, neither protein expression of megalin nor AT1 receptor, in the different experimental groups studied. However, Cd exposure did induce the expression of the tubular injury marker Kim-1 and decreased

  7. [The increase in receptor-mediated endocytosis of drugs in the composition of nanoparticles with the address fragment].

    PubMed

    Kostryukova, L V; Sanzhakov, M A; Ignatov, D V; Prozorovskyi, V N; Druzhilovskaya, O S; Kasatkina, E S; Medvedeva, N V; Ipatova, O M

    2016-03-01

    It is known that disorders in the cell functioning of the organs/tissues is accompanied by increased expression of certain receptors. A modern approach to improve the specificity of the drug accumulation in the affected area is to construct the delivery nanosystems with the address fragments. Active tagged transport may help to reduce the dose of the drug, minimizing the impact on healthy cells and organs (reduced adverse events). This approach is particularly important in oncology because of the high toxicity of the drugs used. In this work we have obtained and characterized the pharmaceutical composition of doxorubicin and chlorine e6 into colloidal nanoparticles with synthesized previously targeted conjugates based on folic acid and biotin. On the cell culture Hep G2 it was shown an increase in the internalization of drugs when they were introduced in the incubation medium in the form of drug compositions with transport nanosystems and targeted fragments. PMID:27420624

  8. Windpipe Controls Drosophila Intestinal Homeostasis by Regulating JAK/STAT Pathway via Promoting Receptor Endocytosis and Lysosomal Degradation

    PubMed Central

    Li, Min; Wu, Longfei; Wang, Guolun; Baeg, Gyeong-Hun; You, Jia; Li, Zhouhua; Lin, Xinhua

    2015-01-01

    The adult intestinal homeostasis is tightly controlled by proper proliferation and differentiation of intestinal stem cells. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is essential for the regulation of adult stem cell activities and maintenance of intestinal homeostasis. Currently, it remains largely unknown how JAK/STAT signaling activities are regulated in these processes. Here we have identified windpipe (wdp) as a novel component of the JAK/STAT pathway. We demonstrate that Wdp is positively regulated by JAK/STAT signaling in Drosophila adult intestines. Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions. Conversely, ectopic expression of Wdp inhibits JAK/STAT signaling activity. Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation. Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis. PMID:25923769

  9. Endocytosis via galactose receptors in vivo. Ligand size directs uptake by hepatocytes and/or liver macrophages

    SciTech Connect

    Schlepper-Schaefer, J.; Huelsmann, D.; Djovkar, A.; Meyer, H.E.; Herbertz, L.; Kolb, H.; Kolb-Bachofen, V.

    1986-01-01

    The intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues is rat liver was followed. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au/sub 5/), 17 nm (Au/sub 17/), 50 nm (Au/sub 50/) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au/sub 5/ in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc.

  10. Synaptic Vesicle Endocytosis

    PubMed Central

    Saheki, Yasunori; De Camilli, Pietro

    2012-01-01

    Neurons can sustain high rates of synaptic transmission without exhausting their supply of synaptic vesicles. This property relies on a highly efficient local endocytic recycling of synaptic vesicle membranes, which can be reused for hundreds, possibly thousands, of exo-endocytic cycles. Morphological, physiological, molecular, and genetic studies over the last four decades have provided insight into the membrane traffic reactions that govern this recycling and its regulation. These studies have shown that synaptic vesicle endocytosis capitalizes on fundamental and general endocytic mechanisms but also involves neuron-specific adaptations of such mechanisms. Thus, investigations of these processes have advanced not only the field of synaptic transmission but also, more generally, the field of endocytosis. This article summarizes current information on synaptic vesicle endocytosis with an emphasis on the underlying molecular mechanisms and with a special focus on clathrin-mediated endocytosis, the predominant pathway of synaptic vesicle protein internalization. PMID:22763746

  11. Apo- and holo-lactoferrin are both internalized by lactoferrin receptor via clathrin-mediated endocytosis but differentially affect ERK-signaling and cell proliferation in Caco-2 cells

    PubMed Central

    Jiang, Rulan; Lopez, Veronica; Kelleher, Shannon L.; Lönnerdal, Bo

    2011-01-01

    Lactoferrin (Lf) is a major iron-binding and multi-functional protein in exocrine fluids such as breast milk and mucosal secretions. The functions of Lf appear dependent upon the iron-saturation of the Lf protein and are postulated to be mediated through Lf internalization by a Lf receptor (LfR). However, mechanisms by which LfR mediates Lf internalization in enterocytes are unknown. We now demonstrate that a LfR previously cloned from the small intestine mediates Lf endocytosis in a human enterocyte model (Caco-2 cells). LfR was detected at the plasma membrane by cell surface biotinylation; both apo-Lf and holo-Lf uptake were significantly inhibited in cells transfected with LfR siRNA. Treatments of hypertonic sucrose and clathrin siRNA and co-immunoprecipitation of LfR with clathrin adaptor AP2 indicate that LfR regulates Lf endocytosis via clathrin-mediated endocytosis. Although both iron-free Lf (apo-Lf) and iron-saturated Lf (holo-Lf) enter Caco-2 cells via a similar mechanism and no significant differences were observed in the binding and uptake of apo- and holo-Lf in Caco-2 cells, apo-Lf but not holo-Lf stimulates proliferation of Caco-2 cells. Interestingly, apo-Lf stimulated extracellular signal-regulated mitogen-activated protein kinase (ERK) cascade to a significantly greater extent than holo-Lf and the apo-Lf induced proliferation was significantly inhibited by an ERK cascade inhibitor (U0126) and clathrin siRNA. Taken together, our data suggest that LfR is a major pathway through which Lf is taken up by enterocytes, which occurs independently of iron saturation through clathrin-mediated endocytosis. The differential effects of apo- and holo-Lf are not due to differences in cellular internalization mechanisms. PMID:21935933

  12. Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities

    PubMed Central

    Nair, Padma; Yamamoto, Takashi; Largent-Milnes, Tally M.; Cowell, Scott; Kulkarni, Vinod; Moye, Sharif; Navratilova, Edita; Davis, Peg; Ma, Shou-Wu; Vanderah, Todd W.; Lai, Josephine; Porreca, Frank; Hruby, Victor J.

    2013-01-01

    The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr1-DAla2-Gly3-Phe4-Gly5-Trp6-O-[3′,5′-Bzl(CF3)2]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo. PMID:23899615

  13. Endocytosis of apolipoprotein A-V by members of the low density lipoprotein receptor and the VPS10p domain receptor families.

    PubMed

    Nilsson, Stefan K; Christensen, Stine; Raarup, Merete K; Ryan, Robert O; Nielsen, Morten S; Olivecrona, Gunilla

    2008-09-19

    Apolipoprotein A-V (apoA-V) is present in low amounts in plasma and has been found to modulate triacylglycerol levels in humans and in animal models. ApoA-V displays affinity for members of the low density lipoprotein receptor (LDL-R) gene family, known as the classical lipoprotein receptors, including LRP1 and SorLA/LR11. In addition to LDL-A binding repeats, the mosaic receptor SorLA/LR11 also possesses a Vps10p domain. Here we show that apoA-V also binds to sortilin, a receptor from the Vsp10p domain gene family that lacks LDL-A repeats. Binding of apoA-V to sortilin was competed by neurotensin, a ligand that binds specifically to the Vps10p domain. To investigate the biological fate of receptor-bound apoA-V, binding experiments were conducted with cultured human embryonic kidney cells transfected with either SorLA/LR11 or sortilin. Compared with nontransfected cells, apoA-V binding to SorLA/LR11- and sortilin-expressing cells was markedly enhanced. Internalization experiments, live imaging studies, and fluorescence resonance energy transfer analyses demonstrated that labeled apoA-V was rapidly internalized, co-localized with receptors in early endosomes, and followed the receptors through endosomes to the trans-Golgi network. The observed decrease of fluorescence signal intensity as a function of time during live imaging experiments suggested ligand uncoupling in endosomes with subsequent delivery to lysosomes for degradation. This interpretation was supported by experiments with (125)I-labeled apoA-V, demonstrating clear differences in degradation between transfected and nontransfected cells. We conclude that apoA-V binds to receptors possessing LDL-A repeats and Vsp10p domains and that apoA-V is internalized into cells via these receptors. This could be a mechanism by which apoA-V modulates lipoprotein metabolism in vivo. PMID:18603531

  14. Effects of the delta-opioid agonist SNC80 on the abuse liability of methadone in rhesus monkeys: a behavioral economic analysis

    PubMed Central

    Banks, Matthew L.; Roma, Peter G.; Folk, John E.; Rice, Kenner C.

    2012-01-01

    Rationale Delta-opioid agonists enhance the antinociceptive efficacy of methadone and other mu-opioid agonists. However, relatively little is known about the degree to which delta agonists might enhance the abuse-related effects of mu agonists. Objective This study used a behavioral economic approach to examine effects of the delta agonist SNC80 [(+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethylbenzamide] on the reinforcing effects of methadone in a drug self-administration assay. Interactions between SNC80 and cocaine were also examined for comparison. Methods Rhesus monkeys (n=4), surgically implanted with indwelling intravenous catheters, were tested in two phases. In phase 1, drug self-administration dose-effect curves for methadone (0.0032–0.1 mg/kg/injection (inj)) and cocaine (0.0032–0.32 mg/kg/inj) alone were determined under a fixed-ratio 10 (FR 10) schedule of reinforcement. In phase 2, FR values were increased every 3 days (FR 1–FR 1800) during availability of methadone alone (0.032 mg/kg/inj) and in combination with varying proportions of SNC80 (0.1:1, 0.3:1, and 0.9:1 SNC80/methadone) or of cocaine alone (0.032 mg/kg/inj) and in combination with varying proportions of SNC80 (0.33:1, 1:1, and 3:1 SNC80/ cocaine). Demand curves related drug intake to FR price, and measures of reinforcement were derived. Results Methadone and cocaine alone each functioned as a reinforcer. SNC80 did not alter measures of reinforcement for either methadone or cocaine. Conclusions SNC80 at proportions previously shown to enhance methadone-induced antinociception did not enhance the abuse-related effects of methadone. These results support the proposition that delta agonists may selectively enhance mu agonist analgesic effects without enhancing mu agonist abuse liability. PMID:21369752

  15. Endocytosis of Nanomedicines

    PubMed Central

    Sahay, Gaurav; Alakhova, Daria Y; Kabanov, Alexander V

    2010-01-01

    Novel nanomaterials are being developed to improve diagnosis and therapy of diseases through effective delivery of drugs, biopharmaceutical molecules and imaging agents to target cells in disease sites. Such diagnostic and therapeutic nanomaterials, also termed “nanomedicines”, often require site-specific cellular entry to deliver their payload to subcellular locations hidden beneath cell membranes. Nanomedicines can employ multiple pathways for cellular entry, which are currently insufficiently understood. This review, first, classifies various mechanisms of endocytosis available to nanomedicines including phagocytosis and pinocytosis through clathrin-dependent and clathrin-independent pathways. Second, it describes the current experimental tools to study endocytosis of nanomedicines. Third, it provides specific examples from recent literature and our own work on endocytosis of nanomedicines. Finally, these examples are used to ascertain 1) the role of particle size, shape, material composition, surface chemistry and/or charge for utilization of a selected pathway(s); 2) the effect of cell type on the processing of nanomedicines; 3) the effect of nanomaterial-cell interactions on the processes of endocytosis, the fate of the nanomedicines and the resulting cellular responses. This review will be useful to a diverse audience of students and scientists who are interested in understanding endocytosis of nanomedicines. PMID:20226220

  16. Adapting for endocytosis: roles for endocytic sorting adaptors in directing neural development

    PubMed Central

    Yap, Chan Choo; Winckler, Bettina

    2015-01-01

    Proper cortical development depends on the orchestrated actions of a multitude of guidance receptors and adhesion molecules and their downstream signaling. The levels of these receptors on the surface and their precise locations can greatly affect guidance outcomes. Trafficking of receptors to a particular surface locale and removal by endocytosis thus feed crucially into the final guidance outcomes. In addition, endocytosis of receptors can affect downstream signaling (both quantitatively and qualitatively) and regulated endocytosis of guidance receptors is thus an important component of ensuring proper neural development. We will discuss the cell biology of regulated endocytosis and the impact on neural development. We focus our discussion on endocytic accessory proteins (EAPs) (such as numb and disabled) and how they regulate endocytosis and subsequent post-endocytic trafficking of their cognate receptors (such as Notch, TrkB, β-APP, VLDLR, and ApoER2). PMID:25904845

  17. Synaptic actions of neuropeptide FF in the rat parabrachial nucleus: interactions with opioid receptors.

    PubMed

    Chen, X; Zidichouski, J A; Harris, K H; Jhamandas, J H

    2000-08-01

    The pontine parabrachial nucleus (PBN) receives both opioid and Neuropeptide FF (NPFF) projections from the lower brain stem and/or the spinal cord. Because of this anatomical convergence and previous evidence that NPFF displays both pro- and anti-opioid activities, this study examined the synaptic effects of NPFF in the PBN and the mechanisms underlying these effects using an in vitro brain slice preparation and the nystatin-perforated patch-clamp recording technique. Under voltage-clamp conditions, NPFF reversibly reduced the evoked excitatory postsynaptic currents (EPSCs) in a dose-dependent fashion. This effect was not accompanied by apparent changes in the holding current, the current-voltage relationship or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced inward currents in the PBN cells. When a paired-pulse protocol was used, NPFF increased the ratio of these synaptic currents. Analysis of miniature EPSCs showed that NPFF caused a rightward shift in the frequency-distribution curve, whereas the amplitude-distribution curve remained unchanged. Collectively, these experiments indicate that NPFF reduces the evoked EPSCs through a presynaptic mechanism of action. The synaptic effects induced by NPFF (5 microM) could not be blocked by the specific mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (1 microM), but application of delta-opioid receptor antagonist Tyr-Tic-Phe-Phe (5 microM) almost completely prevented effects of NPFF. Moreover, the delta-opioid receptor agonist, Deltorphin (1 microM), mimicked the effects as NPFF and also occluded NPFF's actions on synaptic currents. These results indicate that NPFF modulates excitatory synaptic transmission in the PBN through an interaction with presynaptic delta-opioid receptors. These observations provide a cellular basis for NPFF enhancement of the antinociceptive effects consequent to central activation of delta-opioid receptors. PMID:10938301

  18. Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages

    PubMed Central

    Shim, Do-Wan; Heo, Kang-Hyuck; Kim, Young-Kyu; Sim, Eun-Jeong; Kang, Tae-Bong; Choi, Jae-Wan; Sim, Dae-Won; Cheong, Sun-Hee; Lee, Seung-Hong; Bang, Jeong-Kyu; Won, Hyung-Sik; Lee, Kwang-Ho

    2015-01-01

    Antimicrobial peptides (AMPs), also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1β, IL-6, INF-β, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4)- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation. PMID:26017270

  19. Rapid agonist-induced loss of sup 125 I-. beta. -endorphin opioid receptor sites in NG108-15, but not SK-N-SH neuroblastoma cells

    SciTech Connect

    Cone, R.I.; Lameh, J.; Sadee, W. )

    1991-01-01

    The authors have measured {mu} and {delta} opioid receptor sites on intact SK-N-SH and NG108-15 neuroblastoma cells, respectively, in culture. Use of {sup 125}I-{beta}-endorphin ({beta}E) as a tracer, together with {beta}E(6-31) to block high-affinity non-opioid binding in both cell lines, permitted the measurement of cell surface {mu} and {delta} opioid receptor sites. Labeling was at {delta} sites in NG108-15 cells and predominantly at {mu} sites in SK-N-SH cells. Pretreatment with the {mu} and {delta} agonist, DADLE, caused a rapid loss of cell surface {delta} receptor sites in NG108-15 cells, but failed to reduce significantly {mu} receptor density in SK-N-SH cells.

  20. An immersed boundary method for endocytosis

    NASA Astrophysics Data System (ADS)

    Tseng, Yu-Hau; Huang, Huaxiong

    2014-09-01

    Endocytosis is one of the cellular functions for capturing (engulfing) vesicles or microorganisms. Understanding the biophysical mechanisms of this cellular process is essential from a bioengineering point of view since it will provide guidance for developing effective targeted drug delivery therapies. In this paper, we propose an immersed boundary (IB) method that can be used to simulate the dynamical process of this important biological function. In our model, membranes of the vesicle and the cell are treated as Canham-Helfrich Hamiltonian interfaces. The membrane-bound molecules are modeled as insoluble surfactants such that the molecules after binding are regarded as a product of a “chemical” reaction. Our numerical examples show that the immersed boundary method is a useful simulation tool for studying endocytosis, where the roles of interfacial energy, fluid flow and viscous dissipation in the success of the endocytosis process can be investigated in detail. A distinct feature of our IB method is the treatment of the two binding membranes that is different from the merging of fluid-fluid interfaces. Another important feature of our method is the strict conservation of membrane-borne receptors and ligands, which is important for predicting the dynamics of the endocytosis process.

  1. Exploiting Endocytosis for Nanomedicines

    PubMed Central

    Akinc, Akin; Battaglia, Giuseppe

    2013-01-01

    In this article, we briefly review the endocytic pathways used by cells, pointing out their defining characteristics and highlighting physical limitations that may direct the internalization of nanoparticles to a subset of these pathways. A more detailed description of these pathways is presented in the literature. We then focus on the endocytosis of nanomedicines and present how various nanomaterial parameters impact these endocytic processes. This topic is an area of active research, motivated by the recognition that an improved understanding of how nanomaterials interact at the molecular, cellular, and whole-organism level will lead to the design of better nanomedicines in the future. Next, we briefly review some of the important nanomedicines already on the market or in clinical development that serve to exemplify how endocytosis can be exploited for medical benefit. Finally, we present some key unanswered questions and remaining challenges to be addressed by the field. PMID:24186069

  2. Endocytosis in enterocytes.

    PubMed

    Zimmer, Klaus-Peter; de Laffolie, Jan; Barone, Maria Vittoria; Naim, Hassan Y

    2016-05-01

    Endocytosis is a fundamental cell biological process, which carries out essential functions in a polarized epithelial cell such as enterocytes provided with a huge surface area of the brush border membrane. Major tasks of enterocytes, which are regulated by endocytic signals, are digestion and absorption of nutrients and drugs/pharmacological agents, barrier permeability to microorganism, toxins and antigens, and transcytotic crosstalk between intestinal lumen and lamina propria cells with access to the circulation.Investigations on inflammatory bowel diseases such as food allergy, celiac disease, Crohn's disease, and ulcerative colitis focus on immune processes originating within enterocytes as antigen presenting cells. Thus the initiation of oral tolerance, that is, the binding of food antigens to MHC class II proteins, might be localized within late endosomes of enterocytes. Furthermore, the late endosomal compartment of enterocytes seems to be involved in the processing of luminal antigens during the pathogenesis of celiac disease and inflammatory bowel diseases. Investigations of inherited diseases such as microvillus inclusion disease have revealed a pathogenetic defect in the autophagocytotic and/or recycling pathway of enterocytes.Our progress in the cell and molecular biological understanding of the endocytosis and the methodical opportunities of translational research offer now new therapeutic options for patients suffering from endocytosis-related diseases of enterocytes. PMID:26993488

  3. Endocytosis and Endosomal Trafficking in Plants.

    PubMed

    Paez Valencia, Julio; Goodman, Kaija; Otegui, Marisa S

    2016-04-29

    Endocytosis and endosomal trafficking are essential processes in cells that control the dynamics and turnover of plasma membrane proteins, such as receptors, transporters, and cell wall biosynthetic enzymes. Plasma membrane proteins (cargo) are internalized by endocytosis through clathrin-dependent or clathrin-independent mechanism and delivered to early endosomes. From the endosomes, cargo proteins are recycled back to the plasma membrane via different pathways, which rely on small GTPases and the retromer complex. Proteins that are targeted for degradation through ubiquitination are sorted into endosomal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery for degradation in the vacuole. Endocytic and endosomal trafficking regulates many cellular, developmental, and physiological processes, including cellular polarization, hormone transport, metal ion homeostasis, cytokinesis, pathogen responses, and development. In this review, we discuss the mechanisms that mediate the recognition and sorting of endocytic and endosomal cargos, the vesiculation processes that mediate their trafficking, and their connection to cellular and physiological responses in plants. PMID:27128466

  4. Differential effects of exercise on brain opioid receptor binding and activation in rats.

    PubMed

    Arida, Ricardo Mario; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abrão; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa

    2015-01-01

    Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms. PMID:25330347

  5. Activation of G protein by opioid receptors: role of receptor number and G-protein concentration.

    PubMed

    Remmers, A E; Clark, M J; Alt, A; Medzihradsky, F; Woods, J H; Traynor, J R

    2000-05-19

    The collision-coupling model for receptor-G-protein interaction predicts that the rate of G-protein activation is dependent on receptor density, but not G-protein levels. C6 cells expressing mu- or delta-opioid receptors, or SH-SY5Y cells, were treated with beta-funaltrexamine (mu) or naltrindole-5'-isothiocyanate (delta) to decrease receptor number. The time course of full or partial agonist-stimulated ¿35SGTPgammaS binding did not vary in C6 cell membranes containing <1-25 pmol/mg mu-opioid receptor, or 1. 4-4.3 pmol/mg delta-opioid receptor, or in SHSY5Y cells containing 0. 16-0.39 pmol/mg receptor. The association of ¿35SGTPgammaS binding was faster in membranes from C6mu cells than from C6delta cells. A 10-fold reduction in functional G-protein, following pertussis toxin treatment, lowered the maximal level of ¿35SGTPgammaS binding but not the association rate. These data indicate a compartmentalization of opioid receptors and G protein at the cell membrane. PMID:10822058

  6. Catching Up with Ultrafast Endocytosis.

    PubMed

    Brockmann, Marisa M; Rosenmund, Christian

    2016-05-01

    The mechanism and speed of endocytosis at central synapses after neurotransmitter release is still under debate. In this issue of Neuron,Delvendahl et al. (2016) propose an ultrafast form of endocytosis after single action potentials (APs) at physiological temperature. PMID:27151632

  7. Myoferlin is critical for endocytosis in endothelial cells

    PubMed Central

    Bernatchez, Pascal N.; Sharma, Arpeeta; Kodaman, Pinar

    2009-01-01

    Myoferlin is a member of the ferlin family of proteins that promotes endomembrane fusion with the plasma membrane in muscle cells and endothelial cells. In addition, myoferlin is necessary for the surface expression of vascular endothelial growth factor receptor 2 through the formation of a protein complex with dynamin-2 (Dyn-2). Since Dyn-2 is necessary for the fission of endocytic vesicles from the plasma membrane, we tested the hypothesis that myoferlin may regulates aspects of receptor-dependent endocytosis. Here we show that myoferlin gene silencing decreases both clathrin and caveolae/raft-dependent endocytosis, whereas ectopic myoferlin expression in COS-7 cells increases endocytosis by up to 125%. Interestingly, we have observed that inhibition of Dyn-2 activity or caveolin-1 (Cav-1) expression impairs endocytosis as well as membrane resealing after injury, indicating that Dyn-2 and Cav-1 also participate in both membrane fission and fusion processes. Mechanistically, myoferlin partially colocalizes with Dyn-2 and Cav-1 and forms a protein complex with Cav-1 solubilized from tissue extracts. Together, these data describe a new role for myoferlin in receptor-dependent endocytosis and an overlapping role for myoferlin-Dyn-2-Cav-1 protein complexes in membrane fusion and fission events. PMID:19494235

  8. Actin and Endocytosis in Budding Yeast

    PubMed Central

    Goode, Bruce L.; Eskin, Julian A.; Wendland, Beverly

    2015-01-01

    Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. PMID:25657349

  9. Actin-Regulator Feedback Interactions during Endocytosis.

    PubMed

    Wang, Xinxin; Galletta, Brian J; Cooper, John A; Carlsson, Anders E

    2016-03-29

    Endocytosis mediated by clathrin, a cellular process by which cells internalize membrane receptors and their extracellular ligands, is an important component of cell signaling regulation. Actin polymerization is involved in endocytosis in varying degrees depending on the cellular context. In yeast, clathrin-mediated endocytosis requires a pulse of polymerized actin and its regulators, which recruit and activate the Arp2/3 complex. In this article, we seek to identify the main protein-protein interactions that 1) cause actin and its regulators to appear in pulses, and 2) determine the effects of key mutations and drug treatments on actin and regulator assembly. We perform a joint modeling/experimental study of actin and regulator dynamics during endocytosis in the budding yeast Saccharomyces cerevisiae. We treat both a stochastic model that grows an explicit three-dimensional actin network, and a simpler two-variable Fitzhugh-Nagumo type model. The models include a negative-feedback interaction of F-actin onto the Arp2/3 regulators. Both models explain the pulse time courses and the effects of interventions on actin polymerization: the surprising increase in the peak F-actin count caused by reduced regulator branching activity, the increase in F-actin resulting from slowing of actin disassembly, and the increased Arp2/3 regulator lifetime resulting from latrunculin treatment. In addition, they predict that decreases in the regulator branching activity lead to increases in accumulation of regulators, and we confirmed this prediction with experiments on yeast harboring mutations in the Arp2/3 regulators, using quantitative fluorescence microscopy. Our experimental measurements suggest that the regulators act quasi-independently, in the sense that accumulation of a particular regulator is most strongly affected by mutations of that regulator, as opposed to the others. PMID:27028652

  10. Cloning and pharmacological characterization of a rat kappa opioid receptor.

    PubMed Central

    Meng, F; Xie, G X; Thompson, R C; Mansour, A; Goldstein, A; Watson, S J; Akil, H

    1993-01-01

    A full-length cDNA was isolated from a rat striatal library by using low-stringency screening with two PCR fragments, one spanning transmembrane domains 3-6 of the mouse delta opioid receptor and the other unidentified but homologous to the mouse delta receptor from rat brain. The novel cDNA had a long open reading frame encoding a protein of 380 residues with 59% identity to the mouse delta receptor and topography consistent with a seven-helix guanine nucleotide-binding protein-coupled receptor. COS-1 cells transfected with the coding region of this clone showed high-affinity binding to kappa opioid receptor-selective ligands such as dynorphin A and U-50,488 and also nonselective opioid ligands such as bremazocine, ethylketocyclazocine, and naloxone. Not bound at all (or bound with low affinity) were dynorphin A-(2-13), enantiomers of naloxone and levophanol [i.e., (+)-naloxone and dextrorphan], and selective mu and delta opioid receptor ligands. Activation of the expressed receptor by kappa receptor agonists led to inhibition of cAMP. Finally, in situ hybridization revealed a mRNA distribution in rat brain that corresponded well to the distribution of binding sites labeled with kappa-selective ligands. These observations indicate that we have cloned a cDNA encoding a rat kappa receptor of the kappa 1 subtype. Images Fig. 3 PMID:8234341

  11. Different subtypes of opioid receptors have different affinities for G-proteins.

    PubMed

    Polastron, J; Jauzac, P

    1994-05-01

    In this work, we have characterized the opioid receptor expressed by the human neuroblastoma cell line SK-N-BE and compared its hydrodynamic behaviour with those of well known opioid receptors: mu-opioid receptor of rabbit cerebellum and delta-opioid receptor of the hybrid cell line NG 108-15. Human neuroblastoma cell line SK-N-BE expresses a substantial amount of opioid receptors (200-300 fmoles/mg of protein). Pharmacological characterization suggests an heterogenous population of receptors and the presence of two delta subtypes which are, at least partially, negatively coupled with adenylate cyclase via a Gi protein. These receptors exist under two different molecular forms and, in this respect, strikingly contrast with the archetypic delta receptors of NG 108-15 hybrid cell line which show only a high molecular weight form and appear more tightly coupled with the G protein. Hydrodynamic behaviour of SK-N-BE opioid receptors is reminiscent of the profile observed with the rabbit cerebellum mu-opioid receptor. This observation is consistent with the presence of two delta-opioid receptors subtypes, one of which exhibiting properties close to those of mu opioid receptors. Taken overall, our results suggest that different types and subtypes of opioid receptors, even if they are coupled to the same inhibitory G protein, are more or less tightly coupled with their transduction proteins and that closely related opioid receptors can form allosterically interacting complexes. PMID:7920183

  12. In Vivo Techniques to Investigate the Internalization Profile of Opioid Receptors

    PubMed Central

    Pradhan, Amynah A.; Tawfik, Vivianne L.; Laboy, Alycia F.; Scherrer, Grégory

    2015-01-01

    G-protein-coupled receptors (GPCRs) regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Receptor internalization is commonly observed following agonist binding and activation. Receptor trafficking events have been well characterized in cell systems, but the in vivo significance of GPCR internalization is still poorly understood. To address this issue, we have developed an innovative knock-in mouse model, where an opioid receptor is directly visible in vivo. These knockin mice express functional fluorescent delta opioid receptors (DOR-eGFP) in place of the endogenous receptor, and these receptors are expressed at physiological levels within their native environment. DOR-eGFP mice have proven to be an extraordinary tool in studying receptor neuroanatomy, real-time receptor trafficking in live neurons, and in vivo receptor internalization. We have used this animal model to determine the relationship between receptor trafficking in neurons and receptor function at a behavioral level. Here, we describe in detail the construction and characterization of this knockin mouse. We also outline how to use these mice to examine the behavioral consequences of agonist-specific trafficking at the delta opioid receptor. These techniques are potentially applicable to any GPCR, and highlight the powerful nature of this imaging tool. PMID:25293318

  13. Notch1 endocytosis is induced by ligand and is required for signal transduction.

    PubMed

    Chapman, G; Major, J A; Iyer, K; James, A C; Pursglove, S E; Moreau, J L M; Dunwoodie, S L

    2016-01-01

    The Notch signalling pathway is widely utilised during embryogenesis in situations where cell-cell interactions are important for cell fate specification and differentiation. DSL ligand endocytosis into the ligand-expressing cell is an important aspect of Notch signalling because it is thought to supply the force needed to separate the Notch heterodimer to initiate signal transduction. A functional role for receptor endocytosis during Notch signal transduction is more controversial. Here we have used live-cell imaging to examine trafficking of the Notch1 receptor in response to ligand binding. Contact with cells expressing ligands induced internalisation and intracellular trafficking of Notch1. Notch1 endocytosis was accompanied by transendocytosis of ligand into the Notch1-expressing signal-receiving cell. Ligand caused Notch1 endocytosis into SARA-positive endosomes in a manner dependent on clathrin and dynamin function. Moreover, inhibition of endocytosis in the receptor-expressing cell impaired ligand-induced Notch1 signalling. Our findings resolve conflicting observations from mammalian and Drosophila studies by demonstrating that ligand-dependent activation of Notch1 signalling requires receptor endocytosis. Endocytosis of Notch1 may provide a force on the ligand:receptor complex that is important for potent signal transduction. PMID:26522918

  14. On the modeling of endocytosis

    NASA Astrophysics Data System (ADS)

    Zhang, Tao; Sknepnek, Rastko; Schwarz, Jennifer; Bowick, Mark

    2013-03-01

    Endocytosis is the primary mechanism by which extracellular material enters the cell. During endocytosis, the cell membrane deforms to surround the extracellular material and draw it into the cell, followed by a pinch-off to produce an internal vesicle. Recent experiments on clathrin-mediated endocytosis all agree that the actin cytoskeleton plays a crucial role in the deformation of the cell membrane. The actin cytoskeleton is a crosslinked network of filaments exerting active forces. However, competing ideas remain as to precisely how the actin cytoskeleton organizes itself to help drive the deformation. To begin to resolve this controversy, we mathematically model clathrin-mediated endocytosis using variational methods and Monte Carlo simulations. In particular, we investigate how the deformation of the cell membrane depends on the organization of the actin cytoskeletal network, and its associated active forces, to rule out one or more of the competing ideas.

  15. Bile acids reduce endocytosis of high-density lipoprotein (HDL) in HepG2 cells.

    PubMed

    Röhrl, Clemens; Eigner, Karin; Fruhwürth, Stefanie; Stangl, Herbert

    2014-01-01

    High-density lipoprotein (HDL) transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence of high concentrations of taurocholate, a natural non-cell-permeable bile acid, in human hepatic HepG2 and HuH7 cells. In contrast, selective cholesteryl-ester (CE) uptake was increased. Taurocholate exerted these effects extracellularly and independently of HDL modification, cell membrane perturbation or blocking of endocytic trafficking. Instead, this reduction of endocytosis and increase in selective uptake was dependent on SR-BI. In addition, cell-permeable bile acids reduced HDL endocytosis by farnesoid X receptor (FXR) activation: chenodeoxycholate and the non-steroidal FXR agonist GW4064 reduced HDL endocytosis, whereas selective CE uptake was unaltered. Reduced HDL endocytosis by FXR activation was independent of SR-BI and was likely mediated by impaired expression of the scavenger receptor cluster of differentiation 36 (CD36). Taken together we have shown that bile acids reduce HDL endocytosis by transcriptional and non-transcriptional mechanisms. Further, we suggest that HDL endocytosis and selective lipid uptake are not necessarily tightly linked to each other. PMID:25010412

  16. Receptor-mediated endocytosis of insulin in lower vertebrates: internalization and intracellular processing of 125I-insulin in isolated hepatocytes of lamprey and frog.

    PubMed

    Lappova, Y L; Leibush, B N

    1995-10-01

    The binding of 125I-insulin to cellular insulin receptors and the internalization of insulin-receptor complexes have been studied in isolated hepatocytes of frog and lamprey. Two classes of binding sites (Kd 10(-9) and 10(-8) M) were found in cells of both species. The molecular weight of the insulin receptor alpha-subunit was 130 kDa in both species. Internalization of bound 125I-insulin in both species was found in the temperature range 0 to 20 degrees. Cells "loaded" with 125I-insulin were used to estimate the fate of the internalized ligand. Release of internalized ligand from frog cells increased at temperatures ranging from 0 to 20 degrees. At 0 degrees the degraded 125I-insulin was 5%, at 5 degrees 7%, and at 20 degrees 17% of total radioactivity accumulated in the medium. In lamprey hepatocytes there was neither radioactivity accumulation in the incubation medium nor release from cells at all temperatures studied. The intracellular degradation of internalized 125I-insulin in frog hepatocytes was much lower than that in lamprey cells. In frog hepatocytes the specific binding of 125I-insulin was increased twofold in the presence of the lysosomal inhibitor chloroquine. In contrast no increase was found in lamprey hepatocytes. In conclusion, the processing pathways of internalized insulin in the cells of ectothermal and endothermal vertebrates are generally similar but in ectothermal animals all events take place at lower temperatures and at lower rates. The peculiarities of insulin processing in lamprey hepatocytes most likely result from the transformation of hepatocytes during the nonfeeding prespawning period. PMID:8575649

  17. Quantitative Analysis of HER2-mediated Effects on HER2 and Epidermal Growth Factor Receptor Endocytosis: DISTRIBUTION OF HOMO- AND HETERODIMERS DEPENDS ON RELATIVE HER2 LEVELS

    SciTech Connect

    Hendriks, Bart S.; Opresko, Lee ); Wiley, H Steven ); Lauffenburger, Douglas A.

    2003-05-15

    Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR) family. Many cell types express multiple EGFR family members (including EGFR, HER2, HER3 and/or HER4) that interact to form an array of homo- and hetero-dimers. Differential trafficking of these receptors should strongly affect signaling through this system by changing substrate access and heterodimerization efficiency. Because of the complexity of these dynamic processes we used a quantitative, computational model to understand this system. As a test case, parameters characterizing EGFR and HER2 interactions were derived using experimental data obtained from mammary epithelial cells constructed to express different levels of HER2. With this data we were able to estimate receptor-specific internalization rate constants and dimer uncoupling rate constants. These parameters were not otherwise experimentally accessible due to the complex system interplay. Our models indicated that HER2:EGFR heterodimers traffic as single entities. Direct experiments using EGF and anti-HER2 and anti-EGFR antibodies using independently derived cell lines confirmed many of the predictions of the model. Furthermore, our model could predict the relationship between HER2 expression levels and the transient distribution of EGFR homodimers and heterodimers. Our results suggest that the levels of HER2 found on normal cells are barely at the threshold necessary to drive efficient heterodimerization. Thus, altering local HER2 concentrations in membrane microdomains could serve as an effective mechanism for regulating HER2 heterodimerization and could explain why HER2 overexpression found in some cancers have such a profound effect on cell physiology.

  18. Nexus of signaling and endocytosis in oncogenesis driven by non-small cell lung cancer-associated epidermal growth factor receptor mutants.

    PubMed

    Chung, Byung Min; Tom, Eric; Zutshi, Neha; Bielecki, Timothy Alan; Band, Vimla; Band, Hamid

    2014-12-10

    Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and aberrant EGFR signaling as a result of receptor overexpression and/or mutation occurs in many types of cancer. Tumor cells in non-small cell lung cancer (NSCLC) patients that harbor EGFR kinase domain mutations exhibit oncogene addiction to mutant EGFR, which confers high sensitivity to tyrosine kinase inhibitors (TKIs). As patients invariably develop resistance to TKIs, it is important to delineate the cell biological basis of mutant EGFR-induced cellular transformation since components of these pathways can serve as alternate therapeutic targets to preempt or overcome resistance. NSCLC-associated EGFR mutants are constitutively-active and induce ligand-independent transformation in nonmalignant cell lines. Emerging data suggest that a number of factors are critical for the mutant EGFR-dependent tumorigenicity, and bypassing the effects of TKIs on these pathways promotes drug resistance. For example, activation of downstream pathways such as Akt, Erk, STAT3 and Src is critical for mutant EGFR-mediated biological processes. It is now well-established that the potency and spatiotemporal features of cellular signaling by receptor tyrosine kinases such as EGFR, as well as the specific pathways activated, is determined by the nature of endocytic traffic pathways through which the active receptors traverse. Recent evidence indicates that NSCLC-associated mutant EGFRs exhibit altered endocytic trafficking and they exhibit reduced Cbl ubiquitin ligase-mediated lysosomal downregulation. More recent work has shown that mutant EGFRs undergo ligand-independent traffic into the endocytic recycling compartment, a behavior that plays a key role in Src pathway activation and oncogenesis. These studies are beginning to delineate the close nexus between signaling and endocytic traffic of EGFR mutants as a key driver of oncogenic processes. Therefore, in this review, we will discuss the links

  19. Cell adhesion defines the topology of endocytosis and signaling

    PubMed Central

    Grossier, Jean-Philippe; Xouri, Georgia; Goud, Bruno; Schauer, Kristine

    2014-01-01

    Preferred sites of endocytosis have been observed in various cell types, but whether they occur randomly or are linked to cellular cues is debated. Here, we quantified the sites of endocytosis of transferrin (Tfn) and epidermal growth factor (EGF) in cells whose adhesion geometry was defined by micropatterns. 3D probabilistic density maps revealed that Tfn was enriched in adhesive sites during uptake, whereas EGF endocytosis was restricted to the dorsal cellular surface. This spatial separation was not due to distributions of corresponding receptors but was regulated by uptake mechanisms. Asymmetric uptake of Tfn resulted from the enrichment of clathrin and adaptor protein 2 at adhesive areas. Asymmetry in EGF uptake was strongly dependent on the actin cytoskeleton and led to asymmetry in EGF receptor activation. Mild alteration of actin dynamics abolished asymmetry in EGF uptake and decreased EGF-induced downstream signaling, suggesting that cellular adhesion cues influence signal propagation. We propose that restriction of endocytosis at distinct sites allows cells to sense their environment in an “outside-in” mechanism. PMID:24366944

  20. Analgesic effect of interferon-alpha via mu opioid receptor in the rat.

    PubMed

    Jiang, C L; Son, L X; Lu, C L; You, Z D; Wang, Y X; Sun, L Y; Cui, R Y; Liu, X Y

    2000-03-01

    Using the tail-flick induced by electro-stimulation as a pain marker, it was found that pain threshold (PT) was significantly increased after injecting interferon-alpha (IFN alpha) into the lateral ventricle of rats. This effect was dosage-dependent and abolished by monoclonal antibody (McAb) to IFN alpha. Naloxone could inhibit the analgesic effect of IFN alpha, suggesting that the analgesic effect of IFN alpha be related to the opioid receptors. Beta-funaltrexamine (beta-FNA), the mu specific receptor antagonist could completely block the analgesic effect of IFN alpha. The selective delta-opioid receptor antagonist, ICI174,864 and the kappa-opioid receptor antagonist, nor-BNI both failed to prevent the analgesic effect of IFN alpha. IFN alpha could significantly inhibit the production of the cAMP stimulated by forskolin in SK-N-SH cells expressing the mu-opioid receptor, not in NG108-15 cells expressing the delta-opioid receptor uniformly. The results obtained provide further evidence for opioid activity of IFN alpha and suggest that this effect is mediated by central opioid receptors of the mu subtype. The evidence is consistent with the hypothesis that multiple actions of cytokines, such as immunoregulatory and neuroregulatory effects, might be mediated by distinct domains of cytokines interacting with different receptors. PMID:10676852

  1. The mother of all endocytosis

    PubMed Central

    2013-01-01

    Massive endocytosis is initiated by a series of steps that involve a sudden influx of calcium ions, changes in mitochondria, and modification of surface proteins by lipids. A better understanding of this process could lead to new approaches to reducing the tissue damage that is caused by heart attacks. PMID:24282238

  2. The mechanosensitive APJ internalization via clathrin-mediated endocytosis: A new molecular mechanism of cardiac hypertrophy.

    PubMed

    He, Lu; Chen, Linxi; Li, Lanfang

    2016-05-01

    The G protein-coupled receptor APJ elicits cellular response to diverse extracellular stimulus. Accumulating evidence reveals that APJ receptor plays a prominent role in the cardiomyocyte adapting to hypertrophic stimulation. At present, it remains obscure that the regulatory mechanism of APJ receptor in myocardial hypertrophy. The natural endogenous ligands apelin and Elabela as well as agonists maintain high affinity for the APJ receptor and drive its internalization. Ligand-activated receptor internalization is mainly performed by clathrin-mediated endocytic pathway. Simultaneously, clathrin-mediated endocytosis takes participate in the occurrence and development of cardiac hypertrophy. In this study, we hypothesize that natural ligands and agonists induce the mechanosensitive APJ internalization via clathrin-mediated endocytosis. APJ internalization may contribute to the development of cardiac hypertrophy. The mechanosensitive APJ internalization via clathrin-mediated endocytosis may be a new molecular mechanism of cardiac hypertrophy. PMID:27063076

  3. Clathrin- and Dynamin-Independent Endocytosis of FGFR3 – Implications for Signalling

    PubMed Central

    Haugsten, Ellen Margrethe; Zakrzewska, Malgorzata; Brech, Andreas; Pust, Sascha; Olsnes, Sjur; Sandvig, Kirsten; Wesche, Jørgen

    2011-01-01

    Endocytosis of tyrosine kinase receptors can influence both the duration and the specificity of the signal emitted. We have investigated the mechanisms of internalization of fibroblast growth factor receptor 3 (FGFR3) and compared it to that of FGFR1 which is internalized predominantly through clathrin-mediated endocytosis. Interestingly, we observed that FGFR3 was internalized at a slower rate than FGFR1 indicating that it may use a different endocytic mechanism than FGFR1. Indeed, after depletion of cells for clathrin, internalization of FGFR3 was only partly inhibited while endocytosis of FGFR1 was almost completely abolished. Similarly, expression of dominant negative mutants of dynamin resulted in partial inhibition of the endocytosis of FGFR3 whereas internalization of FGFR1 was blocked. Interfering with proposed regulators of clathrin-independent endocytosis such as Arf6, flotillin 1 and 2 and Cdc42 did not affect the endocytosis of FGFR1 or FGFR3. Furthermore, depletion of clathrin decreased the degradation of FGFR1 resulting in sustained signalling. In the case of FGFR3, both the degradation and the signalling were only slightly affected by clathrin depletion. The data indicate that clathrin-mediated endocytosis is required for efficient internalization and downregulation of FGFR1 while FGFR3, however, is internalized by both clathrin-dependent and clathrin-independent mechanisms. PMID:21779335

  4. Systematic analysis of endocytosis by cellular perturbations.

    PubMed

    Kühling, Lena; Schelhaas, Mario

    2014-01-01

    Endocytosis is an essential process of eukaryotic cells that facilitates numerous cellular and organismal functions. The formation of vesicles from the plasma membrane serves the internalization of ligands and receptors and leads to their degradation or recycling. A number of distinct mechanisms have been described over the years, several of which are only partially characterized in terms of mechanism and function. These are often referred to as novel endocytic pathways. The pathways differ in their mode of uptake and in their intracellular destination. Here, an overview of the set of cellular proteins that facilitate the different pathways is provided. Further, the approaches to distinguish between the pathways by different modes of perturbation are critically discussed, emphasizing the use of genetic tools such as dominant negative mutant proteins. PMID:24947372

  5. ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling

    PubMed Central

    Mercier, Vincent; Laporte, Marine H.; Destaing, Olivier; Blot, Béatrice; Blouin, Cédric M.; Pernet-Gallay, Karin; Chatellard, Christine; Saoudi, Yasmina; Albiges-Rizo, Corinne; Lamaze, Christophe; Fraboulet, Sandrine; Petiot, Anne; Sadoul, Rémy

    2016-01-01

    The molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE. PMID:27244115

  6. ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling.

    PubMed

    Mercier, Vincent; Laporte, Marine H; Destaing, Olivier; Blot, Béatrice; Blouin, Cédric M; Pernet-Gallay, Karin; Chatellard, Christine; Saoudi, Yasmina; Albiges-Rizo, Corinne; Lamaze, Christophe; Fraboulet, Sandrine; Petiot, Anne; Sadoul, Rémy

    2016-01-01

    The molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE. PMID:27244115

  7. Human podocytes perform polarized, caveolae-dependent albumin endocytosis

    PubMed Central

    Dobrinskikh, Evgenia; Okamura, Kayo; Kopp, Jeffrey B.; Doctor, R. Brian

    2014-01-01

    The renal glomerulus forms a selective filtration barrier that allows the passage of water, ions, and small solutes into the urinary space while restricting the passage of cells and macromolecules. The three layers of the glomerular filtration barrier include the vascular endothelium, glomerular basement membrane (GBM), and podocyte epithelium. Podocytes are capable of internalizing albumin and are hypothesized to clear proteins that traverse the GBM. The present study followed the fate of FITC-labeled albumin to establish the mechanisms of albumin endocytosis and processing by podocytes. Confocal imaging and total internal reflection fluorescence microscopy of immortalized human podocytes showed FITC-albumin endocytosis occurred preferentially across the basal membrane. Inhibition of clathrin-mediated endocytosis and caveolae-mediated endocytosis demonstrated that the majority of FITC-albumin entered podocytes through caveolae. Once internalized, FITC-albumin colocalized with EEA1 and LAMP1, endocytic markers, and with the neonatal Fc receptor, a marker for transcytosis. After preloading podocytes with FITC-albumin, the majority of loaded FITC-albumin was lost over the subsequent 60 min of incubation. A portion of the loss of albumin occurred via lysosomal degradation as pretreatment with leupeptin, a lysosomal protease inhibitor, partially inhibited the loss of FITC-albumin. Consistent with transcytosis of albumin, preloaded podocytes also progressively released FITC-albumin into the extracellular media. These studies confirm the ability of podocytes to endocytose albumin and provide mechanistic insight into cellular mechanisms and fates of albumin handling in podocytes. PMID:24573386

  8. High-density lipoprotein endocytosis in endothelial cells

    PubMed Central

    Fruhwürth, Stefanie; Pavelka, Margit; Bittman, Robert; Kovacs, Werner J; Walter, Katharina M; Röhrl, Clemens; Stangl, Herbert

    2013-01-01

    AIM: To describe the way stations of high-density lipoprotein (HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescence microscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type I mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrin-coated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis. PMID:24340136

  9. Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

    PubMed Central

    Fekri, Farnaz; Delos Santos, Ralph Christian; Karshafian, Raffi

    2016-01-01

    Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted

  10. Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms.

    PubMed

    Fekri, Farnaz; Delos Santos, Ralph Christian; Karshafian, Raffi; Antonescu, Costin N

    2016-01-01

    Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted

  11. Numb: "Adapting" notch for endocytosis.

    PubMed

    Jafar-Nejad, Hamed; Norga, Koenraad; Bellen, Hugo

    2002-08-01

    During sensory organ precursor divisions in Drosophila, the numb gene product segregates asymmetrically into one of the two daughter cells, to which it confers a specific fate by inhibiting Notch signaling. In this issue of Developmental Cell, Berdnik et al. show that Numb recruits alpha-Adaptin and that this physical interaction plays a role in downregulating Notch, presumably by stimulating endocytosis of Notch. PMID:12194846

  12. Endocytosis of Viruses and Bacteria

    PubMed Central

    Cossart, Pascale; Helenius, Ari

    2014-01-01

    Of the many pathogens that infect humans and animals, a large number use cells of the host organism as protected sites for replication. To reach the relevant intracellular compartments, they take advantage of the endocytosis machinery and exploit the network of endocytic organelles for penetration into the cytosol or as sites of replication. In this review, we discuss the endocytic entry processes used by viruses and bacteria and compare the strategies used by these dissimilar classes of pathogens. PMID:25085912

  13. The multiple facets of opioid receptor function: implications for addiction

    PubMed Central

    Lutz, Pierre-Eric; Kieffer, Brigitte L.

    2013-01-01

    Addiction is characterized by altered reward processing, disrupted emotional responses and poor decision-making. Beyond a central role in drug reward, increasing evidence indicate that opioid receptors are more generally involved in all these processes. Recent studies establish the mu opioid receptor as a main player in social reward, which attracts increasing attention in psychiatric research. There is growing interest in blocking the kappa opioid receptor to prevent relapse, and alleviate the negative affect of withdrawal. The delta opioid receptor emerges as a potent mood enhancer, whose involvement in addiction is less clear. All three opioid receptors are likely implicated in addiction-depression comorbidity, and understanding of their roles in cognitive deficits associated to drug abuse is only beginning. PMID:23453713

  14. Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis

    PubMed Central

    Urano, Daisuke; Phan, Nguyen; Jones, Janice C.; Yang, Jing; Huang, Jirong; Grigston, Jeffrey; Taylor, J. Philip; Jones, Alan M.

    2012-01-01

    Signal transduction typically begins by ligand-dependent activation of a concomitant partner which is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (Regulator of G protein Signaling 1), which is the prototype of a seven transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required both for G protein-mediated sugar signaling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis. PMID:22940907

  15. Synthetic and Receptor Signaling Explorations of the Mitragyna Alkaloids: Mitragynine as an Atypical Molecular Framework for Opioid Receptor Modulators.

    PubMed

    Kruegel, Andrew C; Gassaway, Madalee M; Kapoor, Abhijeet; Váradi, András; Majumdar, Susruta; Filizola, Marta; Javitch, Jonathan A; Sames, Dalibor

    2016-06-01

    Mu-opioid receptor agonists represent mainstays of pain management. However, the therapeutic use of these agents is associated with serious side effects, including potentially lethal respiratory depression. Accordingly, there is a longstanding interest in the development of new opioid analgesics with improved therapeutic profiles. The alkaloids of the Southeast Asian plant Mitragyna speciosa, represented by the prototypical member mitragynine, are an unusual class of opioid receptor modulators with distinct pharmacological properties. Here we describe the first receptor-level functional characterization of mitragynine and related natural alkaloids at the human mu-, kappa-, and delta-opioid receptors. These results show that mitragynine and the oxidized analogue 7-hydroxymitragynine, are partial agonists of the human mu-opioid receptor and competitive antagonists at the kappa- and delta-opioid receptors. We also show that mitragynine and 7-hydroxymitragynine are G-protein-biased agonists of the mu-opioid receptor, which do not recruit β-arrestin following receptor activation. Therefore, the Mitragyna alkaloid scaffold represents a novel framework for the development of functionally biased opioid modulators, which may exhibit improved therapeutic profiles. Also presented is an enantioselective total synthesis of both (-)-mitragynine and its unnatural enantiomer, (+)-mitragynine, employing a proline-catalyzed Mannich-Michael reaction sequence as the key transformation. Pharmacological evaluation of (+)-mitragynine revealed its much weaker opioid activity. Likewise, the intermediates and chemical transformations developed in the total synthesis allowed the elucidation of previously unexplored structure-activity relationships (SAR) within the Mitragyna scaffold. Molecular docking studies, in combination with the observed chemical SAR, suggest that Mitragyna alkaloids adopt a binding pose at the mu-opioid receptor that is distinct from that of classical opioids. PMID

  16. Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors

    PubMed Central

    Abrami, Laurence; Bischofberger, Mirko; Kunz, Béatrice; Groux, Romain; van der Goot, F. Gisou

    2010-01-01

    The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a β-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin. PMID:20221438

  17. The coiled-coil domain of EHD2 mediates inhibition of LeEix2 endocytosis and signaling.

    PubMed

    Bar, Maya; Sharfman, Miya; Schuster, Silvia; Avni, Adi

    2009-01-01

    Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis. PMID:19936242

  18. The Coiled-Coil Domain of EHD2 Mediates Inhibition of LeEix2 Endocytosis and Signaling

    PubMed Central

    Bar, Maya; Sharfman, Miya; Schuster, Silvia; Avni, Adi

    2009-01-01

    Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis. PMID:19936242

  19. The architectural relationship of components controlling mast cell endocytosis

    PubMed Central

    Cleyrat, Cédric; Darehshouri, Anza; Anderson, Karen L.; Page, Christopher; Lidke, Diane S.; Volkmann, Niels; Hanein, Dorit; Wilson, Bridget S.

    2013-01-01

    Summary Eukaryotic cells use multiple routes for receptor internalization. Here, we examine the topographical relationships of clathrin-dependent and clathrin-independent endocytic structures on the plasma membranes of leukemia-derived mast cells. The high affinity IgE receptor (FcεRI) utilizes both pathways, whereas transferrin receptor serves as a marker for the classical clathrin-mediated endocytosis pathway. Both receptors were tracked by live-cell imaging in the presence or absence of inhibitors that established their differential dependence on specific endocytic adaptor proteins. The topology of antigen-bound FcεRI, clathrin, dynamin, Arf6 and Eps15-positive structures were analyzed by 2D and 3D immunoelectron microscopy techniques, revealing their remarkable spatial relationships and unique geometry. We conclude that the mast cell plasma membrane has multiple specialized domains for endocytosis. Their close proximity might reflect shared components, such as lipids and adaptor proteins, that facilitate inward membrane curvature. Intersections between these specialized domains might represent sorting stations that direct cargo to specific endocytic pathways. PMID:23986485

  20. Transferrin: Endocytosis and Cell Signaling in Parasitic Protozoa.

    PubMed

    Reyes-López, Magda; Piña-Vázquez, Carolina; Serrano-Luna, Jesús

    2015-01-01

    Iron is the fourth most abundant element on Earth and the most abundant metal in the human body. This element is crucial for life because almost all organisms need iron for several biological activities. This is the case with pathogenic organisms, which are at the vanguard in the battle with the human host for iron. The latest regulates Fe concentration through several iron-containing proteins, such as transferrin. The transferrin receptor transports iron to each cell that needs it and maintains it away from pathogens. Parasites have developed several strategies to obtain iron as the expression of specific transferrin receptors localized on plasma membrane, internalized through endocytosis. Signal transduction pathways related to the activation of the receptor have functional importance in proliferation. The study of transferrin receptors and other proteins with action in the signaling networks is important because these proteins could be used as therapeutic targets due to their specificity or to differences with the human counterpart. In this work, we describe proteins that participate in signal transduction processes, especially those that involve transferrin endocytosis, and we compare these processes with those found in T. brucei, T. cruzi, Leishmania spp., and E. histolytica parasites. PMID:26090431

  1. Transferrin: Endocytosis and Cell Signaling in Parasitic Protozoa

    PubMed Central

    Serrano-Luna, Jesús

    2015-01-01

    Iron is the fourth most abundant element on Earth and the most abundant metal in the human body. This element is crucial for life because almost all organisms need iron for several biological activities. This is the case with pathogenic organisms, which are at the vanguard in the battle with the human host for iron. The latest regulates Fe concentration through several iron-containing proteins, such as transferrin. The transferrin receptor transports iron to each cell that needs it and maintains it away from pathogens. Parasites have developed several strategies to obtain iron as the expression of specific transferrin receptors localized on plasma membrane, internalized through endocytosis. Signal transduction pathways related to the activation of the receptor have functional importance in proliferation. The study of transferrin receptors and other proteins with action in the signaling networks is important because these proteins could be used as therapeutic targets due to their specificity or to differences with the human counterpart. In this work, we describe proteins that participate in signal transduction processes, especially those that involve transferrin endocytosis, and we compare these processes with those found in T. brucei, T. cruzi, Leishmania spp., and E. histolytica parasites. PMID:26090431

  2. Nodal-mediated epigenesis requires dynamin-mediated endocytosis

    PubMed Central

    Ertl, Robin P.; Robertson, Anthony J.; Saunders, Diane; Coffman, James A.

    2011-01-01

    Nodal proteins are diffusible morphogens that drive pattern formation via short-range feedback activation coupled to long-range Lefty-mediated inhibition. In the sea urchin embryo, specification of the secondary (oral-aboral) axis occurs via zygotic expression of nodal, which is localized to the prospective oral ectoderm at early blastula stage. In mid-blastula stage embryos treated with low micromolar nickel or zinc, nodal expression expands progressively beyond the confines of this localized domain to encompass the entire equatorial circumference of the embryo, producing radialized embryos lacking an oral-aboral axis. RNAseq analysis of embryos treated with nickel, zinc or cadmium (which does not radialize embryos) showed that several genes involved in endocytosis were similarly perturbed by nickel and zinc but not cadmium. Inhibiting dynamin, a GTPase required for receptor-mediated endocytosis, phenocopies the effects of nickel and zinc, suggesting that dynamin-mediated endocytosis is required as a sink to limit the range of Nodal signaling. PMID:21337468

  3. The function of EHD2 in endocytosis and defense signaling is affected by SUMO.

    PubMed

    Bar, Maya; Schuster, Silvia; Leibman, Meirav; Ezer, Ran; Avni, Adi

    2014-03-01

    Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulates many cellular processes. SUMOylation has been shown to regulate cellular localization and function of a variety of proteins, in some cases affecting nuclear import or export. We have previously characterized two EHDs (EH domain containing proteins) in Arabidospis and showed their involvement in plant endocytosis. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and the leucine rich repeat receptor like protein LeEix2, an effect that requires and intact coiled-coil domain. Inhibition of endocytosis of LeEix2 by EHD2 is effective in inhibiting defense responses mediated by the LeEix2 receptor in response to its ligand EIX. In the present work we demonstrate that SUMOylation of EHD2 appears to be required for EHD2-induced inhibition of LeEix2 endocytosis. Indeed, we found that a mutant form of EHD2, possessing a defective SUMOylation site, has an increased nuclear abundance, can no longer be SUMOylated and is no longer effective in inhibiting LeEix2 endocytosis or defense signaling in response to EIX. PMID:24154852

  4. Opioid receptors and legal highs: Salvia divinorum and Kratom.

    PubMed

    Babu, Kavita M; McCurdy, Christopher R; Boyer, Edward W

    2008-02-01

    Salvia divinorum and Mitragyna speciosa ("Kratom"), two unscheduled dietary supplements whose active agents are opioid receptor agonists, have discrete psychoactive effects that have contributed to their increasing popularity. Salvia divinorum contains the highly selective kappa- opioid receptor agonist salvinorin A; this compound produces visual hallucinations and synesthesia. Mitragynine, the major alkaloid identified from Kratom, has been reported as a partial opioid agonist producing similar effects to morphine. An interesting minor alkaloid of Kratom, 7-hydroxymitragynine, has been reported to be more potent than morphine. Both Kratom alkaloids are reported to activate supraspinal mu- and delta- opioid receptors, explaining their use by chronic narcotics users to ameliorate opioid withdrawal symptoms. Despite their widespread Internet availability, use of Salvia divinorum and Kratom represents an emerging trend that escapes traditional methods of toxicologic monitoring. The purpose of this article is to familiarize toxicologists and poison control specialists with these emerging psychoactive dietary supplements. PMID:18259963

  5. Biotinylated human. beta. -endorphins as probes for the opioid receptor

    SciTech Connect

    Hochhaus, G.; Gibson, B.W.; Sadee, W.

    1988-01-05

    The reaction of human ..beta..-endorphin and biotinyl N-hydroxysuccinimide with or without spacer arm, afforded a series of products that were separated by high performance liquid chromatography (HPLC). Liquid secondary ion mass spectrometry of the biotinylated products and their tryptic digests produced abundant protonated molecular ions (MH/sup +/), which specified the number and location of biotinylation. Between 1 and 4 biotinyl residues were incorporated per human ..beta..-endorphin molecule, at Lys-9, -19, -24, -28, and -29, but not at the amino-terminal Try-1. Three HPLC fractions were isolated for receptor binding studies monobiotinylation of Lys-9, Lys-19, and a mixture of Lys-24, Lys-28, and Lys-29 derivatives. IC/sub 50/ values for binding to ..mu.. and delta opioid receptor sites were 3-8 times higher for monobiotinylated derivatives than for the parent human ..beta..-endorphin. Association with avidin decreased opioid receptor affinities for the C/sub 6/ spacer derivative biotinylated at position Lys-9, which is close to the (1-5) enkephalin receptor region. In contrast, avidin did not affect or even increased apparent affinities to ..mu.. and delta sites for derivatives biotinylated at the ..cap alpha..-helical part of the molecule (Lys-19, -24, -28, and -29). Biotinylated human ..beta..-endorphins also bound to low affinity nonopioid binding sites on NG-108-15 cells; however, affinities to these sites were considerably reduced when derivatives were bound to avidin. The ability of biotinylated human ..beta..-endorphin to cross-link the ..mu.. and delta opioid receptors to avidin allows application of the biotin-avidin system as a molecular probe of the opioid receptor.

  6. Ca2+ and calmodulin initiate all forms of endocytosis during depolarization at a nerve terminal

    PubMed Central

    Xu, Jianhua; Fan, Junmei; Xue, Lei; Melicoff, Ernestina; Adachi, Roberto; Bai, Li; Wu, Ling-Gang

    2016-01-01

    Although endocytosis maintains synaptic transmission, how endocytosis is initiated is unclear. We found that calcium influx initiated all forms of endocytosis at a single nerve terminal in rodents, including clathrin-dependent slow endocytosis, bulk endocytosis, rapid endocytosis and endocytosis overshoot (excess endocytosis), with each being evoked with a correspondingly higher calcium threshold. As calcium influx increased, endocytosis gradually switched from very slow endocytosis to slow endocytosis to bulk endocytosis to rapid endocytosis and to endocytosis overshoot. The calcium-induced endocytosis rate increase was a result of the speeding up of membrane invagination and fission. Pharmacological experiments suggested that the calcium sensor mediating these forms of endocytosis is calmodulin. In addition to its role in recycling vesicles, calcium/calmodulin-initiated endocytosis facilitated vesicle mobilization to the readily releasable pool, probably by clearing fused vesicle membrane at release sites. Our findings provide a unifying mechanism for the initiation of various forms of endocytosis that are critical in maintaining exocytosis. PMID:19633667

  7. Rab5 Isoforms Specifically Regulate Different Modes of Endocytosis in Leishmania.

    PubMed

    Rastogi, Ruchir; Verma, Jitender Kumar; Kapoor, Anjali; Langsley, Gordon; Mukhopadhyay, Amitabha

    2016-07-01

    Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a:N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptor-mediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a(+/-) and LdRab5b(+/-) cells. LdRab5a(+/-) Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b(+/-) cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania. PMID:27226564

  8. Effects of Endosomal Photodamage on Membrane Recycling and Endocytosis

    PubMed Central

    Kessel, David; Santiago, Ann Marie; Andrzejak, Michelle

    2011-01-01

    The flux of receptor-independent endocytosis can be estimated by addition of wortmannin to cell cultures. Membrane influx is unaffected but traffic out of late endosomes is impaired, resulting in a substantial enlargement of these organelles. Using the 1c1c7 murine hepatoma, we investigated the effect of endosomal photodamage on this endocytic pathway. We previously reported that photodamage catalyzed by the lysosomal photosensitizer NPe6 prevented wortmannin-induced endosomal swelling, indicating an earlier block in the process. In this study, we show that endosomal photodamage, initiated by photodamage from an asymmetrically-substituted porphine or a phthalocyanine, also prevents wortmannin-induced endosomal swelling, even when the PDT dose is insufficient to cause endosomal disruption. As the PDT dose is increased, endosomal breakage occurs, as does apoptosis and cell death. Very high PDT doses result in necrosis. We propose that photodamage to endosomes results in alterations in the endosomal structure such that influx of new material is inhibited and receptor-independent endocytosis is prevented. In an additional series of studies, we found that the swollen late endosomes induced by wortmannin are unable to retain previously accumulated fluorescent probes or photosensitizers. PMID:21208213

  9. 3D modeling, ligand binding and activation studies of the cloned mouse delta, mu; and kappa opioid receptors.

    PubMed

    Filizola, M; Laakkonen, L; Loew, G H

    1999-11-01

    Refined 3D models of the transmembrane domains of the cloned delta, mu and kappa opioid receptors belonging to the superfamily of G-protein coupled receptors (GPCRs) were constructed from a multiple sequence alignment using the alpha carbon template of rhodopsin recently reported. Other key steps in the procedure were relaxation of the 3D helix bundle by unconstrained energy optimization and assessment of the stability of the structure by performing unconstrained molecular dynamics simulations of the energy optimized structure. The results were stable ligand-free models of the TM domains of the three opioid receptors. The ligand-free delta receptor was then used to develop a systematic and reliable procedure to identify and assess putative binding sites that would be suitable for similar investigation of the other two receptors and GPCRs in general. To this end, a non-selective, 'universal' antagonist, naltrexone, and agonist, etorphine, were used as probes. These ligands were first docked in all sites of the model delta opioid receptor which were sterically accessible and to which the protonated amine of the ligands could be anchored to a complementary proton-accepting residue. Using these criteria, nine ligand-receptor complexes with different binding pockets were identified and refined by energy minimization. The properties of all these possible ligand-substrate complexes were then examined for consistency with known experimental results of mutations in both opioid and other GPCRs. Using this procedure, the lowest energy agonist-receptor and antagonist-receptor complexes consistent with these experimental results were identified. These complexes were then used to probe the mechanism of receptor activation by identifying differences in receptor conformation between the agonist and the antagonist complex during unconstrained dynamics simulation. The results lent support to a possible activation mechanism of the mouse delta opioid receptor similar to that recently

  10. Clathrin light chains' role in selective endocytosis influences antibody isotype switching.

    PubMed

    Wu, Shuang; Majeed, Sophia R; Evans, Timothy M; Camus, Marine D; Wong, Nicole M L; Schollmeier, Yvette; Park, Minjong; Muppidi, Jagan R; Reboldi, Andrea; Parham, Peter; Cyster, Jason G; Brodsky, Frances M

    2016-08-30

    Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor β receptor 2 (TGFβR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the β2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules. PMID:27540116

  11. Pak1 phosphorylation enhances cortactin-N-WASP interaction in clathrin-caveolin-independent endocytosis.

    PubMed

    Grassart, Alexandre; Meas-Yedid, Vannary; Dufour, Alexandre; Olivo-Marin, Jean-Christophe; Dautry-Varsat, Alice; Sauvonnet, Nathalie

    2010-08-01

    Growing evidence indicates that kinases are central to the regulation of endocytic pathways. Previously, we identified p21-activated kinase 1 (Pak1) as the first specific regulator of clathrin- and caveolae-independent endocytosis used by the interleukin 2 receptor subunit (IL-2R). Here, we address the mechanism by which Pak1 regulates IL-2Rbeta endocytosis. First, we show that Pak1 phosphorylates an activator of actin polymerization, cortactin, on its serine residues 405 and 418. Consistently, we observe a specific inhibition of IL-2Rbeta endocytosis when cells overexpress a cortactin, wherein these serine residues have been mutated. In addition, we show that the actin polymerization enhancer, neuronal Wiskott-Aldrich syndrome protein (N-WASP), is involved in IL-2Rbeta endocytosis. Strikingly, we find that Pak1 phosphorylation of cortactin on serine residues 405 and 418 increases its association with N-WASP. Thus, Pak1, by controlling the interaction between cortactin and N-WASP, could regulate the polymerization of actin during clathrin-independent endocytosis. PMID:20444238

  12. Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal β-catenin/Armadillo.

    PubMed

    Gagliardi, Maria; Hernandez, Ana; McGough, Ian J; Vincent, Jean-Paul

    2014-11-15

    A key step in the canonical Wnt signalling pathway is the inhibition of GSK3β, which results in the accumulation of nuclear β-catenin (also known as CTNNB1), and hence regulation of target genes. Evidence suggests that endocytosis is required for signalling, yet its role and the molecular understanding remains unclear. A recent and controversial model suggests that endocytosis contributes to Wnt signalling by causing the sequestration of the ligand-receptor complex, including LRP6 and GSK3 to multivesicular bodies (MVBs), thus preventing GSK3β from accessing β-catenin. Here, we use specific inhibitors (Dynasore and Dyngo-4a) to confirm the essential role of endocytosis in Wnt/Wingless signalling in human and Drosophila cells. However, we find no evidence that, in Drosophila cells or wing imaginal discs, LRP6/Arrow traffics to MVBs or that MVBs are required for Wnt/Wingless signalling. Moreover, we show that activation of signalling through chemical blockade of GSK3β is prevented by endocytosis inhibitors, suggesting that endocytosis impacts on Wnt/Wingless signalling downstream of the ligand-receptor complex. We propose that, through an unknown mechanism, endocytosis boosts the resting pool of β-catenin upon which GSK3β normally acts. PMID:25236598

  13. Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal β-catenin/Armadillo

    PubMed Central

    Gagliardi, Maria; Hernandez, Ana; McGough, Ian J.; Vincent, Jean-Paul

    2014-01-01

    ABSTRACT A key step in the canonical Wnt signalling pathway is the inhibition of GSK3β, which results in the accumulation of nuclear β-catenin (also known as CTNNB1), and hence regulation of target genes. Evidence suggests that endocytosis is required for signalling, yet its role and the molecular understanding remains unclear. A recent and controversial model suggests that endocytosis contributes to Wnt signalling by causing the sequestration of the ligand–receptor complex, including LRP6 and GSK3 to multivesicular bodies (MVBs), thus preventing GSK3β from accessing β-catenin. Here, we use specific inhibitors (Dynasore and Dyngo-4a) to confirm the essential role of endocytosis in Wnt/Wingless signalling in human and Drosophila cells. However, we find no evidence that, in Drosophila cells or wing imaginal discs, LRP6/Arrow traffics to MVBs or that MVBs are required for Wnt/Wingless signalling. Moreover, we show that activation of signalling through chemical blockade of GSK3β is prevented by endocytosis inhibitors, suggesting that endocytosis impacts on Wnt/Wingless signalling downstream of the ligand–receptor complex. We propose that, through an unknown mechanism, endocytosis boosts the resting pool of β-catenin upon which GSK3β normally acts. PMID:25236598

  14. Ultrafast endocytosis at mouse hippocampal synapses

    NASA Astrophysics Data System (ADS)

    Watanabe, Shigeki; Rost, Benjamin R.; Camacho-Pérez, Marcial; Davis, M. Wayne; Söhl-Kielczynski, Berit; Rosenmund, Christian; Jorgensen, Erik M.

    2013-12-01

    To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20s after fusion by the assembly of clathrin scaffolds or in approximately 1s by the reversal of fusion pores via `kiss-and-run' endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy--`flash-and-freeze' electron microscopy. Docked vesicles fuse and collapse into the membrane within 30ms of the stimulus. Compensatory endocytosis occurs within 50 to 100ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover, it is 200-fold faster than clathrin-mediated endocytosis. It is likely that `ultrafast endocytosis' is specialized to restore the surface area of the membrane rapidly.

  15. Clathrin-Independent Pathways of Endocytosis

    PubMed Central

    Mayor, Satyajit; Parton, Robert G.; Donaldson, Julie G.

    2014-01-01

    There are many pathways of endocytosis at the cell surface that apparently operate at the same time. With the advent of new molecular genetic and imaging tools, an understanding of the different ways by which a cell may endocytose cargo is increasing by leaps and bounds. In this review we explore pathways of endocytosis that occur in the absence of clathrin. These are referred to as clathrin-independent endocytosis (CIE). Here we primarily focus on those pathways that function at the small scale in which some have distinct coats (caveolae) and others function in the absence of specific coated intermediates. We follow the trafficking itineraries of the material endocytosed by these pathways and finally discuss the functional roles that these pathways play in cell and tissue physiology. It is likely that these pathways will play key roles in the regulation of plasma membrane area and tension and also control the availability of membrane during cell migration. PMID:24890511

  16. Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions

    PubMed Central

    Watanabe, Shigeki; Liu, Qiang; Davis, M Wayne; Hollopeter, Gunther; Thomas, Nikita; Jorgensen, Nels B; Jorgensen, Erik M

    2013-01-01

    Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis. DOI: http://dx.doi.org/10.7554/eLife.00723.001 PMID:24015355

  17. Listeria monocytogenes Internalin B Activates Junctional Endocytosis to Accelerate Intestinal Invasion

    PubMed Central

    Pentecost, Mickey; Kumaran, Jyothi; Ghosh, Partho; Amieva, Manuel R.

    2010-01-01

    Listeria monocytogenes (Lm) uses InlA to invade the tips of the intestinal villi, a location at which cell extrusion generates a transient defect in epithelial polarity that exposes the receptor for InlA, E-cadherin, on the cell surface. As the dying cell is removed from the epithelium, the surrounding cells reorganize to form a multicellular junction (MCJ) that Lm exploits to find its basolateral receptor and invade. By examining individual infected villi using 3D-confocal imaging, we uncovered a novel role for the second major invasin, InlB, during invasion of the intestine. We infected mice intragastrically with isogenic strains of Lm that express or lack InlB and that have a modified InlA capable of binding murine E-cadherin and found that Lm lacking InlB invade the same number of villi but have decreased numbers of bacteria within each infected villus tip. We studied the mechanism of InlB action at the MCJs of polarized MDCK monolayers and find that InlB does not act as an adhesin, but instead accelerates bacterial internalization after attachment. InlB locally activates its receptor, c-Met, and increases endocytosis of junctional components, including E-cadherin. We show that MCJs are naturally more endocytic than other sites of the apical membrane, that endocytosis and Lm invasion of MCJs depends on functional dynamin, and that c-Met activation by soluble InlB or hepatocyte growth factor (HGF) increases MCJ endocytosis. Also, in vivo, InlB applied through the intestinal lumen increases endocytosis at the villus tips. Our findings demonstrate a two-step mechanism of synergy between Lm's invasins: InlA provides the specificity of Lm adhesion to MCJs at the villus tips and InlB locally activates c-Met to accelerate junctional endocytosis and bacterial invasion of the intestine. PMID:20485518

  18. The basic amino acids in the coiled-coil domain of CIN85 regulate its interaction with c-Cbl and phosphatidic acid during epidermal growth factor receptor (EGFR) endocytosis

    PubMed Central

    2014-01-01

    Background During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain. Results Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation. Conclusions As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation. PMID

  19. Activity-dependent acceleration of endocytosis at a central synapse.

    PubMed

    Wu, Wei; Xu, Jianhua; Wu, Xin-Sheng; Wu, Ling-Gang

    2005-12-14

    Accumulated evidence indicates the existence of rapid and slow endocytosis at many synapses. It has been proposed that rapid endocytosis is activated by intense stimulation when vesicle recycling needs to be speeded up to supply vesicles at hippocampal synapses. However, the evidence, as obtained with imaging techniques, which are somewhat indirect in indicating rapid endocytosis, is controversial. Furthermore, a slower time course of endocytosis is often found after more intense nerve activity, casting doubt on the role of rapid endocytosis at synapses. Here, we addressed this issue at a mammalian central synapse, the calyx of Held, using a capacitance measurement technique that provides a higher time resolution than imaging techniques. We found that rapid endocytosis with a time constant of approximately 1-2 s was activated during intense nerve activity. Reducing the presynaptic calcium current or buffering the intracellular calcium with EGTA significantly inhibited rapid endocytosis, suggesting that calcium triggers rapid endocytosis. During intense stimulation, rapid endocytosis retrieved up to approximately eight vesicles per second per active zone, approximately eightfold larger than reported in the hippocampus, and thus played a dominant role during and within 3 s after intense stimulation. Slow endocytosis became dominant 3 s after intense stimulation likely because of the fall of the intracellular calcium level that deactivated rapid endocytosis. These results underscore the importance of calcium-triggered rapid endocytosis, which offers the nerve terminal the plasticity to speed up vesicle cycling during intense nerve activity. PMID:16354926

  20. METHODS TO QUANTIFY ENDOCYTOSIS: A REVIEW

    EPA Science Inventory

    Endocytosis is a process whereby extracellular matter is transported in bulk to the cell's interior. To accomplish this the cell extends portions of the plasma membrane which surround the mass to be ingested. As these cytoplasmic projections meet, they fuse. The endocytic vacuole...

  1. Zero tolerance: amphipathic helices in endocytosis.

    PubMed

    Wood, Laura A; Royle, Stephen J

    2015-04-20

    Endocytosis is the physical battle to form a new vesicle in the face of counteracting forces, such as membrane tension. Skruzny et al. (2015) and Miller et al. (2015) now shed light on endocytic proteins that bear a "Helix 0" and on the proteins' role in the struggle to make clathrin-coated vesicles. PMID:25898162

  2. Hyaluronic acid binding, endocytosis and degradation by sinusoidal liver endothelial cells

    SciTech Connect

    McGary, C.T.

    1988-01-01

    The binding, endocytosis, and degradation of {sup 125}I-hyaluronic acid ({sup 125}I-HA) by liver endothelial cells (LEC) was studied under several conditions. The dissociation of receptor-bound {sup 125}I-HA was rapid, with a half time of {approx}31 min and a K{sub off} of 6.3 {times} 10{sup {minus}4}/sec. A large reversible increase in {sup 125}I-HA binding to LEC at pH 5.0 was due to an increase in the observed affinity of the binding interaction. Pronase digestion suggested the protein nature of the receptor and the intracellular location of the digitonin exposed binding activity. Binding and endocytosis occur in the presence of 10 mM EGTA indicating that divalent cations are not required for receptor function. To study the degradation of {sup 125}I-HA by LEC, a cetylpyridinium chloride (CPC) precipitation assay was characterized. The minimum HA length required for precipitation was elucidated. The fate of the LEC HA receptor after endocytosis was examined.

  3. Developmental Function of Nm23/awd - A Mediator of Endocytosis

    PubMed Central

    Nallamothu, Gouthami; Dammai, Vincent; Hsu, Tien

    2009-01-01

    The metastasis suppressor gene Nm23 is highly conserved from yeast to human, implicating a critical developmental function. Studies in cultured mammalian cells have identified several potential functions, but many have not been directly verified in vivo. Here we summarize the studies on the Drosophila homologue of the Nm23 gene, named abnormal wing discs (awd), which shares 78% amino acid identity with the human Nm23-H1 and H2 isoforms. These studies confirmed that awd gene encodes a nucleoside diphosphate kinase, and provided strong evidence of a role for awd in regulating cell differentiation and motility via regulation of growth factor receptor signaling. The latter function is mainly mediated by control of endocytosis. This review provides a historical account of the discovery and subsequent analyses of the awd gene. We will also discuss the possible molecular function of the Awd protein that underlies the endocytic function. PMID:19373545

  4. Molecular Mechanisms of B Cell Antigen Gathering and Endocytosis.

    PubMed

    Hoogeboom, Robbert; Tolar, Pavel

    2016-01-01

    Generation of high-affinity, protective antibodies requires B cell receptor (BCR) signaling, as well as antigen internalization and presentation to helper T cells. B cell antigen internalization is initiated by antigen capture, either from solution or from immune synapses formed on the surface of antigen-presenting cells, and proceeds via clathrin-dependent endocytosis and intracellular routing to late endosomes. Although the components of this pathway are still being discovered, it has become clear that antigen internalization is actively regulated by BCR signaling at multiple steps and, vice versa, that localization of the BCR along the endocytic pathway modulates signaling. Accordingly, defects in BCR internalization or trafficking contribute to enhanced B cell activation in models of autoimmune diseases and in B cell lymphomas. In this review, we discuss how BCR signaling complexes regulate each of the steps of this endocytic process and why defects along this pathway manifest as hyperactive B cell responses in vivo. PMID:26336965

  5. Molecular characterization of opioid receptors

    SciTech Connect

    Howard, A.D.

    1986-01-01

    The aim of this research was to purify and characterize active opioid receptors and elucidate molecular aspects of opioid receptor heterogeneity. Purification to apparent homogeneity of an opioid binding protein from bovine caudate was achieved by solubilization in the non-ionic detergent, digitonin, followed by sequential chromatography on the opiate affinity matrix, ..beta..-naltrexylethylenediamine-CH-Sepharose 4B, and on the lectine affinity matrix, wheat germ agglutinin-agarose. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) followed by autoradiography revealed that radioiodinated purified receptor gave a single band. Purified receptor preparations showed a specific activity of 12,000-15,000 fmol of opiate bound per mg of protein. Radioiodinated human beta-endorphin (/sup 125/I-beta-end/sub H/) was used as a probe to investigate the ligand binding subunits of mu and delta opioid receptors. /sup 125/I-beta-end/sub H/ was shown to bind to a variety of opioid receptor-containing tissues with high affinity and specificity with preference for mu and delta sites, and with little, if any, binding to kappa sites. Affinity crosslinking techniques were employed to covalently link /sup 125/I-beta-end/sub H/ to opioid receptors, utilizing derivatives of bis-succinimidyl esters that are bifunctional crosslinkers with specificities for amino and sulfhydryl groups. This, and competition experiments with high type-selective ligands, permitted the assignment of two labeled peptides to their receptor types, namely a peptide of M/sub r/ = 65,000 for mu receptors and one of M/sub r/ = 53,000 for delta receptors.

  6. Microtubule Motors Power Plasma Membrane Tubulation in Clathrin-Independent Endocytosis

    PubMed Central

    Day, Charles A; Baetz, Nicholas W; Copeland, Courtney A; Kraft, Lewis J; Han, Bing; Tiwari, Ajit; Drake, Kimberly R; De Luca, Heidi; Chinnapen, Daniel J-F; Davidson, Michael W; Holmes, Randall K; Jobling, Michael G; Schroer, Trina A; Lencer, Wayne I; Kenworthy, Anne K

    2015-01-01

    How the plasma membrane is bent to accommodate clathrin-independent endocytosis remains uncertain. Recent studies suggest Shiga and cholera toxin induce membrane curvature required for their uptake into clathrin-independent carriers by binding and cross-linking multiple copies of their glycosphingolipid receptors on the plasma membrane. But it remains unclear if toxin-induced sphingolipid crosslinking provides sufficient mechanical force for deforming the plasma membrane, or if host cell factors also contribute to this process. To test this, we imaged the uptake of cholera toxin B-subunit into surface-derived tubular invaginations. We found that cholera toxin mutants that bind to only one glycosphingolipid receptor accumulated in tubules, and that toxin binding was entirely dispensable for membrane tubulations to form. Unexpectedly, the driving force for tubule extension was supplied by the combination of microtubules, dynein and dynactin, thus defining a novel mechanism for generating membrane curvature during clathrin-independent endocytosis. PMID:25690058

  7. Enhanced endocytosis of nano-curcumin in nasopharyngeal cancer cells: An atomic force microscopy study

    NASA Astrophysics Data System (ADS)

    Prasanth, R.; Nair, Greshma; Girish, C. M.

    2011-10-01

    Recent studies in drug development have shown that curcumin can be a good competent due to its improved anticancer, antioxidant, anti-proliferative, and anti-inflammatory activities. A detailed real time characterization of drug (curcumin)-cell interaction is carried out in human nasopharyngeal cancer cells using atomic force microscopy. Nanocurcumin shows an enhanced uptake over micron sized drugs attributed to the receptor mediated route. Cell membrane stiffness plays a critical role in the drug endocytosis in nasopharyngeal cancer cells.

  8. Long-term potentiation decay and memory loss are mediated by AMPAR endocytosis.

    PubMed

    Dong, Zhifang; Han, Huili; Li, Hongjie; Bai, Yanrui; Wang, Wei; Tu, Man; Peng, Yan; Zhou, Limin; He, Wenting; Wu, Xiaobin; Tan, Tao; Liu, Mingjing; Wu, Xiaoyan; Zhou, Weihui; Jin, Wuyang; Zhang, Shu; Sacktor, Todd Charlton; Li, Tingyu; Song, Weihong; Wang, Yu Tian

    2015-01-01

    Long-term potentiation (LTP) of synaptic strength between hippocampal neurons is associated with learning and memory, and LTP dysfunction is thought to underlie memory loss. LTP can be temporally and mechanistically classified into decaying (early-phase) LTP and nondecaying (late-phase) LTP. While the nondecaying nature of LTP is thought to depend on protein synthesis and contribute to memory maintenance, little is known about the mechanisms and roles of decaying LTP. Here, we demonstrated that inhibiting endocytosis of postsynaptic α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs) prevents LTP decay, thereby converting it into nondecaying LTP. Conversely, restoration of AMPAR endocytosis by inhibiting protein kinase Mζ (PKMζ) converted nondecaying LTP into decaying LTP. Similarly, inhibition of AMPAR endocytosis prolonged memory retention in normal animals and reduced memory loss in a murine model of Alzheimer's disease. These results strongly suggest that an active process that involves AMPAR endocytosis mediates the decay of LTP and that inhibition of this process can prolong the longevity of LTP as well as memory under both physiological and pathological conditions. PMID:25437879

  9. Dynamin 2–dependent endocytosis is required for normal megakaryocyte development in mice

    PubMed Central

    Bender, Markus; Giannini, Silvia; Grozovsky, Renata; Jönsson, Terese; Christensen, Hilary; Pluthero, Fred G.; Ko, Amy; Mullally, Ann; Kahr, Walter H. A.; Hoffmeister, Karin M.

    2015-01-01

    Dynamins are highly conserved large GTPases (enzymes that hydrolyze guanosine triphosphate) involved in endocytosis and vesicle transport, and mutations in the ubiquitous and housekeeping dynamin 2 (DNM2) have been associated with thrombocytopenia in humans. To determine the role of DNM2 in thrombopoiesis, we generated Dnm2fl/fl Pf4-Cre mice specifically lacking DNM2 in the megakaryocyte (MK) lineage. Dnm2fl/fl Pf4-Cre mice had severe macrothrombocytopenia with moderately accelerated platelet clearance. Dnm2-null bone marrow MKs had altered demarcation membrane system formation in vivo due to defective endocytic pathway, and fetal liver–derived Dnm2-null MKs formed proplatelets poorly in vitro, showing that DNM2-dependent endocytosis plays a major role in MK membrane formation and thrombopoiesis. Endocytosis of the thrombopoietin receptor Mpl was impaired in Dnm2-null platelets, causing constitutive phosphorylation of the tyrosine kinase JAK2 and elevated circulating thrombopoietin levels. MK-specific DNM2 deletion severely disrupted bone marrow homeostasis, as reflected by marked expansion of hematopoietic stem and progenitor cells, MK hyperplasia, myelofibrosis, and consequent extramedullary hematopoiesis and splenomegaly. Taken together, our data demonstrate that unrestrained MK growth and proliferation results in rapid myelofibrosis and establishes a previously unrecognized role for DNM2-dependent endocytosis in megakaryopoiesis, thrombopoiesis, and bone marrow homeostasis. PMID:25468568

  10. An endocytosis pathway initiated through neuropilin-1 and regulated by nutrient availability

    PubMed Central

    Pang, Hong-Bo; Braun, Gary B.; Friman, Tomas; Aza-Blanc, Pedro; Ruidiaz, Manuel E.; Sugahara, Kazuki N.; Teesalu, Tambet; Ruoslahti, Erkki

    2014-01-01

    Neuropilins (NRPs) are trans-membrane receptors involved in axon guidance and vascular development. Many growth factors and other signaling molecules bind to NRPs through a C-terminal, basic sequence motif (C-end Rule or CendR motif). Peptides with this motif (CendR peptides) are taken up into cells by endocytosis. Tumor-homing CendR peptides penetrate through tumor tissue and have shown utility in enhancing drug delivery into tumors. Here we show, using RNAi screening and subsequent validation studies, that NRP1-mediated endocytosis of CendR peptides is distinct from known endocytic pathways. Ultrastructurally, CendR endocytosis resembles macropinocytosis, but is mechanistically different. We also show that nutrient-sensing networks such as mTOR signaling regulate CendR endocytosis and subsequent intercellular transport of CendR cargo, both of which are stimulated by nutrient depletion. As CendR is a bulk transport pathway, our results suggest a role for it in nutrient transport; CendR-enhanced drug delivery then makes use of this natural pathway. PMID:25277522

  11. KIF13B enhances the endocytosis of LRP1 by recruiting LRP1 to caveolae

    PubMed Central

    Kanai, Yoshimitsu; Wang, Daliang

    2014-01-01

    Multifunctional low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) recognizes and internalizes a large number of diverse ligands, including LDL and factor VIII. However, little is known about the regulation of LRP1 endocytosis. Here, we show that a microtubule-based motor protein, KIF13B, in an unexpected and unconventional function, enhances caveolin-dependent endocytosis of LRP1. KIF13B was highly expressed in the liver and was localized on the sinusoidal plasma membrane of hepatocytes. KIF13B knockout (KO) mice showed elevated levels of serum cholesterol and factor VIII, and KO MEFs showed decreased uptake of LDL. Exogenous KIF13B, initially localized on the plasma membrane with caveolae, was translocated to the vesicles in the cytoplasm with LRP1 and caveolin-1. KIF13B bound to hDLG1 and utrophin, which, in turn, bound to LRP1 and caveolae, respectively. These linkages were required for the KIF13B-enhanced endocytosis of LRP1. Thus, we propose that KIF13B, working as a scaffold, recruits LRP1 to caveolae via LRP1–hDLG1–KIF13B–utrophin–caveolae linkage and enhances the endocytosis of LRP1. PMID:24469637

  12. Light-regulated stapled peptides to inhibit protein-protein interactions involved in clathrin-mediated endocytosis.

    PubMed

    Nevola, Laura; Martín-Quirós, Andrés; Eckelt, Kay; Camarero, Núria; Tosi, Sébastien; Llobet, Artur; Giralt, Ernest; Gorostiza, Pau

    2013-07-22

    Control of membrane traffic: Photoswitchable inhibitors of protein-protein interactions were applied to photoregulate clathrin-mediated endocytosis (CME) in living cells. Traffic light (TL) peptides acting as "stop" and "go" signals for membrane traffic can be used to dissect the role of CME in receptor internalization and in cell growth, division, and differentiation. PMID:23775788

  13. Sigma 1 receptor modulation of G-protein-coupled receptor signaling: potentiation of opioid transduction independent from receptor binding.

    PubMed

    Kim, Felix J; Kovalyshyn, Ivanka; Burgman, Maxim; Neilan, Claire; Chien, Chih-Cheng; Pasternak, Gavril W

    2010-04-01

    sigma Ligands modulate opioid actions in vivo, with agonists diminishing morphine analgesia and antagonists enhancing the response. Using human BE(2)-C neuroblastoma cells that natively express opioid receptors and human embryonic kidney (HEK) cells transfected with a cloned mu opioid receptor, we now demonstrate a similar modulation of opioid function, as assessed by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding, by sigma(1) receptors. sigma Ligands do not compete opioid receptor binding. Administered alone, neither sigma agonists nor antagonists significantly stimulated [(35)S]GTP gamma S binding. Yet sigma receptor selective antagonists, but not agonists, shifted the EC(50) of opioid-induced stimulation of [(35)S]GTP gamma S binding by 3- to 10-fold to the left. This enhanced potency was seen without a change in the efficacy of the opioid, as assessed by the maximal stimulation of [(35)S]GTP gamma S binding. sigma(1) Receptors physically associate with mu opioid receptors, as shown by coimmunoprecipitation studies in transfected HEK cells, implying a direct interaction between the proteins. Thus, sigma receptors modulate opioid transduction without influencing opioid receptor binding. RNA interference knockdown of sigma(1) in BE(2)-C cells also potentiated mu opioid-induced stimulation of [(35)S]GTP gamma S binding. These modulatory actions are not limited to mu and delta opioid receptors. In mouse brain membrane preparations, sigma(1)-selective antagonists also potentiated both opioid receptor and muscarinic acetylcholine receptor-mediated stimulation of [(35)S]GTP gamma S binding, suggesting a broader role for sigma receptors in modulating G-protein-coupled receptor signaling. PMID:20089882

  14. Multiscale perspectives of virus entry via endocytosis

    PubMed Central

    2013-01-01

    Most viruses take advantage of endocytic pathways to gain entry into host cells and initiate infections. Understanding of virus entry via endocytosis is critically important for the design of antiviral strategies. Virus entry via endocytosis is a complex process involving hundreds of cellular proteins. The entire process is dictated by events occurring at multiple time and length scales. In this review, we discuss and evaluate the available means to investigate virus endocytic entry, from both experimental and theoretical/numerical modeling fronts, and highlight the importance of multiscale features. The complexity of the process requires investigations at a systems biology level, which involves the combination of different experimental approaches, the collaboration of experimentalists and theorists across different disciplines, and the development of novel multiscale models. PMID:23734580

  15. Exocytosis and endocytosis: modes, functions, and coupling mechanisms.

    PubMed

    Wu, Ling-Gang; Hamid, Edaeni; Shin, Wonchul; Chiang, Hsueh-Cheng

    2014-01-01

    Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progress in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis. PMID:24274740

  16. Exocytosis and Endocytosis: Modes, Functions, and Coupling Mechanisms*

    PubMed Central

    Wu, Ling-Gang; Hamid, Edaeni; Shin, Wonchul; Chiang, Hsueh-Cheng

    2016-01-01

    Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progress in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis. PMID:24274740

  17. Identification of opioid receptor subtypes in antinociceptive actions of supraspinally-administered mitragynine in mice.

    PubMed

    Thongpradichote, S; Matsumoto, K; Tohda, M; Takayama, H; Aimi, N; Sakai, S; Watanabe, H

    1998-01-01

    Mitragynine (MG), a major alkaloidal constituent extracted from the plant Mitragyna speciosa Korth, is known to exert an opioid-like activity. Our previous study showed the involvement of opioid systems in the antinociceptive activity of MG in the tail-pinch and hot-plate tests in mice. In the present study, to clarify the opioid receptor subtypes involved in the antinociceptive action of MG, we investigated the effects of selective antagonists for mu-, delta- and kappa- opioid receptors on antinociception caused by the intracerebroventricular (i.c.v.) injection of MG in the tail-pinch and hot-plate tests in mice. The coadministration of a selective mu-opioid antagonist, cyprodime (1-10 microg, i.c.v.) and the pretreatment with a selective mu1-opioid antagonist naloxonazine (1-3 microg, i.c.v.) significantly antagonized the antinociceptive activities of MG (10 microg, i.c.v.) and morphine (MOR, 3 microg, i.c.v.) in the tail-pinch and hot-plate tests. Naltrindole (1-5 ng, i.c.v.), a selective delta-opioid antagonist, also blocked the effects of MG (10 microg, i.c.v.) without affecting MOR (3 microg, i.c.v.) antinociception. Nor-binaltorphimine, a selective kappa-opioid antagonist, significantly attenuated MG (10 microg, i.c.v.) antinociception in the tail-pinch test but not in the hot-plate test at the dose (1 microg, i.c.v.) that antagonized the antinociceptive effects of the selective kappa-opioid agonist U50,488H in both tests, while it had no effect on MOR antinociception in either tests. These results suggest that antinociception caused by i.c.v. MG is dominantly mediated by mu- and delta-opioid receptor subtypes, and that the selectivity of MG for the supraspinal opioid receptor subtypes differs from that of MOR in mice. PMID:9585164

  18. Role of flotillins in the endocytosis of GPCR in salivary gland epithelial cells.

    PubMed

    Park, Moon-Yong; Kim, Nahyun; Wu, Li-Ling; Yu, Guang-Yan; Park, Kyungpyo

    2016-08-01

    Endocytosis has numerous functions in cellular homeostasis. Defects in the endocytic pathway of receptors may lead to dysfunction of salivary gland secretion. Therefore, elucidating the complex mechanisms of endocytosis may facilitate solutions for disease treatment and prevention. The muscarinic type 3 receptor (M3R), a G-protein-coupled receptor (GPCR) located in the plasma membrane, is involved in numerous physiological activities such as smooth muscle contraction and saliva secretion. M3R enters cells through clathrin-mediated endocytosis (CME), while flotillins (flot-1 and -2), highly conserved proteins residing in lipid-raft microdomains, make use of clathrin-independent endocytosis (CIE) for their internalization. Since these two proteins use two discrete pathways for endocytic entry, the association of flotillins with CME is poorly understood. We examined whether flotillins play a role in CME of M3R using immunoblotting, immunocytochemistry, confocal immunofluorescence microscopy, co-immunoprecipitation, and RNA interference techniques in secretory epithelial cells. Upon stimulation with a cholinergic agonist, M3R, flot-1, and flot-2 each internalized from the plasma membrane into intracellular sites. The addition of chlorpromazine and cytochalasin D, well-known inhibitors of CME, inhibited internalization of M3R via CME. Filipin III and methyl-β-cyclodextrin (mβCD) acting as lipid raft inhibitors disrupted internalization of flot-1/2 via CIE. Interestingly, filipin III and mβCD slightly reduced expression level of M3R whereas chlorpromazine and cytochalasin D did not affect internalization of the flotillin isoforms. M3R and flot-1/2 colocalized and interacted with each other as they entered the cytosol during limited periods of incubation. Moreover, knockdown of flot-1 or -2 by flotillin-specific siRNA prevented internalization and reduced the endocytic efficiency of M3R. Our results suggest that flot-1 and -2 are partially involved in CME of M3R by

  19. The pleckstrin homology domain of phospholipase D1 accelerates EGFR endocytosis by increasing the expression of the Rab5 effector, rabaptin-5

    PubMed Central

    Park, Mi Hee; Choi, Kang-Yell; Min, Do Sik

    2015-01-01

    Endocytosis is differentially regulated by hypoxia-inducible factor-1α (HIF-1α) and phospholipase D (PLD). However, the relationship between HIF-1α and PLD in endocytosis is unknown. HIF-1α is degraded through the prolyl hydroxylase (PHD)/von Hippel–Lindau (VHL) ubiquitination pathway in an oxygen-dependent manner. Here, we show that PLD1 recovers the decrease in epidermal growth factor receptor (EGFR) endocytosis induced by HIF-1α independent of lipase activity via the Rab5-mediated endosome fusion pathway. EGF-induced interaction of PLD1 with HIF-1α, PHD and VHL may contribute to EGFR endocytosis. The pleckstrin homology domain (PH) of PLD1 itself promotes degradation of HIF-1α, then accelerates EGFR endocytosis via upregulation of rabaptin-5 and suppresses tumor progression. These findings reveal a novel role of the PLD1-PH domain as a positive regulator of endocytosis and provide a link between PLD1 and HIF-1α in the EGFR endocytosis pathway. PMID:26680696

  20. Rift Valley Fever Virus Strain MP-12 Enters Mammalian Host Cells via Caveola-Mediated Endocytosis

    PubMed Central

    Harmon, Brooke; Schudel, Benjamin R.; Maar, Dianna; Kozina, Carol; Ikegami, Tetsuro; Tseng, Chien-Te Kent

    2012-01-01

    Rift Valley fever virus (RVFV) is a zoonotic pathogen capable of causing serious morbidity and mortality in both humans and livestock. The lack of efficient countermeasure strategies, the potential for dispersion into new regions, and the pathogenesis in humans and livestock make RVFV a serious public health concern. The receptors, cellular factors, and entry pathways used by RVFV and other members of the family Bunyaviridae remain largely uncharacterized. Here we provide evidence that RVFV strain MP-12 uses dynamin-dependent caveola-mediated endocytosis for cell entry. Caveolae are lipid raft domains composed of caveolin (the main structural component), cholesterol, and sphingolipids. Caveola-mediated endocytosis is responsible for the uptake of a wide variety of host ligands, as well as bacteria, bacterial toxins, and a number of viruses. To determine the cellular entry mechanism of RVFV, we used small-molecule inhibitors, RNA interference (RNAi), and dominant negative (DN) protein expression to inhibit the major mammalian cell endocytic pathways. Inhibitors and RNAi specific for macropinocytosis and clathrin-mediated endocytosis had no effect on RVFV infection. In contrast, inhibitors of caveola-mediated endocytosis, and RNAi targeted to caveolin-1 and dynamin, drastically reduced RVFV infection in multiple cell lines. Expression of DN caveolin-1 also reduced RVFV infection significantly, while expression of DN EPS15, a protein required for the assembly of clathrin-coated pits, and DN PAK-1, an obligate mediator of macropinocytosis, had no significant impact on RVFV infection. These results together suggest that the primary mechanism of RVFV MP-12 uptake is dynamin-dependent, caveolin-1-mediated endocytosis. PMID:22993156

  1. The role played by endocytosis in albumin-induced secretion of TGF-beta1 by proximal tubular epithelial cells.

    PubMed

    Diwakar, Ramaswamy; Pearson, Alex L; Colville-Nash, Paul; Brunskill, Nigel J; Dockrell, Mark E C

    2007-05-01

    Proteinuria predicts the decline of renal function in chronic kidney disease. Reducing albuminuria has been shown to be associated with a reduction in this rate of decline. Proximal tubular epithelial cells (PTECs), when exposed to albumin produce matrix proteins, proinflammatory and profibrotic cytokines like TGF-beta(1). Some of these effects are dependent on endocytosis of albumin by PTECs. However, conditions like diabetic nephropathy, believed to be associated with reduced albumin endocytosis, are associated with interstitial fibrosis. Moreover, megalin, the putative albumin binding receptor in PTECs, has potential signaling motifs in its cytoplasmic domain, suggesting its ability to signal in response to ligand binding from the apical surface of PTECs. Hence, we looked to see whether albumin-induced secretion of TGF-beta(1) by PTECs is dependent on albumin endocytosis or whether it could occur in the absence of albumin endocytosis. We studied the production of TGF-beta(1) in two accepted models of PTECs, opossum kidney cells and human kidney cell clone-8 cells, with widely varying degrees of endocytosis. We then studied the effect of inhibiting albumin endocytosis with various inhibitors on albumin-induced TGF-beta(1) secretion. Our results indicate that albumin-induced TGF-beta(1) secretion by PTECs does not require albumin endocytosis and therefore the mechanism for the induction of some profibrotic responses by albumin may differ from those required for some of the inflammatory responses. Moreover, we found that albumin-induced TGF-beta(1) secretion by PTECs is not dependent on its interaction with megalin. PMID:17213467

  2. Iterative endocytosis of transferrin by K562 cells.

    PubMed Central

    Young, S P; Bomford, A

    1994-01-01

    The effect of iron on the exocytosis of transferrin by K562 cells was studied by first allowing the cells to endocytose apotransferrin or diferric transferrin. Subsequent release of the apotransferrin was very rapid with a t 1/2 of 3.01 min, compared with 5.5 min for diferric transferrin. Release of apotransferrin was slowed by the weak base methylamine, t 1/2 8.0 min, but the effect of this agent was substantially greater when iron-transferrin was used, t 1/2 18.65 min, suggesting that methylamine affects both iron removal and receptor recycling. Release of iron-transferrin could be accelerated to a rate comparable with that of apotransferrin by addition of the permeant iron-chelator desferrioxamine. The difference in the rates of release of different forms of the protein could be explained by the re-endocytosis of the iron-rich protein, a process detected by the accelerated release of transferrin when the cells were washed in medium at pH 5.5 containing an iron-chelator or treated with a protease-containing medium to digest transferrin accessible at the cell surface. It appears that in cells incubated under control conditions, re-endocytosis of transferrin, which is incompletely depleted of iron, occurs and that a transferrin molecule may make two passes through the cell before all the iron is removed. This mechanism helps to explain why very little iron-transferrin is released from cells and why the efficiency of the iron uptake process is so high. PMID:8129715

  3. Mib1 modulates dynamin 2 recruitment via Snx18 to promote Dll1 endocytosis for efficient Notch signaling.

    PubMed

    Okano, Makoto; Matsuo, Hiromi; Nishimura, Yuya; Hozumi, Katsuto; Yoshioka, Saho; Tonoki, Ayako; Itoh, Motoyuki

    2016-05-01

    Notch signaling regulates normal development and tissue homeostasis. Ligand endocytosis plays critical roles in Notch signaling activation. Endocytic proteins such as epsin and dynamin participate in Notch ligand activity by mediating Notch ligand endocytosis. The ubiquitin ligase Mib1 also plays essential roles in Notch signaling via Notch ligand ubiquitination. However, the molecular links between Mib1 and endocytic proteins have not been fully defined. Here, we show that Mib1 is involved in dynamin 2 recruitment to Dll1 and that Snx18, which interacts with dynamin 2, modestly regulates Dll1 endocytosis. Furthermore, the ubiquitin ligase activity of Mib1 is induced by Notch ligand-receptor interactions. Mib1 promotes the interaction between dynamin 2 and Snx18 in an ubiquitin ligase activity-dependent manner. These results suggest that Mib1 modulates dynamin recruitment by regulating the interaction between Snx18 and dynamin 2, thereby helping to ensure the efficient signaling activity of Notch ligands. PMID:26923255

  4. Dynamic spectrin/ankyrin-G microdomains promote lateral membrane assembly by opposing endocytosis

    PubMed Central

    Jenkins, Paul M.; He, Meng; Bennett, Vann

    2015-01-01

    Current physical models for plasma membranes emphasize dynamic 10- to 300-nm compartments at thermodynamic equilibrium but subject to thermal fluctuations. However, epithelial lateral membranes contain micrometer-sized domains defined by an underlying membrane skeleton composed of spectrin and its partner ankyrin-G. We demonstrate that these spectrin/ankyrin-G domains exhibit local microtubule-dependent movement on a time scale of minutes and encounter most of the lateral membranes within an hour. Spectrin/ankyrin-G domains exclude clathrin and clathrin-dependent cargo, and inhibit both receptor-mediated and bulk endocytosis. Moreover, inhibition of endocytosis fully restores lateral membrane height in spectrin- or ankyrin-G–depleted cells. These findings support a non-equilibrium cellular-scale model for epithelial lateral membranes, where spectrin/ankyrin-G domains actively patrol the plasma membrane, analogous to “window washers,” and promote columnar morphology by blocking membrane uptake. PMID:26523289

  5. Eph/ephrin molecules—a hub for signaling and endocytosis

    PubMed Central

    Pitulescu, Mara E.; Adams, Ralf H.

    2010-01-01

    The development, homeostasis, and regeneration of complex organ systems require extensive cell–cell communication to ensure that different cells proliferate, migrate, differentiate, assemble, and function in a coordinated and timely fashion. Eph receptor tyrosine kinases and their ephrin ligands are critical regulators of cell contact-dependent signaling and patterning. Eph/ephrin binding can lead to very diverse biological readouts such as adhesion versus repulsion, or increased versus decreased motility. Accordingly, depending on cell type and context, a limited and conserved set of receptor–ligand interactions is translated into a large variety of downstream signaling processes. Recent evidence indicates that the endocytosis of Eph/ephrin molecules, together with the internalization of various associated tissue-specific effectors, might be one of the key principles responsible for such highly diverse and adaptable biological roles. Here, we summarize recent insights into Eph/ephrin signaling and endocytosis in three biological systems; i.e., the brain, intestine, and vasculature. PMID:21078817

  6. Effects of mitragynine on cAMP formation mediated by delta-opiate receptors in NG108-15 cells.

    PubMed

    Tohda, M; Thongpraditchote, S; Matsumoto, K; Murakami, Y; Sakai, S; Aimi, N; Takayama, H; Tongroach, P; Watanabe, H

    1997-04-01

    Mitragynine, a major constituent of the young leaves of Mitragyna speciosa KORTH., has been reported to exert antinociceptive activity in mice. To determine the mechanism the influence of mitragynine on cAMP content was measured in NG108-15 cells which possess delta opioid receptors and alpha 2B-adrenoceptors. Mitragynine inhibited the forskolin-stimulated cAMP content in a concentration dependent manner as well as morphine and noradrenaline. Mitragynine- and morphine-induced inhibition of cAMP content were blocked by naloxone. Although idazoxane inhibited noradrenaline-induced inhibition of the cAMP content, idazoxane had no effect on mitragynine-induced inhibition. These results suggest that mitragynine acts directly on opioid receptors, but not on alpha 2-adrenoceptors, to show antinociceptive activity. PMID:9145205

  7. Shank2 Regulates Renal Albumin Endocytosis

    PubMed Central

    Dobrinskikh, Evgenia; Lewis, Linda; Doctor, R Brian; Okamura, Kayo; Lee, Min Goo; Altmann, Christopher; Faubel, Sarah; Kopp, Jeffrey B; Blaine, Judith

    2015-01-01

    Albuminuria is a strong and independent predictor of kidney disease progression but the mechanisms of albumin handling by the kidney remain to be fully defined. Previous studies have shown that podocytes endocytose albumin. Here we demonstrate that Shank2, a large scaffolding protein originally identified at the neuronal postsynaptic density, is expressed in podocytes in vivo and in vitro and plays an important role in albumin endocytosis in podocytes. Knockdown of Shank2 in cultured human podocytes decreased albumin uptake, but the decrease was not statistically significant likely due to residual Shank2 still present in the knockdown podocytes. Complete knockout of Shank2 in podocytes significantly diminished albumin uptake in vitro. Shank2 knockout mice develop proteinuria by 8 weeks of age. To examine albumin handling in vivo in wild-type and Shank2 knockout mice we used multiphoton intravital imaging. While FITC-labeled albumin was rapidly seen in the renal tubules of wild-type mice after injection, little albumin was seen in the tubules of Shank2 knockout mice indicating dysregulated renal albumin trafficking in the Shank2 knockouts. We have previously found that caveolin-1 is required for albumin endocytosis in cultured podocytes. Shank2 knockout mice had significantly decreased expression and altered localization of caveolin-1 in podocytes suggesting that disruption of albumin endocytosis in Shank2 knockouts is mediated via caveolin-1. In summary, we have identified Shank2 as another component of the albumin endocytic pathway in podocytes. PMID:26333830

  8. Cell mobility after endocytosis of carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Pirbhai, Massooma; Flores, Thomas; Jedlicka, Sabrina; Rotkin, Slava

    2013-03-01

    Directed cell movement plays a crucial role in cellular behaviors such as neuronal cell division, cell migration, and cell differentiation. There is evidence in preclinical in vivo studies that small fields have successfully been used to enhance regrowth of damages spinal cord axons but with a small success rate. Fortunately, the evolution of functional biomaterials and nanotechnology may provide promising solutions for enhancing the application of electric fields in guiding neuron migration and neurogenesis within the central nervous system. In this work, we studied how endocytosis and subsequent retention of carbon nanotubes affects the mobility of cells under the influence of an electric field, including the directed cell movement.

  9. Nanomechanics of magnetically driven cellular endocytosis

    NASA Astrophysics Data System (ADS)

    Zablotskii, V.; Lunov, O.; Dejneka, A.; Jastrabík, L.; Polyakova, T.; Syrovets, T.; Simmet, Th.

    2011-10-01

    Being essential for many pharmacodynamic and pharmacokinetic processes and playing a crucial role in regulating substrate detachment that enables cellular locomotion, endocytotic mechanisms in many aspects still remain a mystery and therefore can hardly be controlled. Here, we report on experimental and modeling studies of the magnetically assisted endocytosis of functionalized superparamagnetic iron oxide nanoparticles by prostate cancer cells (PC-3) and characterize the time and force scales of the cellular uptake machinery. The results indicate how the cellular uptake rate could be controlled by applied magnetic field, membrane elasticity, and nanoparticle magnetic moment.

  10. Kinetics of virus entry by endocytosis

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2015-04-01

    Entry of virions into the host cells is either endocytotic or fusogenic. In both cases, it occurs via reversible formation of numerous relatively weak bonds resulting in wrapping of a virion by the host membrane with subsequent membrane rupture or scission. The corresponding kinetic models are customarily focused on the formation of bonds and do not pay attention to the energetics of the whole process, which is crucially dependent, especially in the case of endocytosis, on deformation of actin filaments forming the cytoskeleton of the host cell. The kinetic model of endocytosis, proposed by the author, takes this factor into account and shows that the whole process can be divided into a rapid initial transient stage and a long steady-state stage. The entry occurs during the latter stage and can be described as a first-order reaction. Depending on the details of the dependence of the grand canonical potential on the number of bonds, the entry can be limited either by the interplay of bond formation and membrane rupture (or scission) or by reaching a maximum of this potential.

  11. Exocytosis and endocytosis in juxtaglomerular cells.

    PubMed

    Friis, U G; Jensen, B L; Hansen, P B; Andreasen, D; Skøtt, O

    2000-01-01

    The cellular events related to secretion of renin are not well understood. Here we review some of the evidence that has led to the current understanding of renin secretion as a process that involves exocytosis as the predominant mode of secretion. This is based on the observation of occasional fusion events between secretory granules and cell membrane and measurement of intermittent secretion of renin from single afferent arterioles, with a renin content of each secretion episode that corresponds to the renin content of one secretory granule. More recently it has been demonstrated that the afferent arterioles lose a large number of renin granules after acute stimulation without changing the average granular volume. Current electrophysiological techniques have now permitted direct measurements of cell membrane capacitance in juxtaglomerular (JG) cells as a measure of net addition (exocytosis) or removal (endocytosis) of membrane material. With this technique we have shown that cAMP, which is a vasodilator and stimulates renin secretion, enhances net exocytosis at low concentrations, while at higher concentrations membrane retrieval processes are also stimulated. We suggest that both exocytosis and endocytosis are regulated processes in the JG-cells and both may be important for the long-term control of renin secretion at the single cell level. PMID:10691785

  12. PLD1 regulates Xenopus convergent extension movements by mediating Frizzled7 endocytosis for Wnt/PCP signal activation.

    PubMed

    Lee, Hyeyoon; Lee, Seung Joon; Kim, Gun-Hwa; Yeo, Inchul; Han, Jin-Kwan

    2016-03-01

    Phospholipase D (PLD) is involved in the regulation of receptor-associated signaling, cell movement, cell adhesion and endocytosis. However, its physiological role in vertebrate development remains poorly understood. In this study, we show that PLD1 is required for the convergent extension (CE) movements during Xenopus gastrulation by activating Wnt/PCP signaling. Xenopus PLD1 protein is specifically enriched in the dorsal region of Xenopus gastrula embryo and loss or gain-of-function of PLD1 induce defects in gastrulation and CE movements. These defective phenotypes are due to impaired regulation of Wnt/PCP signaling pathway. Biochemical and imaging analysis using Xenopus tissues reveal that PLD1 is required for Fz7 receptor endocytosis upon Wnt11 stimulation. Moreover, we show that Fz7 endocytosis depends on dynamin and regulation of GAP activity of dynamin by PLD1 via its PX domain is crucial for this process. Taken together, our results suggest that PLD1 acts as a new positive mediator of Wnt/PCP signaling by promoting Wnt11-induced Fz7 endocytosis for precise regulation of Xenopus CE movements. PMID:26806705

  13. HARE-Mediated Endocytosis of Hyaluronan and Heparin Is Targeted by Different Subsets of Three Endocytic Motifs

    PubMed Central

    Pandey, Madhu S.; Harris, Edward N.; Weigel, Paul H.

    2015-01-01

    The hyaluronan (HA) receptor for endocytosis (HARE) is a multifunctional recycling clearance receptor for 14 different ligands, including HA and heparin (Hep), which bind to discrete nonoverlapping sites. Four different functional endocytic motifs (M) in the cytoplasmic domain (CD) target coated pit mediated uptake: (YSYFRI2485 (M1), FQHF2495 (M2), NPLY2519 (M3), and DPF2534 (M4)). We previously found (Pandey et al. J. Biol. Chem. 283, 21453, 2008) that M1, M2, and M3 mediate endocytosis of HA. Here we assessed the ability of HARE variants with a single-motif deletion or containing only a single motif to endocytose HA or Hep. Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs. Surprisingly, a HARE CD variant with only M3 internalized both HA and Hep, whereas variants with either M2 or M4 alone did not endocytose either ligand. Internalization of HA and Hep by HARE CD mutants was dynamin-dependent and was inhibited by hyperosmolarity, confirming clathrin-mediated endocytosis. The results indicate a complicated relationship among multiple CD motifs that target coated pit uptake and a more fundamental role for motif M3. PMID:25883656

  14. Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis.

    PubMed

    Wang, Changhe; Wang, Yeshi; Hu, Meiqin; Chai, Zuying; Wu, Qihui; Huang, Rong; Han, Weiping; Zhang, Claire Xi; Zhou, Zhuan

    2016-01-01

    Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin-11 (Syt11), a non-Ca(2+)-binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin-mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin-coated pits and bulk endocytosis-like structures increase on the plasma membrane in Syt11-knockdown neurons. Structural-functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis. PMID:26589353

  15. Differential requirements for actin during yeast and mammalian endocytosis.

    PubMed

    Aghamohammadzadeh, Soheil; Ayscough, Kathryn R

    2009-08-01

    Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination. PMID:19597484

  16. Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons☆

    PubMed Central

    Nusshold, Christoph; Uellen, Andreas; Bernhart, Eva; Hammer, Astrid; Damm, Sabine; Wintersperger, Andrea; Reicher, Helga; Hermetter, Albin; Malle, Ernst; Sattler, Wolfgang

    2013-01-01

    Neuronal sphingolipids (SL) play important roles during axonal extension, neurotrophic receptor signaling and neurotransmitter release. Many of these signaling pathways depend on the presence of specialized membrane microdomains termed lipid rafts. Sphingomyelin (SM), one of the main raft constituents, can be formed de novo or supplied from exogenous sources. The present study aimed to characterize fluorescently-labeled SL turnover in a murine neuronal cell line (CATH.a). Our results demonstrate that at 4 °C exogenously added BODIPY-SM accumulates exclusively at the plasma membrane. Treatment of cells with bacterial sphingomyelinase (SMase) and back-exchange experiments revealed that 55–67% of BODIPY-SM resides in the outer leaflet of the plasma membrane. Endocytosis of BODIPY-SM occurs via caveolae with part of internalized BODIPY-fluorescence ending up in the Golgi and the ER. Following endocytosis BODIPY-SM undergoes hydrolysis, a reaction substantially faster than BODIPY-SM synthesis from BODIPY-ceramide. RNAi demonstrated that both, acid (a)SMase and neutral (n)SMases contribute to BODIPY-SM hydrolysis. Finally, high-density lipoprotein (HDL)-associated BODIPY-SM was efficiently taken up by CATH.a cells. Our findings indicate that endocytosis of exogenous SM occurs almost exclusively via caveolin-dependent pathways, that both, a- and nSMases equally contribute to neuronal SM turnover and that HDL-like particles might represent physiological SM carriers/donors in the brain. PMID:23973266

  17. The Lowe Syndrome Protein OCRL1 Is Required for Endocytosis in the Zebrafish Pronephric Tubule

    PubMed Central

    Oltrabella, Francesca; Pietka, Grzegorz; Ramirez, Irene Barinaga-Rementeria; Mironov, Aleksandr; Starborg, Toby; Drummond, Iain A.; Hinchliffe, Katherine A.; Lowe, Martin

    2015-01-01

    Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the underlying basis for the renal tubulopathy seen in both human disorders, of which a hallmark is low molecular weight proteinuria, is currently unknown. Here, we show that deficiency in OCRL1 causes a defect in endocytosis in the zebrafish pronephric tubule, a model for the mammalian renal tubule. This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway. We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells. Cell polarity within the pronephric tubule is unaffected in mutant embryos. The OCRL1-deficient embryos exhibit a mild ciliogenesis defect, but this cannot account for the observed impairment of endocytosis. Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K. These results indicate for the first time that OCRL1 is required for endocytic trafficking in vivo, and strongly support the hypothesis that endocytic defects are responsible for the renal tubulopathy in Lowe syndrome and Dent-2 disease. Moreover, our results reveal PIP5K as a potential therapeutic target for Lowe syndrome and Dent-2 disease. PMID:25838181

  18. Unconventional EGF-induced ERK1/2-mediated Kv1.3 endocytosis.

    PubMed

    Martínez-Mármol, Ramón; Comes, Núria; Styrczewska, Katarzyna; Pérez-Verdaguer, Mireia; Vicente, Rubén; Pujadas, Lluís; Soriano, Eduardo; Sorkin, Alexander; Felipe, Antonio

    2016-04-01

    The potassium channel Kv1.3 plays roles in immunity, neuronal development and sensory discrimination. Regulation of Kv1.3 by kinase signaling has been studied. In this context, EGF binds to specific receptors (EGFR) and triggers tyrosine kinase-dependent signaling, which down-regulates Kv1.3 currents. We show that Kv1.3 undergoes EGF-dependent endocytosis. This EGF-mediated mechanism is relevant because is involved in adult neural stem cell fate determination. We demonstrated that changes in Kv1.3 subcellular distribution upon EGFR activation were due to Kv1.3 clathrin-dependent endocytosis, which targets the Kv1.3 channels to the lysosomal degradative pathway. Interestingly, our results further revealed that relevant tyrosines and other interacting motifs, such as PDZ and SH3 domains, were not involved in the EGF-dependent Kv1.3 internalization. However, a new, and yet undescribed mechanism, of ERK1/2-mediated threonine phosphorylation is crucial for the EGF-mediated Kv1.3 endocytosis. Our results demonstrate that EGF triggers the down-regulation of Kv1.3 activity and its expression at the cell surface, which is important for the development and migration of adult neural progenitors. PMID:26542799

  19. Unconventional EGF-induced ERK1/2-mediated Kv1.3 endocytosis

    PubMed Central

    Martínez-Mármol, Ramón; Comes, Núria; Styrczewska, Katarzyna; Pérez-Verdaguer, Mireia; Vicente, Rubén; Pujadas, Lluís; Soriano, Eduardo; Sorkin, Alexander

    2016-01-01

    The potassium channel Kv1.3 plays roles in immunity, neuronal development and sensory discrimination. Regulation of Kv1.3 by kinase signaling has been studied. In this context, EGF binds to specific receptors (EGFR) and triggers tyrosine kinase-dependent signaling, which down-regulates Kv1.3 currents. We show that Kv1.3 undergoes EGF-dependent endocytosis. This EGF-mediated mechanism is relevant because is involved in adult neural stem cell fate determination. We demonstrated that changes in Kv1.3 subcellular distribution upon EGFR activation were due to Kv1.3 clathrin-dependent endocytosis, which targets the Kv1.3 channels to the lysosomal degradative pathway. Interestingly, our results further revealed that relevant tyrosines and other interacting motifs, such as PDZ and SH3 domains, were not involved in the EGF-dependent Kv1.3 internalization. However, a new, and yet undescribed mechanism, of ERK1/2-mediated threonine phosphorylation is crucial for the EGF-mediated Kv1.3 endocytosis. Our results demonstrate that EGF triggers the down-regulation of Kv1.3 activity and its expression at the cell surface, which is important for the development and migration of adult neural progenitors. PMID:26542799

  20. Endocytosis of heat-denatured albumin by cultured rat Kupffer cells

    SciTech Connect

    Brouwer, A.; Knook, D.L.

    1982-10-01

    Purified Kupffer cells were obtained by centrifugal elutriation of sinusoidal cells isolated by pronase treatment of the rat liver. The endocytosis of radioactively labeled heat-aggregated colloidal albumin (CA /sup 125/I) was investigated in maintenance cultures of the purified Kupffer cells. The endocytic capacity of the cells was studied during 4 days of culture. Maximum uptake was observed after 24 hr of culture, with a gradual decline during the following days. When the uptake was measured after incubation with increasing concentrations of CA /sup 125/I, a saturation effect was observed. This finding and the observed high rate of uptake are strong indications that receptor sites on the cell membrane are involved in the mechanism of endocytosis. The uptake of CA /sup 125/I by Kupffer cells was inhibited by the metabolic inhibitors fluoride and antimycin A, indicating that endocytosis of CA /sup 125/I is dependent on energy derived from both glycolysis and mitochondrial respiration. The mechanism of internalization may also require the action of microfilaments as well as intact microtubules, since both cytochalasin B and colchicine inhibited the uptake of CA /sup 125/I. The intracellular degradation of CA /sup 125/I by Kupffer cells was strongly inhibited by chloroquine but not by colchicine. The degradation of ingested CA /sup 125/I occurred within the Kupffer cell lysosomes.

  1. Ouabain uptake by endocytosis in isolated guinea pig atria

    SciTech Connect

    Nunez-Duran, H.; Riboni, L.; Ubaldo, E.; Kabela, E.; Barcenas-Ruiz, L. Instituto Nacional de Cardiologia, Mexico DF )

    1988-10-01

    Mammalian cells specifically internalize some molecular species through receptor-mediated endocytosis (RME). The authors have used four different experimental protocols to investigate whether ouabain enters cardiac cells of guinea pig atrium through this pathway. First, by electron microscope morphometry the authors found that ouabain increased endocytic vesicles in atrial cells. Second, by scintillation counting they found that ({sup 3}H)ouabain uptake by the tissue is decreased by three treatments that decrease RME, i.e., NH{sub 4}Cl, trifluoperazine, and 16 mM (K{sup +}){sub 0}. Third, by radioautography at the electron microscope level, they checked that in preceding experiments ({sup 3}H)ouabain was washed out of plasma membrane after 60-min rinse and interiorized into the cardiac cells. Fourth, isometric tension recordings showed that the positive inotropic effect of ouabain was diminished in the presence of inhibitors, whereas that of a hydrophobic analogue, ouabagenin, was not affected. These results suggest that ouabain enters cardiac cells through RME and also that an intracellular site may, at least in part, be responsible for its inotropic effect.

  2. Characterization and visualization of rat and guinea pig brain. kappa. opioid receptors: Evidence for. kappa. sub 1 and. kappa. sub 2 opioid receptors

    SciTech Connect

    Zukin, R.S.; Eghbali, M.; Olive, D.; Unterwald, E.M.; Tempel, A. )

    1988-06-01

    {kappa} opioid receptors ({kappa} receptors) have been characterized in homogenates of guinea pig and rat brain under in vitro binding conditions. {kappa} receptors were labeled by using the tritiated prototypic {kappa} opioid ethylketocyclazocine under conditions in which {mu} and {delta} opioid binding was suppressed. In the case of guinea pig brain membranes, a single population of high-affinity {kappa} opioid receptor sites was observed. In contrast, in the case of rat brain, two populations of {kappa} sites were observed. To test the hypothesis that the high- and low-affinity {kappa} sites represent two distinct {kappa} receptor subtypes, a series of opioids were tested for their abilities to compete for binding to the two sites. U-69,593 and Cambridge 20 selectively displaced the high-affinity {kappa} site in both guinea pig and rat tissue, but were inactive at the rat-brain low-affinity site. Other {kappa} opioid drugs competed for binding to both sites, but with different rank orders of potency. Quantitative light microscopy in vitro autoradiography was used to visualize the neuroanatomical pattern of {kappa} receptors in rat and guinea pig brain. The distribution patterns of the two {kappa} receptor subtypes of rat brain were clearly different. Collectively, these data provide direct evidence for the presence of two {kappa} receptor subtypes; the U-69,593-sensitive, high-affinity {kappa}{sub 1} site predominates in guinea pig brain, and the U-69,593-insensitive, low-affinity {kappa}{sub 2} site predominates in rat brain.

  3. Visualization of multiple opioid-receptor types in rat striatum after specific mesencephalic lesions

    SciTech Connect

    Eghbali, M.; Santoro, C.; Paredes, W.; Gardner, E.L.; Zukin, R.S.

    1987-09-01

    In order to gain insight into a possible modulatory role for ..mu.., delta, and kappa opioid receptors of the nigrostriatal dopaminergic pathway, the authors investigated the topographical organization of the receptors with respect to pre- and postsynaptic membranes. Dopaminergic terminals projecting from the substantia nigra to the corpus striatum were destroyed by unilateral injection of 6-hydroxydopamine into the susbstantia nigra. Quantitative receptor assays using highly specific radioligands were used to measure the density of striatal ..mu.., delta, and kappa receptors before and after denervation. Quantitative in vitro autoradiography was used to visualize the neuroanatomical pattern of receptors on lesioned and nonlesioned sides of the brain under the light microscope. Loss of ..mu.. receptors in striatal patches was striking in the ventro-lateral areas of the striatum, whereas the most notable loss of delta receptors was found in the central striatum. Other brain areas did not differ significantly in ..mu.. receptor density between the lesioned and nonlesioned sides, as determined by autoradiography. These findings suggest that a high percentage of ..mu.. and delta receptors in the striatum are located on the nigrostriatal dopaminergic terminals and support the concept of a modulatory role for ..mu.. and delta opioid peptides in the nigrostriatal dopaminergic pathway.

  4. Bradykinin Release Avoids High Molecular Weight Kininogen Endocytosis

    PubMed Central

    Nascimento, Fabio D.; Souza, Daianne S. P.; Araujo, Mariana S.; Souza, Sinval E. G.; Sampaio, Misako U.; Nader, Helena B.; Tersariol, Ivarne L. S.; Motta, Guacyara

    2015-01-01

    Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. The present

  5. Role of endocytosis and cathepsin-mediated activation in Nipah virus entry

    SciTech Connect

    Diederich, Sandra; Thiel, Lena; Maisner, Andrea

    2008-06-05

    The recent discovery that the Nipah virus (NiV) fusion protein (F) is activated by endosomal cathepsin L raised the question if NiV utilize pH- and protease-dependent mechanisms of entry. We show here that the NiV receptor ephrin B2, virus-like particles and infectious NiV are internalized from the cell surface. However, endocytosis, acidic pH and cathepsin-mediated cleavage are not necessary for the initiation of infection of new host cells. Our data clearly demonstrate that proteolytic activation of the NiV F protein is required before incorporation into budding virions but not after virus entry.

  6. Synucleins Regulate the Kinetics of Synaptic Vesicle Endocytosis

    PubMed Central

    Vargas, Karina J.; Makani, Sachin; Davis, Taylor; Westphal, Christopher H.; Castillo, Pablo E.

    2014-01-01

    Genetic and pathological studies link α-synuclein to the etiology of Parkinson's disease (PD), but the normal function of this presynaptic protein remains unknown. α-Synuclein, an acidic lipid binding protein, shares high sequence identity with β- and γ-synuclein. Previous studies have implicated synucleins in synaptic vesicle (SV) trafficking, although the precise site of synuclein action continues to be unclear. Here we show, using optical imaging, electron microscopy, and slice electrophysiology, that synucleins are required for the fast kinetics of SV endocytosis. Slowed endocytosis observed in synuclein null cultures can be rescued by individually expressing mouse α-, β-, or γ-synuclein, indicating they are functionally redundant. Through comparisons to dynamin knock-out synapses and biochemical experiments, we suggest that synucleins act at early steps of SV endocytosis. Our results categorize α-synuclein with other familial PD genes known to regulate SV endocytosis, implicating this pathway in PD. PMID:25009269

  7. Kidney Injury Molecule-1 Enhances Endocytosis of Albumin in Renal Proximal Tubular Cells.

    PubMed

    Zhao, Xueying; Jiang, Chen; Olufade, Rebecca; Liu, Dong; Emmett, Nerimiah

    2016-04-01

    Receptor-mediated endocytosis plays an important role in albumin reabsorption by renal proximal tubule epithelial cells. Kidney injury molecule-1 (KIM-1) is a scavenger receptor that is upregulated on the apical membrane of proximal tubules in proteinuric kidney disease. In this study, we examined the cellular localization and functional role of KIM-1 in cultured renal tubule epithelial cells (TECs). Confocal immunofluorescence microscopy reveals intracellular and cell surface localization of KIM-1 in primary renal TECs. Albumin stimulation resulted in a redistribution of KIM-1 and tight junction protein zonula occludens-1 in primary TEC monolayer. An increase in albumin internalization was observed in both primary TECs expressing endogenous KIM-1 and rat kidney cell line (NRK-52E) overexpressing exogenous KIM-1. KIM-1-induced albumin accumulation was abolished by its specific antibody. Moreover, endocytosed KIM-1 and its cargo proteins were delivered from endosomes to lysosomes for degradation in a clathrin-dependent pathway. Supportive evidence includes (1) detection of KIM-1 in Rab5-positive early endosomes, Rab7-positive late endosomes/multivesicular bodies, and LAMP1-positive lysosomes, (2) colocalization of KIM-1 and clathrin in the intracellular vesicles, and (3) blockade of KIM-1-mediated albumin internalization by chlorpromazine, an inhibitor of clathrin-dependent endocytosis. KIM-1 expression was upregulated by albumin but downregulated by transforming growth factor-β1. Taken together, our data indicate that KIM-1 increases albumin endocytosis in renal tubule epithelial cells, at least partially via a clathrin-dependent mechanism. J. Cell. Physiol. 231: 896-907, 2016. © 2015 Wiley Periodicals, Inc. PMID:26332568

  8. Retrovirus Entry by Endocytosis and Cathepsin Proteases

    PubMed Central

    Kubo, Yoshinao; Hayashi, Hideki; Matsuyama, Toshifumi; Sato, Hironori; Yamamoto, Naoki

    2012-01-01

    Retroviruses include infectious agents inducing severe diseases in humans and animals. In addition, retroviruses are widely used as tools to transfer genes of interest to target cells. Understanding the entry mechanism of retroviruses contributes to developments of novel therapeutic approaches against retrovirus-induced diseases and efficient exploitation of retroviral vectors. Entry of enveloped viruses into host cell cytoplasm is achieved by fusion between the viral envelope and host cell membranes at either the cell surface or intracellular vesicles. Many animal retroviruses enter host cells through endosomes and require endosome acidification. Ecotropic murine leukemia virus entry requires cathepsin proteases activated by the endosome acidification. CD4-dependent human immunodeficiency virus (HIV) infection is thought to occur via endosomes, but endosome acidification is not necessary for the entry whereas entry of CD4-independent HIVs, which are thought to be prototypes of CD4-dependent viruses, is low pH dependent. There are several controversial results on the retroviral entry pathways. Because endocytosis and endosome acidification are complicatedly controlled by cellular mechanisms, the retrovirus entry pathways may be different in different cell lines. PMID:23304142

  9. Comparison of the butyrate effects on neurotransmitter receptors in neurohybrids NG108-15 and NCB-20 cells

    SciTech Connect

    Zhu, X.Z.; Chuang, D.M.

    1987-08-31

    The authors previous study demonstrated that long term treatment of NCB-20 cells with sodium butyrate resulted in a marked increase in the density of delta-opioid receptors with a much lesser effect on muscarinic cholinergic and no effect on alpha/sub 2/-adrenergic receptors. In the present study the authors investigated the effect of sodium butyrate on these three types of receptors in NG108-15 cells whose neuroblastoma parent is the same as that of NCB-20 cells. Long term treatment of NG108-15 cells with sodium butyrate (0.5 mM) induced a 2-fold increase in the density of the specific binding of /sup 3/H-clonidine. A comparable increase in the number of binding sites was detected when /sup 3/H-yohimbine was used as the receptor ligand. The butyrate-induced increase in the alpha/sub 2/-adrenergic receptor binding could be totally abolished by treatment with a protein synthesis inhibitor, cycloheximide, suggesting that synthesis of receptor protein is involved. The same butyrate treatment had no significant effect on opioid and muscarinic cholinergic receptor bindings. Thus, butyrate effects on the expression of these three types of receptors in NG108-15 and NCB-20 cells are dramatically different. These data suggest that induction by butyrate of neurotransmitter receptors requires concerted action of genetic factors of both parents of the neurohybrids. 22 references, 2 figures, 2 tables.

  10. Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling

    PubMed Central

    Fujishiro, Mitsuhiro; Okamura, Tomohisa; Fujio, Keishi; Okazaki, Hiroaki; Nomura, Seitaro; Takeda, Norifumi; Harada, Mutsuo; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Yamamoto, Kazuhiko; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays. PMID:26020647

  11. CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance

    PubMed Central

    Rajaiah, Rajesh; Perkins, Darren J.; Ireland, Derek D. C.; Vogel, Stefanie N.

    2015-01-01

    Dimerization of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) heterodimers is critical for both MyD88- and TIR-domain–containing adapter-inducing IFN-β (TRIF)-mediated signaling pathways. Recently, Zanoni et al. [(2011) Cell 147(4):868–880] reported that cluster of differentiation 14 (CD14) is required for LPS-/Escherichia coli- induced TLR4 internalization into endosomes and activation of TRIF-mediated signaling in macrophages. We confirmed their findings with LPS but report here that CD14 is not required for receptor endocytosis and downstream signaling mediated by TLR4/MD2 agonistic antibody (UT12) and synthetic small-molecule TLR4 ligands (1Z105) in murine macrophages. CD14 deficiency completely ablated the LPS-induced TBK1/IRF3 signaling axis that mediates production of IFN-β in murine macrophages without affecting MyD88-mediated signaling, including NF-κB, MAPK activation, and TNF-α and IL-6 production. However, neither the MyD88- nor TRIF-signaling pathways and their associated cytokine profiles were altered in the absence of CD14 in UT12- or 1Z105-treated murine macrophages. Eritoran (E5564), a lipid A antagonist that binds the MD2 “pocket,” completely blocked LPS- and 1Z105-driven, but not UT12-induced, TLR4 dimerization and endocytosis. Furthermore, TLR4 endocytosis is induced in macrophages tolerized by exposure to either LPS or UT12 and is independent of CD14. These data indicate that TLR4 receptor endocytosis and the TRIF-signaling pathway are dissociable and that TLR4 internalization in macrophages can be induced by UT12, 1Z105, and during endotoxin tolerance in the absence of CD14. PMID:26106158

  12. Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis

    PubMed Central

    Fogelgren, Ben; Zuo, Xiaofeng; Buonato, Janine M.; Vasilyev, Aleksandr; Baek, Jeong-In; Choi, Soo Young; Chacon-Heszele, Maria F.; Palmyre, Aurélien; Polgar, Noemi; Drummond, Iain; Park, Kwon Moo; Lazzara, Matthew J.

    2014-01-01

    Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury. PMID:25298525

  13. Endocytosis of Gene Delivery Vectors: From Clathrin-dependent to Lipid Raft-mediated Endocytosis

    PubMed Central

    El-Sayed, Ayman; Harashima, Hideyoshi

    2013-01-01

    The ideal nonviral vector delivers its nucleic acid cargo to a specific intracellular target. Vectors enter cells mainly through endocytosis and are distributed to various intracellular organelles. Recent advances in microscopy, lipidomics, and proteomics confirm that the cell membrane is composed of clusters of lipids, organized in the form of lipid raft domains, together with non-raft domains that comprise a generally disordered lipid milieu. The binding of a nonviral vector to either region can determine the pathway for its endocytic uptake and subsequent intracellular itinerary. Given this model of the cell membrane structure, endocytic pathways should be reclassified in relation to lipid rafts. In this review, we attempt to assess the currently recognized endocytic pathways in mammalian cells. The endocytic pathways are classified in relation to the membrane regions that make up the primary endocytic vesicles. This review covers the well-recognized clathrin-mediated endocytosis (CME), phagocytosis, and macropinocytosis in addition to the less addressed pathways that take place in lipid rafts. These include caveolae-mediated, flotillin-dependent, GTPase regulator associated with focal adhesion kinase-1 (GRAF1)-dependent, adenosine diphosphate-ribosylation factor 6 (Arf6)-dependent, and RhoA-dependent endocytic pathways. We summarize the regulators associated with each uptake pathway and methods for interfering with these regulators are discussed. The fate of endocytic vesicles resulting from each endocytic uptake pathway is highlighted. PMID:23587924

  14. /sup 3/H)-(H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) ((/sup 3/H)CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain

    SciTech Connect

    Hawkins, K.N.; Knapp, R.J.; Lui, G.K.; Gulya, K.; Kazmierski, W.; Wan, Y.P.; Pelton, J.T.; Hruby, V.J.; Yamamura, H.I.

    1989-01-01

    The cyclic, conformationally restricted octapeptide (3H)-(H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) ((3H)CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 10(8) M-1 min-1 and 2.49 x 10(8) M-1 min-1 at 25 and 37 degrees C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10(-2) min-1 and 1.03 x 10(-1) min-1 at 25 and 37 degrees C, respectively. Saturation isotherms of (3H)CTOP binding to rat brain membranes gave apparent Kd values of 0.16 and 0.41 nM at 25 and 37 degrees C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37 degrees C, respectively. (3H)CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on (3H)CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide (3H)-(D-Pen2,D-Pen5)enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific (3H)CTOP binding was inhibited by a large number of opioid or opiate ligands.

  15. Activity profiles of dalargin and its analogues in mu-, delta- and kappa-opioid receptor selective bioassays.

    PubMed

    Pencheva, N; Pospisek, J; Hauzerova, L; Barth, T; Milanov, P

    1999-10-01

    1. To elucidate the structural features ensuring action of [D-Ala2, Leu5]-enkephalyl-Arg (dalargin), a series of dalargin analogues were tested for their effectiveness in depressing electrically-evoked contractions of the guinea-pig myenteric plexus-longitudinal muscle preparations (mu- and kappa-opioid receptors) and the vasa deferentia of the hamster (delta-opioid receptors), mouse (mu-, delta- and kappa-opioid receptors), rat (similar to mu-opioid receptors) and rabbit (kappa-opioid receptors). The naloxone KB values in the myenteric plexus were also obtained. 2. [L-Ala2]-dalargin was 19 times less potent than dalargin, and its pharmacological activity was peptidase-sensitive. The ratio of delta-activity to mu-activity for [L-Ala2]-dalargin was 6.78, and KB was 7.9 nM. This emphasizes the role that D-configuration of Ala2 plays in determining the active folding of dalargin molecule as well as in conferring resistance to peptidases. 3. [Met5]-dalargin was equipotent to dalargin in the myenteric plexus, but was more potent in the vasa deferentia of hamster and mouse (KB=5.5 nM). Leu5 and the interdependence of Leu5 and D-Ala2 are of importance for the selectivity of dalargin for mu-opioid receptors. 4. Dalarginamide was more potent and selective for mu-opioid receptors than dalargin, whilst dalarginethylamide, though equipotent to dalarginamide in the myenteric plexus, was more potent at delta-opioid receptors (KB=5.0 nM). [D-Phe4]-dalarginamide and N-Me-[D-Phe4]-dalarginamide were inactive indicating the contribution of L-configuration of Phe4 to the pharmacological potency of dalargin. 5. N-Me-[L-Phe4]-dalarginamide possessed the highest potency and selectivity for mu-opioid receptors (the ratio of delta-activity to mu-activity was 0.00053; KB=2.6 nM). The CONH2 terminus combined with the N-methylation of L-Phe4 increased the potency and selectivity of dalargin for mu-opioid receptors. PMID:10516634

  16. Does the kappa opioid receptor system contribute to pain aversion?

    PubMed Central

    Cahill, Catherine M.; Taylor, Anna M. W.; Cook, Christopher; Ong, Edmund; Morón, Jose A.; Evans, Christopher J.

    2014-01-01

    The kappa opioid receptor (KOR) and the endogenous peptide-ligand dynorphin have received significant attention due the involvement in mediating a variety of behavioral and neurophysiological responses, including opposing the rewarding properties of drugs of abuse including opioids. Accumulating evidence indicates this system is involved in regulating states of motivation and emotion. Acute activation of the KOR produces an increase in motivational behavior to escape a threat, however, KOR activation associated with chronic stress leads to the expression of symptoms indicative of mood disorders. It is well accepted that KOR can produce analgesia and is engaged in chronic pain states including neuropathic pain. Spinal studies have revealed KOR-induced analgesia in reversing pain hypersensitivities associated with peripheral nerve injury. While systemic administration of KOR agonists attenuates nociceptive sensory transmission, this effect appears to be a stress-induced effect as anxiolytic agents, including delta opioid receptor agonists, mitigate KOR agonist-induced analgesia. Additionally, while the role of KOR and dynorphin in driving the dysphoric and aversive components of stress and drug withdrawal has been well characterized, how this system mediates the negative emotional states associated with chronic pain is relatively unexplored. This review provides evidence that dynorphin and the KOR system contribute to the negative affective component of pain and that this receptor system likely contributes to the high comorbidity of mood disorders associated with chronic neuropathic pain. PMID:25452729

  17. Expression of a dominant interfering dynamin mutant in 3T3L1 adipocytes inhibits GLUT4 endocytosis without affecting insulin signaling.

    PubMed

    Kao, A W; Ceresa, B P; Santeler, S R; Pessin, J E

    1998-09-25

    To examine the role of clathrin-coated vesicle endocytosis in insulin receptor signaling and GLUT4 trafficking, we used recombinant adenovirus to express a dominant interfering mutant of dynamin (K44A/dynamin) in 3T3L1 adipocytes. Functional expression of K44A/dynamin, as measured by inhibition of transferrin receptor internalization, did not affect insulin-stimulated insulin receptor autophosphorylation, Shc tyrosine phosphorylation, or mitogen-activated protein kinase activation. Although the tyrosine phosphorylation of insulin receptor substrate-1 was slightly reduced, correlating with a 25% decrease in insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, insulin-stimulated Akt kinase activation was unaffected. In contrast, expression of K44A/dynamin resulted in the cell-surface accumulation of GLUT4 under basal conditions and an inhibition of GLUT4 endocytosis without affecting insulin-stimulated GLUT4 exocytosis. These data demonstrate that disruption of clathrin-mediated endocytosis does not significantly perturb insulin receptor signal transduction pathways. Furthermore, K44A/dynamin expression causes an accumulation of GLUT4 at the cell surface, suggesting that GLUT4 vesicles exist in at least two distinct intracellular compartments, one that undergoes continuous recycling and a second that is responsive to insulin. PMID:9738014

  18. (/sup 3/H)Ethylketocyclazocine binding to mouse brain membranes: evidence for a kappa opioid receptor type

    SciTech Connect

    Garzon, J.; Sanchez-Blazquez, P.; Lee, N.M.

    1984-10-01

    The binding of the putative kappa agonist ethylketocyclazocine (EKC) to synaptosomal membranes of mouse brain was studied. This benzomorphan was able to bind to different opioid receptors. A portion of this binding was not inhibited by the agonist naloxone, even at high concentrations (10 microM). This population of receptors, to which opioate alkaloids and opiod peptides display very low affinity, is probably the sigma receptor. Another class of binding sites was identified by the simultaneous addition of the selective agonists Sandoz FK-33824 and D-Ala2-D-Leu5-enkephalin, which blocked the access of EKC to mu and delta opioid receptors, respectively, leaving a portion of naloxone-displaceable benzomorphan binding still detectable. Analysis of this remaining binding revealed a small population of receptors of high affinity, the kappa receptor. Therefore, EKC binds to the mu, delta, kappa and sigma receptors in the mouse brain, with similar affinities for the mu and kappa (0.22 and 0.15 nM). These results confirm the existence of a kappa opioid receptor type in the mouse brain.

  19. Involvement of peripheral mu opioid receptors in scratching behavior in mice.

    PubMed

    Yamamoto, Atsuki; Sugimoto, Yukio

    2010-12-15

    Pruritus is a common adverse effect of opioid treatment. However, the mechanism by which pruritus is induced by opioid administration is unclear. In this study, we examined the effects of the intradermal injection of loperamide, a peripherally restricted opioid receptor agonist, on the itch sensation. When injected intradermally into the rostral part of the back in mice, loperamide elicited scratching behavior. We also examined the effects of the selective mu opioid receptor agonist [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin acetate (DAMGO), the selective delta opioid receptor agonist [d-Pen(2,5)]-enkephalin (DPDPE), and the selective kappa opioid receptor agonist U-50488H on scratching behavior in mice in order to determine which subtype is involved in opioid-induced pruritus. Following intradermal injection into the rostral part of the back in mice, DAMGO elicited scratching behavior, while DPDPE and U-50488H did not. This suggests that peripheral mu opioid activation elicits the itch sensation. Next, we focused on the treatment of opioid-induced itch sensation without central adverse effects. Naloxone methiodide is a peripherally restricted opioid receptor antagonist. In the present study, naloxone methiodide significantly suppressed scratching behavior induced by loperamide and DAMGO. These findings suggest that mu opioid receptors play a primary role in peripheral pruritus and that naloxone methiodide may represent a possible remedy for opioid-induced itching. PMID:20863827

  20. Stonin1 mediates endocytosis of the proteoglycan NG2 and regulates focal adhesion dynamics and cell motility

    PubMed Central

    Feutlinske, Fabian; Browarski, Marietta; Ku, Min-Chi; Trnka, Philipp; Waiczies, Sonia; Niendorf, Thoralf; Stallcup, William B.; Glass, Rainer; Krause, Eberhard; Maritzen, Tanja

    2015-01-01

    Cellular functions, ranging from focal adhesion (FA) dynamics and cell motility to tumour growth, are orchestrated by signals cells receive from outside via cell surface receptors. Signalling is fine-tuned by the exo–endocytic cycling of these receptors to control cellular responses such as FA dynamics, which determine cell motility. How precisely endocytosis regulates turnover of the various cell surface receptors remains unclear. Here we identify Stonin1, an endocytic adaptor of unknown function, as a regulator of FA dynamics and cell motility, and demonstrate that it facilitates the internalization of the oncogenic proteoglycan NG2, a co-receptor of integrins and platelet-derived growth factor receptor. Embryonic fibroblasts obtained from Stonin1-deficient mice display a marked surface accumulation of NG2, increased cellular signalling and defective FA disassembly as well as altered cellular motility. These data establish Stonin1 as a specific adaptor for the endocytosis of NG2 and as an important factor for FA dynamics and cell migration. PMID:26437238

  1. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

    PubMed Central

    Zhang, Jifeng; Tan, Minghui; Yin, Yichen; Ren, Bingyu; Jiang, Nannan; Guo, Guoqing; Chen, Yuan

    2015-01-01

    Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca2+ channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV) endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons. PMID:26682072

  2. Vesicle formation and endocytosis: function, machinery, mechanisms, and modeling.

    PubMed

    Parkar, Nihal S; Akpa, Belinda S; Nitsche, Ludwig C; Wedgewood, Lewis E; Place, Aaron T; Sverdlov, Maria S; Chaga, Oleg; Minshall, Richard D

    2009-06-01

    Vesicle formation provides a means of cellular entry for extracellular substances and for recycling of membrane constituents. Mechanisms governing the two primary endocytic pathways (i.e., caveolae- and clathrin-mediated endocytosis, as well as newly emerging vesicular pathways) have become the focus of intense investigation to improve our understanding of nutrient, hormone, and drug delivery, as well as opportunistic invasion of pathogens. In this review of endocytosis, we broadly discuss the structural and signaling proteins that compose the molecular machinery governing endocytic vesicle formation (budding, invagination, and fission from the membrane), with some regard for the specificity observed in certain cell types and species. Important biochemical functions of endocytosis and diseases caused by their disruption also are discussed, along with the structures of key components of endocytic pathways and their known mechanistic contributions. The mechanisms by which principal components of the endocytic machinery are recruited to the plasma membrane, where they interact to induce vesicle formation, are discussed, together with computational approaches used to simulate simplified versions of endocytosis with the hope of clarifying aspects of vesicle formation that may be difficult to determine experimentally. Finally, we pose several unanswered questions intended to stimulate further research interest in the cell biology and modeling of endocytosis. PMID:19113823

  3. Vesicle Formation and Endocytosis: Function, Machinery, Mechanisms, and Modeling

    PubMed Central

    Parkar, Nihal S.; Akpa, Belinda S.; Nitsche, Ludwig C.; Wedgewood, Lewis E.; Place, Aaron T.; Sverdlov, Maria S.; Chaga, Oleg

    2009-01-01

    Abstract Vesicle formation provides a means of cellular entry for extracellular substances and for recycling of membrane constituents. Mechanisms governing the two primary endocytic pathways (i.e., caveolae- and clathrin-mediated endocytosis, as well as newly emerging vesicular pathways) have become the focus of intense investigation to improve our understanding of nutrient, hormone, and drug delivery, as well as opportunistic invasion of pathogens. In this review of endocytosis, we broadly discuss the structural and signaling proteins that compose the molecular machinery governing endocytic vesicle formation (budding, invagination, and fission from the membrane), with some regard for the specificity observed in certain cell types and species. Important biochemical functions of endocytosis and diseases caused by their disruption also are discussed, along with the structures of key components of endocytic pathways and their known mechanistic contributions. The mechanisms by which principal components of the endocytic machinery are recruited to the plasma membrane, where they interact to induce vesicle formation, are discussed, together with computational approaches used to simulate simplified versions of endocytosis with the hope of clarifying aspects of vesicle formation that may be difficult to determine experimentally. Finally, we pose several unanswered questions intended to stimulate further research interest in the cell biology and modeling of endocytosis. Antioxid. Redox Signal. 11, 1301–1312. PMID:19113823

  4. Ankyrin-G Inhibits Endocytosis of Cadherin Dimers.

    PubMed

    Cadwell, Chantel M; Jenkins, Paul M; Bennett, Vann; Kowalczyk, Andrew P

    2016-01-01

    Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Therefore, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. However, the mechanisms that control junctional plasticity are not fully understood. Vascular endothelial cadherin (VE-cadherin) is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β- and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of cadherin and stabilizes it at the plasma membrane. Here we report that induced cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions. PMID:26574545

  5. Raft endocytosis of AMF regulates mitochondrial dynamics through Rac1 signaling and the Gp78 ubiquitin ligase.

    PubMed

    Shankar, Jay; Kojic, Liliana D; St-Pierre, Pascal; Wang, Peter T C; Fu, Min; Joshi, Bharat; Nabi, Ivan R

    2013-08-01

    Gp78 is a cell surface receptor that also functions as an E3 ubiquitin ligase in the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. The Gp78 ligand, the glycolytic enzyme phosphoglucose isomerase (PGI; also called autocrine motility factor, AMF), functions as a cytokine upon secretion by tumor cells. AMF is internalized through a PI3K- and dynamin-dependent raft endocytic pathway to the smooth ER; however, the relationship between AMF and Gp78 ubiquitin ligase activity remains unclear. AMF uptake to the smooth ER is inhibited by the dynamin inhibitor, dynasore, is reduced in Gp78 knockdown cells and induces the dynamin-dependent downregulation of its cell surface receptor. AMF uptake is Rac1-dependent and is inhibited by expression of dominant-negative Rac1 and the Rac1 inhibitor NSC23766, and is therefore distinct from Cdc42- and RhoA-dependent raft endocytic pathways. AMF stimulates Rac1 activation, but this is reduced by dynasore treatment and is absent in Gp78-knockdown cells; therefore, AMF activities require Gp78-mediated endocytosis. AMF also prevents Gp78-induced degradation of the mitochondrial fusion proteins, mitofusin 1 and 2 in a dynamin-, Rac1- and phosphoinositide 3-kinase (PI3K)-dependent manner. Gp78 induces mitochondrial clustering and fission in a manner dependent on GP78 ubiquitin ligase activity, and this is also reversed by uptake of AMF. The raft-dependent endocytosis of AMF, therefore, promotes Rac1-PI3K signaling that feeds back to promote AMF endocytosis and also inhibits the ability of Gp78 to target the mitofusins for degradation, thereby preventing Gp78-dependent mitochondrial fission. Through regulation of an ER-localized ubiquitin ligase, the raft-dependent endocytosis of AMF represents an extracellular regulator of mitochondrial fusion and dynamics. PMID:23690547

  6. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells

    PubMed Central

    Coffey, Sam; Costacou, Tina; Orchard, Trevor; Erkan, Elif

    2015-01-01

    Diabetes mellitus (DM) has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA) has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt) in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160) and cytoplasmic tail of megalin. Mice with type 1 DM (T1D) displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications) study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN) at an earlier stage. PMID:26465605

  7. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    PubMed

    Coffey, Sam; Costacou, Tina; Orchard, Trevor; Erkan, Elif

    2015-01-01

    Diabetes mellitus (DM) has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA) has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt) in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160) and cytoplasmic tail of megalin. Mice with type 1 DM (T1D) displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications) study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN) at an earlier stage. PMID:26465605

  8. Biotinylated human beta-endorphins as probes for the opioid receptor.

    PubMed

    Hochhaus, G; Gibson, B W; Sadée, W

    1988-01-01

    The reaction of human beta-endorphin and biotinyl N-hydroxysuccinimide with or without spacer arm, afforded a series of products that were separated by high performance liquid chromatography (HPLC). Liquid secondary ion mass spectrometry of the biotinylated products and their tryptic digests produced abundant protonated molecular ions (MH+), which specified the number and location of biotinylation. Between 1 and 4 biotinyl residues were incorporated per human beta-endorphin molecule, at Lys-9, -19, -24, -28, and -29, but not at the amino-terminal Tyr-1. Three HPLC fractions were isolated for receptor binding studies with monobiotinylation of Lys-9 (B1 beta and B1X beta; X = C6 spacer arm), Lys-19 (B1 gamma), and a mixture of Lys-24, Lys-28, and Lys-29 derivatives (B1 alpha, BX1 alpha). All derivatives displayed tight binding to avidin, and no dissociation from avidin was detectable over several hours at 0 degrees C for the derivatives (BX1 alpha) tested. IC50 values for binding to mu and delta opioid receptor sites were 3-8 times higher for monobiotinylated derivatives than for the parent human beta-endorphin (IC50,mu = 1.5 nM, IC50,delta = 1.3 nM). Association with avidin decreased opioid receptor affinities for the C6 spacer derivative biotinylated at position Lys-9, which is close to the (1-5) enkephalin receptor region. In contrast, avidin did not affect or even increased apparent affinities to mu and delta sites for derivatives biotinylated at the alpha-helical part of the molecule (Lys-19, -24, -28, and -29). Thus, when bound to avidin, the biotinylated human beta-endorphin derivatives with spacer arm (BX1 alpha), substituted near the carboxyl terminal (Lys-24, -28, and -29), displayed mu binding affinities equal to and delta binding affinities only four times lower than underivatized human beta-endorphin. Biotinylated human beta-endorphins also bound to low affinity nonopioid binding sites on NG-108-15 cells; however, affinities to these sites were

  9. Endocytosis and exocytosis of nanoparticles in mammalian cells

    PubMed Central

    Oh, Nuri; Park, Ji-Ho

    2014-01-01

    Engineered nanoparticles that can be injected into the human body hold tremendous potential to detect and treat complex diseases. Understanding of the endocytosis and exocytosis mechanisms of nanoparticles is essential for safe and efficient therapeutic application. In particular, exocytosis is of significance in the removal of nanoparticles with drugs and contrast agents from the body, while endocytosis is of great importance for the targeting of nanoparticles in disease sites. Here, we review the recent research on the endocytosis and exocytosis of functionalized nanoparticles based on various sizes, shapes, and surface chemistries. We believe that this review contributes to the design of safe nanoparticles that can efficiently enter and leave human cells and tissues. PMID:24872703

  10. Phosphoinositides, kinases and adaptors coordinating endocytosis in Trypanosoma brucei

    PubMed Central

    Manna, Paul T; Field, Mark C

    2015-01-01

    In the kinetoplastid parasite Trypanosoma brucei clathrin-mediated endocytosis is essential for survival and aids immune evasion in the mammalian host. The formation of endocytic clathrin coated vesicles in T. brucei is via a unique mechanism owing to an evolutionarily recent loss of the adaptor protein (AP)2 complex, a central hub in endocytic vesicle assembly. Despite this loss, recent studies examining endocytic clathrin coat assembly have highlighted a high degree of conservation between trypanosomes and their mammalian hosts. In particular phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and its putative effectors, TbCALM and TbEpsinR, are central to clathrin-mediated endocytosis in the trypanosome, just as they are in animal cells. In addition to providing insights into the cell biology of T. brucei, these studies also suggest an ancient, possibly pan-eukaryotic connection between PtdIns(4,5)P2 and endocytosis. PMID:27064836

  11. Imaging the Dynamics of Endocytosis in Live Mammalian Tissues

    PubMed Central

    Weigert, Roberto

    2014-01-01

    In mammalian cells, endocytosis plays a pivotal role in regulating several basic cellular functions. Up to now, the dynamics and the organization of the endocytic pathways have been primarily investigated in reductionist model systems such as cell and organ cultures. Although these experimental models have been fully successful in unraveling the endocytic machinery at a molecular level, our understanding of the regulation and the role of endocytosis in vivo has been limited. Recently, advancements in intravital microscopy have made it possible to extend imaging in live animals to subcellular structures, thus revealing new aspects of the molecular machineries regulating membrane trafficking that were not previously appreciated in vitro. Here, we focus on the use of intravital microscopy to study endocytosis in vivo, and discuss how this approach will allow addressing two fundamental questions: (1) how endocytic processes are organized in mammalian tissues, and (2) how they contribute to organ physiopathology. PMID:24691962

  12. SUMOylation of Syntaxin1A regulates presynaptic endocytosis

    PubMed Central

    Craig, Tim J.; Anderson, Dina; Evans, Ashley J.; Girach, Fatima; Henley, Jeremy M.

    2015-01-01

    Neurotransmitter release from the presynaptic terminal is under very precise spatial and temporal control. Following neurotransmitter release, synaptic vesicles are recycled by endocytosis and refilled with neurotransmitter. During the exocytosis event leading to release, SNARE proteins provide most of the mechanical force for membrane fusion. Here, we show one of these proteins, Syntaxin1A, is SUMOylated near its C-terminal transmembrane domain in an activity-dependent manner. Preventing SUMOylation of Syntaxin1A reduces its interaction with other SNARE proteins and disrupts the balance of synaptic vesicle endo/exocytosis, resulting in an increase in endocytosis. These results indicate that SUMOylation regulates the emerging role of Syntaxin1A in vesicle endocytosis, which in turn, modulates neurotransmitter release and synaptic function. PMID:26635000

  13. Diacylglycerol Guides the Hopping of Clathrin-Coated Pits along Microtubules for Exo-Endocytosis Coupling.

    PubMed

    Yuan, Tianyi; Liu, Lin; Zhang, Yongdeng; Wei, Lisi; Zhao, Shiqun; Zheng, Xiaolu; Huang, Xiaoshuai; Boulanger, Jerome; Gueudry, Charles; Lu, Jingze; Xie, Lihan; Du, Wen; Zong, Weijian; Yang, Lu; Salamero, Jean; Liu, Yanmei; Chen, Liangyi

    2015-10-12

    Many receptor-mediated endocytic processes are mediated by constitutive budding of clathrin-coated pits (CCPs) at spatially randomized sites before slowly pinching off from the plasma membrane (60-100 s). In contrast, clathrin-mediated endocytosis (CME) coupled with regulated exocytosis in excitable cells occurs at peri-exocytic sites shortly after vesicle fusion (∼10 s). The molecular mechanism underlying this spatiotemporal coupling remains elusive. We show that coupled endocytosis makes use of pre-formed CCPs, which hop to nascent fusion sites nearby following vesicle exocytosis. A dynamic cortical microtubular network, anchored at the cell surface by the cytoplasmic linker-associated protein on microtubules and the LL5β/ELKS complex on the plasma membrane, provides the track for CCP hopping. Local diacylglycerol gradients generated upon exocytosis guide the direction of hopping. Overall, the CCP-cytoskeleton-lipid interaction demonstrated here mediates exocytosis-coupled fast recycling of both plasma membrane and vesicular proteins, and it is required for the sustained exocytosis during repetitive stimulations. PMID:26439397

  14. Increased expression of endocytosis-Related proteins in rat hippocampus following 10-day electroconvulsive seizure treatment.

    PubMed

    Enomoto, Shingo; Shimizu, Kunio; Nibuya, Masashi; Toda, Hiroyuki; Yoshino, Aihide; Suzuki, Eiji; Kondo, Takashi; Fukuda, Hiroshi

    2016-06-15

    Although electroconvulsive therapy (ECT) is clinically used for severe depression and drug-resistant Parkinson's disease, its exact biological background and mechanism have not yet been fully elucidated. Two potential explanations have been presented so far to explain the increased neuroplastic and resilient profiles of multiple ECT administrations. One is the alteration of central neurotransmitter receptor densities and the other is the expressional upregulation of brain derived neurotrophic factor in various brain regions with enhanced hippocampal neurogenesis and mossy fiber sprouting. In the present report, western blot analyses revealed significantly upregulated expression of various endocytosis-related proteins following 10-day electroconvulsive seizure (ECS) treatment in rat hippocampal homogenates and hippocampal lipid raft fractions extracted using an ultracentrifugation procedure. Upregulated proteins included endocytosis-related scaffolding proteins (caveolin-1, flotillin-1, and heavy and light chains of clathrin) and small GTPases (Rab5, Rab7, Rab11, and Rab4) specifically expressed on various types of endosomes. Two scaffolding proteins, caveolin-1 and flotillin-1, were also increased in the lipid raft fraction. Together with our previous finding of increased autophagy-related proteins in the hippocampal region, the present results suggest membrane trafficking machinery is enhanced following 10-day ECS treatment. We consider that the membrane trafficking machinery that transports functional proteins in the neuronal cells and from or into the synaptic membranes is one of the new candidates supporting the cellular and behavioral neuroplastic profiles of ECS treatments in animal experiments and ECT administrations in clinical settings. PMID:27177725

  15. Synaptic vesicles are “primed” for fast clathrin-mediated endocytosis at the ribbon synapse

    PubMed Central

    Pelassa, Ilaria; Zhao, CongJian; Pasche, Mathias; Odermatt, Benjamin; Lagnado, Leon

    2014-01-01

    Retrieval of synaptic vesicles can occur 1–10 s after fusion, but the role of clathrin during this process has been unclear because the classical mode of clathrin-mediated endocytosis (CME) is an order of magnitude slower, as during retrieval of surface receptors. Classical CME is thought to be rate-limited by the recruitment of clathrin, which raises the question: how is clathrin recruited during synaptic vesicle recycling? To investigate this question we applied total internal reflection fluorescence microscopy (TIRFM) to the synaptic terminal of retinal bipolar cells expressing fluorescent constructs of clathrin light-chain A. Upon calcium influx we observed a fast accumulation of clathrin within 100 ms at the periphery of the active zone. The subsequent loss of clathrin from these regions reflected endocytosis because the application of a potent clathrin inhibitor Pitstop2 dramatically slowed down this phase by ~3 fold. These results indicate that clathrin-dependent retrieval of synaptic vesicles is unusually fast, most probably because of a “priming” step involving a state of association of clathrin with the docked vesicle and with the endosomes and cisternae surrounding the ribbons. Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) showed that the majority of clathrin is moving with the same kinetics as synaptic vesicle proteins. Together, these results indicate that the fast endocytic mechanism operating to retrieve synaptic vesicles differs substantially from the classical mode of CME operating via formation of a coated pit. PMID:25520613

  16. Kinetics of cellular uptake of viruses and nanoparticles via clathrin-mediated endocytosis.

    PubMed

    Banerjee, Anand; Berezhkovskii, Alexander; Nossal, Ralph

    2016-02-01

    Several viruses exploit clathrin-mediated endocytosis to gain entry into host cells. This process is also used extensively in biomedical applications to deliver nanoparticles (NPs) to diseased cells. The internalization of these nano-objects is controlled by the assembly of a clathrin-containing protein coat on the cytoplasmic side of the plasma membrane, which drives the invagination of the membrane and the formation of a cargo-containing endocytic vesicle. Current theoretical models of receptor-mediated endocytosis of viruses and NPs do not explicitly take coat assembly into consideration. In this paper we study cellular uptake of viruses and NPs with a focus on coat assembly. We characterize the internalization process by the mean time between the binding of a particle to the membrane and its entry into the cell. Using a coarse-grained model which maps the stochastic dynamics of coat formation onto a one-dimensional random walk, we derive an analytical formula for this quantity. A study of the dependence of the mean internalization time on NP size shows that there is an upper bound above which this time becomes extremely large, and an optimal size at which it attains a minimum. Our estimates of these sizes compare well with experimental data. We also study the sensitivity of the obtained results on coat parameters to identify factors which significantly affect the internalization kinetics. PMID:26871680

  17. Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2.

    PubMed

    De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

    2016-02-01

    Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions. PMID:26779610

  18. Kinetics of cellular uptake of viruses and nanoparticles via clathrin-mediated endocytosis

    NASA Astrophysics Data System (ADS)

    Banerjee, Anand; Berezhkovskii, Alexander; Nossal, Ralph

    2016-02-01

    Several viruses exploit clathrin-mediated endocytosis to gain entry into host cells. This process is also used extensively in biomedical applications to deliver nanoparticles (NPs) to diseased cells. The internalization of these nano-objects is controlled by the assembly of a clathrin-containing protein coat on the cytoplasmic side of the plasma membrane, which drives the invagination of the membrane and the formation of a cargo-containing endocytic vesicle. Current theoretical models of receptor-mediated endocytosis of viruses and NPs do not explicitly take coat assembly into consideration. In this paper we study cellular uptake of viruses and NPs with a focus on coat assembly. We characterize the internalization process by the mean time between the binding of a particle to the membrane and its entry into the cell. Using a coarse-grained model which maps the stochastic dynamics of coat formation onto a one-dimensional random walk, we derive an analytical formula for this quantity. A study of the dependence of the mean internalization time on NP size shows that there is an upper bound above which this time becomes extremely large, and an optimal size at which it attains a minimum. Our estimates of these sizes compare well with experimental data. We also study the sensitivity of the obtained results on coat parameters to identify factors which significantly affect the internalization kinetics.

  19. Endocytosis is essential for dynamic translocation of a syntaxin 1 orthologue during fission yeast meiosis

    PubMed Central

    Kashiwazaki, Jun; Yamasaki, Yuriko; Itadani, Akiko; Teraguchi, Erika; Maeda, Yukari; Shimoda, Chikashi; Nakamura, Taro

    2011-01-01

    Syntaxin is a component of the target soluble N-ethylmaleimide–sensitive factor attachment protein receptor complex, which is responsible for fusion of membrane vesicles at the target membrane. The fission yeast syntaxin 1 orthologue Psy1 is essential for both vegetative growth and spore formation. During meiosis, Psy1 disappears from the plasma membrane (PM) and dramatically relocalizes on the nascent forespore membrane, which becomes the PM of the spore. Here we report the molecular details and biological significance of Psy1 relocalization. We find that, immediately after meiosis I, Psy1 is selectively internalized by endocytosis. In addition, a meiosis-specific signal induced by the transcription factor Mei4 seems to trigger this internalization. The internalization of many PM proteins is facilitated coincident with the initiation of meiosis, whereas Pma1, a P-type ATPase, persists on the PM even during the progression of meiosis II. Ergosterol on the PM is also important for the internalization of PM proteins in general during meiosis. We consider that during meiosis in Schizosaccharomyces pombe cells, the characteristics of endocytosis change, thereby facilitating internalization of Psy1 and accomplishing sporulation. PMID:21832151

  20. Endocytosis and toxicity of clostridial binary toxins depend on a clathrin-independent pathway regulated by Rho-GDI.

    PubMed

    Gibert, Maryse; Monier, Marie-Noëlle; Ruez, Richard; Hale, Martha L; Stiles, Bradley G; Benmerah, Alexandre; Johannes, Ludger; Lamaze, Christophe; Popoff, Michel R

    2011-01-01

    Clostridial binary toxins, such as Clostridium perfringens Iota and Clostridium botulinum C2, are composed of a binding protein (Ib and C2II respectively) that recognizes distinct membrane receptors and mediates internalization of a catalytic protein (Ia and C2-I respectively) with ADP-ribosyltransferase activity that disrupts the actin cytoskeleton. We show here that the endocytic pathway followed by these toxins is independent of clathrin but requires the activity of dynamin and is regulated by Rho-GDI. This endocytic pathway is similar to a recently characterized clathrin-independent pathway followed by the interleukin-2 (IL2) receptor. We found indeed that Ib and C2II colocalized intracellularly with the IL2 receptor but not the transferrin receptor after different times of endocytosis. Accordingly, the intracellular effects of Iota and C2 on the cytoskeleton were inhibited by inactivation of dynamin or by Rho-GDI whereas inhibitors of clathrin-dependent endocytosis had no protective effect. PMID:20846184

  1. Trafficking of Kainate Receptors

    PubMed Central

    Pahl, Steffen; Tapken, Daniel; Haering, Simon C.; Hollmann, Michael

    2014-01-01

    Ionotropic glutamate receptors (iGluRs) mediate the vast majority of excitatory neurotransmission in the central nervous system of vertebrates. In the protein family of iGluRs, kainate receptors (KARs) comprise the probably least well understood receptor class. Although KARs act as key players in the regulation of synaptic network activity, many properties and functions of these proteins remain elusive until now. Especially the precise pre-, extra-, and postsynaptic localization of KARs plays a critical role for neuronal function, as an unbalanced localization of KARs would ultimately lead to dysregulated neuronal excitability. Recently, important advances in the understanding of the regulation of surface expression, function, and agonist-dependent endocytosis of KARs have been achieved. Post-translational modifications like PKC-mediated phosphorylation and SUMOylation have been reported to critically influence surface expression and endocytosis, while newly discovered auxiliary proteins were shown to shape the functional properties of KARs. PMID:25141211

  2. Bladder Uptake of Liposomes after Intravesical Administration Occurs by Endocytosis

    PubMed Central

    Rajaganapathy, Bharathi Raja; Chancellor, Michael B.; Nirmal, Jayabalan; Dang, Loan; Tyagi, Pradeep

    2015-01-01

    Liposomes have been used therapeutically and as a local drug delivery system in the bladder. However, the exact mechanism for the uptake of liposomes by bladder cells is unclear. In the present study, we investigated the role of endocytosis in the uptake of liposomes by cultured human UROtsa cells of urothelium and rat bladder. UROtsa cells were incubated in serum-free media with liposomes containing colloidal gold particles for 2 h either at 37°C or at 4°C. Transmission Electron Microscopy (TEM) images of cells incubated at 37°C found endocytic vesicles containing gold inside the cells. In contrast, only extracellular binding was noticed in cells incubated with liposomes at 4°C. Absence of liposome internalization at 4°C indicates the need of energy dependent endocytosis as the primary mechanism of entry of liposomes into the urothelium. Flow cytometry analysis revealed that the uptake of liposomes at 37°C occurs via clathrin mediated endocytosis. Based on these observations, we propose that clathrin mediated endocytosis is the main route of entry for liposomes into the urothelial layer of the bladder and the findings here support the usefulness of liposomes in intravesical drug delivery. PMID:25811468

  3. Role of turgor pressure in endocytosis in fission yeast

    PubMed Central

    Basu, Roshni; Munteanu, Emilia Laura; Chang, Fred

    2014-01-01

    Yeast and other walled cells possess high internal turgor pressure that allows them to grow and survive in the environment. This turgor pressure, however, may oppose the invagination of the plasma membrane needed for endocytosis. Here we study the effects of turgor pressure on endocytosis in the fission yeast Schizosaccharomyces pombe by time-lapse imaging of individual endocytic sites. Decreasing effective turgor pressure by addition of sorbitol to the media significantly accelerates early steps in the endocytic process before actin assembly and membrane ingression but does not affect the velocity or depth of ingression of the endocytic pit in wild-type cells. Sorbitol also rescues endocytic ingression defects of certain endocytic mutants and of cells treated with a low dose of the actin inhibitor latrunculin A. Endocytosis proceeds after removal of the cell wall, suggesting that the cell wall does not contribute mechanically to this process. These studies suggest that endocytosis is governed by a mechanical balance between local actin-dependent inward forces and opposing forces from high internal turgor pressure on the plasma membrane. PMID:24403609

  4. Opioid receptor selectivity profile change via isosterism for 14-O-substituted naltrexone derivatives.

    PubMed

    Zhang, Yan; Elbegdorj, Orgil; Yuan, Yunyun; Beletskaya, Irina O; Selley, Dana E

    2013-07-01

    Isosterism is commonly used in drug discovery and development to address stability, selectivity, toxicity, pharmacokinetics, and efficacy issues. A series of 14-O-substituted naltrexone derivatives were identified as potent mu opioid receptor (MOR) antagonists with improved selectivity over the kappa opioid receptor (KOR) and the delta opioid receptor (DOR), compared to naltrexone. Since esters are not metabolically very stable under typical physiological conditions, their corresponding amide analogs were thus synthesized and biologically evaluated. Unlike their isosteres, most of these novel ligands seem to be dually selective for the MOR and the KOR over the DOR. The restricted flexibility of the amide bond linkage might be responsible for their altered selectivity profile. However, the majority of the 14-N-substituted naltrexone derivatives produced marginal or no MOR stimulation in the (35)S-GTP[γS] assay, which resembled their ester analogs. The current study thus indicated that the 14-substituted naltrexone isosteres are not bioisosteres since they have distinctive pharmacological profile with the regard to their opioid receptor binding affinity and selectivity. PMID:23721804

  5. Initiation of synapse formation by Wnt-induced MuSK endocytosis

    PubMed Central

    Gordon, Laura R.; Gribble, Katherine D.; Syrett, Camille M.; Granato, Michael

    2012-01-01

    In zebrafish, the MuSK receptor initiates neuromuscular synapse formation by restricting presynaptic growth cones and postsynaptic acetylcholine receptors (AChRs) to the center of skeletal muscle cells. Increasing evidence suggests a role for Wnts in this process, yet how muscle cells respond to Wnt signals is unclear. Here, we show that in vivo, wnt11r and wnt4a initiate MuSK translocation from muscle membranes to recycling endosomes and that this transition is crucial for AChR accumulation at future synaptic sites. Moreover, we demonstrate that components of the planar cell polarity pathway colocalize to recycling endosomes and that this localization is MuSK dependent. Knockdown of several core components disrupts MuSK translocation to endosomes, AChR localization and axonal guidance. We propose that Wnt-induced trafficking of the MuSK receptor to endosomes initiates a signaling cascade to align pre- with postsynaptic elements. Collectively, these findings suggest a general mechanism by which Wnt signals shape synaptic connectivity through localized receptor endocytosis. PMID:22318632

  6. Clathrin-mediated endocytosis of FITC-albumin in alveolar type II epithelial cell line RLE-6TN.

    PubMed

    Yumoto, Ryoko; Nishikawa, Hiromi; Okamoto, Miho; Katayama, Hirokazu; Nagai, Junya; Takano, Mikihisa

    2006-05-01

    We examined mechanisms of FITC-albumin uptake by alveolar type II epithelial cells using cultured RLE-6TN cells. Alkaline phosphatase activity and the expression of cytokeratin 19 mRNA, which are characteristic features of alveolar type II epithelial cells, were detected in RLE-6TN cells. The uptake of FITC-albumin by the cells was time and temperature dependent and showed the saturation kinetics of high- and low-affinity transport systems. FITC-albumin uptake was inhibited by native albumin, by chemically modified albumin, and by metabolic inhibitors and bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase. Confocal laser scanning microscopic analysis after FITC-albumin uptake showed punctate localization of fluorescence in the cells, which was partly localized in lysosomes. FITC-albumin taken up by the cells gradually degraded over time, as shown by fluoroimage analyzer after SDS-PAGE. The uptake of FITC-albumin by RLE-6TN cells was not inhibited by nystatin, indomethacin, or methyl-beta-cyclodextrin (inhibitors of caveolae-mediated endocytosis) but was inhibited by phenylarsine oxide and chlorpromazine (inhibitors of clathrin-mediated endocytosis) in a concentration-dependent manner. Uptake was also inhibited by potassium depletion and hypertonicity, conditions known to inhibit clathrin-mediated endocytosis. These results indicate that the uptake of FITC-albumin in cultured alveolar type II epithelial cells, RLE-6TN, is mediated by clathrin-mediated but not by caveolae-mediated endocytosis, and intracellular FITC-albumin is gradually degraded in lysosomes. Possible receptors involved in this endocytic system are discussed. PMID:16361359

  7. Methods to study endocytic trafficking of the EGF receptor

    PubMed Central

    Pinilla-Macua, Itziar; Sorkin, Alexander

    2016-01-01

    Endocytosis and postendocytic sorting of epidermal growth factor (EGF) receptor (EGFR) are the major regulators of EGFR signaling. EGFR endocytosis and ubiquitin-dependent lysosomal targeting are also considered to be the prototypic experimental system for studying the molecular mechanisms of stimulus-induced and constitutive endocytic trafficking. Therefore, elucidation of the mechanisms of EGFR endocytosis and its regulation of the signaling network is essential not only for better understanding of the EGFR biology but also for defining general regulatory principles in the endocytosis system. Comprehensive analysis of these mechanisms requires quantitative and physiologically relevant methodological approaches for measuring the rates of EGFR internalization, degradation, and recycling. Basic experimental protocols described in this chapter cover a combination of single-cell microscopy and biochemical methods that are used to follow EGF-induced endocytosis of EGFR in real time, measure the kinetic rate parameters of EGFR internalization and recycling, and analyze EGF-dependent ubiquitination and degradation of EGFR. PMID:26360045

  8. Mechanism of α-1 antitrypsin endocytosis by lung endothelium

    PubMed Central

    Sohrab, Sadaf; Petrusca, Daniela N.; Lockett, Angelia D.; Schweitzer, Kelly S.; Rush, Natalia I.; Gu, Yuan; Kamocki, Krzysztof; Garrison, Jana; Petrache, Irina

    2009-01-01

    The integrity of lung alveoli is maintained by proper circulating levels of α-1 antitrypsin (A1AT). Next to cigarette smoking, A1AT deficiency is a major risk factor for lung emphysema development. We recently reported that in addition to neutralizing neutrophil elastases in the extracellular compartment, A1AT is internalized by lung endothelial cells and inhibits apoptosis. We hypothesized that the intracellular uptake of A1AT by endothelial cells may be required for its protective function; therefore, we studied the mechanisms of A1AT internalization by primary rat lung microvascular endothelial cells and the effect of cigarette smoke on this process both in vitro and in vivo (in mice). Purified A1AT was taken up intracellularly by endothelial cells in a time-dependent, dose-dependent, and conformer-specific manner and was detected in the cytoplasm of endothelial cells of nondiseased human lung sections. Despite a critical role for caveoli in endothelial cell endocytosis in general, specific inhibition of clathrin-mediated, but not caveoli-mediated, endocytosis profoundly decreased A1AT internalization and reversed the A1AT’s antiapoptotic action. Further more, A1AT associated with clathrin heavy chains, but not with caveolin-1 in the plasma membrane fraction of endothelial cells. Interestingly, cigarette smoke exposure significantly inhibited A1AT uptake both in endothelial cells and in the mouse lung and altered the intracellular distribution of clathrin heavy chains. Our results suggest that clathrin-mediated endocytosis regulates A1AT intracellular function in the lung endothelium and may be an important determinant of the serpin’s protection against developing cigarette smoke-induced emphysema. Sohrab, S., Petrusca, D. N., Lockett, A. D., Schweitzer, K. S., Rush, N. I., Gu, Y., Kamocki, K., Garrison, J., Petrache, I. Mechanism of α-1 antitrypsin endocytosis by lung endothelium. PMID:19423638

  9. Statin-sensitive endocytosis of albumin by glomerular podocytes.

    PubMed

    Eyre, Jeanette; Ioannou, Kyriakos; Grubb, Blair D; Saleem, Moin A; Mathieson, Peter W; Brunskill, Nigel J; Christensen, Erik I; Topham, Peter S

    2007-02-01

    Glomerular podocytes are critical regulators of glomerular permeability via the slit diaphragm and may play a role in cleaning the glomerular filter. Whether podocytes are able to endocytose proteins is uncertain. We studied protein endocytosis in conditionally immortalized mouse and human podocytes using FITC-albumin by direct quantitative assay and by fluorescence microscopy and electron microscopy in mouse podocytes. Furthermore, in vivo uptake was studied in human, rat, and mouse podocytes. Both mouse and human podocytes displayed specific one-site binding for FITC-albumin with K(d) of 0.91 or 0.44 mg/ml and B(max) of 3.15 or 0.81 microg/mg cell protein, respectively. In addition, they showed avid endocytosis of FITC-albumin with K(m) of 9.48 or 4.5 mg/ml and V(max) of 474.3 or 97.4 microg.mg cell protein(-1).h(-1), respectively. Immunoglobulin and transferrin were inefficient competitors of this process, indicating some specificity for albumin. Accumulation of endocytosed albumin could be demonstrated in intracellular vesicles by fluorescence confocal microscopy and electron microscopy. Endocytosis was sensitive to pretreatment with simvastatin. In vivo accumulation of albumin was found in all three species but was most pronounced in the rat. We conclude that podocytes are able to endocytose protein in a statin-sensitive manner. This function is likely to be highly significant in health and disease. In addition, protein endocytosis by podocytes may represent a useful, measurable phenotypic characteristic against which potentially injurious or beneficial interventions can be assessed. PMID:17032937

  10. Tubular proteinuria in patients with HNF1α mutations: HNF1α drives endocytosis in the proximal tubule.

    PubMed

    Terryn, Sara; Tanaka, Karo; Lengelé, Jean-Philippe; Olinger, Eric; Dubois-Laforgue, Danièle; Garbay, Serge; Kozyraki, Renata; Van Der Smissen, Patrick; Christensen, Erik I; Courtoy, Pierre J; Bellanné-Chantelot, Christine; Timsit, José; Pontoglio, Marco; Devuyst, Olivier

    2016-05-01

    Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor expressed in the liver, pancreas, and proximal tubule of the kidney. Mutations of HNF1α cause an autosomal dominant form of diabetes mellitus (MODY-HNF1A) and tubular dysfunction. To gain insights into the role of HNF1α in the proximal tubule, we analyzed Hnf1a-deficient mice. Compared with wild-type littermates, Hnf1a knockout mice showed low-molecular-weight proteinuria and a 70% decrease in the uptake of β2-microglobulin, indicating a major endocytic defect due to decreased expression of megalin/cubilin receptors. We identified several binding sites for HNF1α in promoters of Lrp2 and Cubn genes encoding megalin and cubilin, respectively. The functional interaction of HNF1α with these promoters was shown in C33 epithelial cells lacking endogenous HNF1α. Defective receptor-mediated endocytosis was confirmed in proximal tubule cells from these knockout mice and could be rescued by transfection of wild-type but not mutant HNF1α. Transfection of human proximal tubule HK2 cells with HNF1α was able to upregulate megalin and cubilin expression and to increase endocytosis of albumin. Low-molecular-weight proteinuria was consistently detected in individuals with HNF1A mutations compared with healthy controls and patients with non-MODY-HNF1A diabetes mellitus. Thus, HNF1α plays a key role in the constitutive expression of megalin and cubilin, hence regulating endocytosis in the proximal tubule of the kidney. These findings provide new insight into the renal phenotype of individuals with mutations of HNF1A. PMID:27083284

  11. Chemokine receptor internalization and intracellular trafficking.

    PubMed

    Neel, Nicole F; Schutyser, Evemie; Sai, Jiqing; Fan, Guo-Huang; Richmond, Ann

    2005-12-01

    The internalization and intracellular trafficking of chemokine receptors have important implications for the cellular responses elicited by chemokine receptors. The major pathway by which chemokine receptors internalize is the clathrin-mediated pathway, but some receptors may utilize lipid rafts/caveolae-dependent internalization routes. This review discusses the current knowledge and controversies regarding these two different routes of endocytosis. The functional consequences of internalization and the regulation of chemokine receptor recycling will also be addressed. Modifications of chemokine receptors, such as palmitoylation, ubiquitination, glycosylation, and sulfation, may also impact trafficking, chemotaxis and signaling. Finally, this review will cover the internalization and trafficking of viral and decoy chemokine receptors. PMID:15998596

  12. Translocation and Endocytosis for Cell-penetrating Peptide Internalization

    PubMed Central

    Jiao, Chen-Yu; Delaroche, Diane; Burlina, Fabienne; Alves, Isabel D.; Chassaing, Gérard; Sagan, Sandrine

    2009-01-01

    Cell-penetrating peptides (CPPs) share the property of cellular internalization. The question of how these peptides reach the cytoplasm of cells is still widely debated. Herein, we have used a mass spectrometry-based method that enables quantification of internalized and membrane-bound peptides. Internalization of the most used CPP was studied at 37 °C (endocytosis and translocation) and 4 °C (translocation) in wild type and proteoglycan-deficient Chinese hamster ovary cells. Both translocation and endocytosis are internalization pathways used by CPP. The choice of one pathway versus the other depends on the peptide sequence (not the number of positive changes), the extracellular peptide concentration, and the membrane components. There is no relationship between the high affinity of these peptides for the cell membrane and their internalization efficacy. Translocation occurs at low extracellular peptide concentration, whereas endocytosis, a saturable and cooperative phenomenon, is activated at higher concentrations. Translocation operates in a narrow time window, which implies a specific lipid/peptide co-import in cells. PMID:19833724

  13. Signaling induced by hop/STI-1 depends on endocytosis

    SciTech Connect

    Americo, Tatiana A.; Chiarini, Luciana B.; Linden, Rafael . E-mail: rlinden@biof.ufrj.br

    2007-06-29

    The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrP{sup C}), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrP{sup C} and activation of the Erk pathway. We tested whether clathrin-mediated endocytosis affects signaling induced by hop/STI-1. Both hyperosmolarity induced by sucrose and monodansyl-cadaverine blocked Erk activity induced by hop/STI-1, without affecting the high basal Akt activity typical of A172. The endocytosis inhibitors also affected the sub-cellular distribution of phosphorylated Erk, consistent with blockade of the latter's activity. The data indicate that signaling induced by hop/STI-1 depends on endocytosis. These findings are consistent with a role of sub-cellular trafficking in signal transduction following engagement by PrP{sup C} by ligands such as hop/STI-1, and may help help unravel both the functions of the prion protein, as well as possible loss-of-function components of prion diseases.

  14. Cholesterol Regulates Multiple Forms of Vesicle Endocytosis at a Mammalian Central Synapse

    PubMed Central

    Yue, Hai-Yuan; Xu, Jianhua

    2015-01-01

    Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from nonspecific effects after cholesterol manipulation. Furthermore, it is unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase (COase) or methyl-β-cyclodextrin (MCD) impaired three different forms of endocytosis, i.e., slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca2+ channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of MCD reduced exocytosis, mainly by decreasing the readily releasable pool (RRP) and the vesicle replenishment after RRP depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses. PMID:25893258

  15. Alpha-adaptin, a marker for endocytosis, is expressed in complex patterns during Drosophila development.

    PubMed Central

    Dornan, S; Jackson, A P; Gay, N J

    1997-01-01

    A Drosophila cDNA encoding a structural homologue of the mammalian coated vesicle component alpha-adaptin (AP2 adaptor complex) has been cloned and sequenced. The mammalian and invertebrate sequences are highly conserved, especially within the amino terminal region, a domain that mediates interactions with other components within the AP2 complex and with specific receptors tails. Mammalian alpha-adaptins are encoded by two genes; however, Drosophila alpha-adaptin has a single gene locus, within polytene bands 21C2-C3 on the left arm of the chromosome 2, closely adjacent to the paired homeobox gene aristaless. There seem to be at least two Drosophila alpha-adaptin transcripts expressed, plausibly by alternative splicing. One of the transcripts is more abundant during early embryogenesis and may be of maternal origin. We have studied the distribution of the alpha-adaptin protein throughout embryogenesis and at the neuromuscular junction of the third instar larva. During cellularization of the blastoderm embryo, the protein is seen between and ahead of the elongating nuclei, and then redistributes to the cell surface during gastrulation. These observations suggest a role for endocytosis in cellularization and are consistent with the finding that dynamin (the shibire gene product), another component of the endocytic mechanism, is required for cellularization. At later stages of embryogenesis, alpha-adaptin is expressed in complex and dynamic patterns. It is strongly induced in elements of the central and peripheral nervous system (e.g., in neuroblasts, the presumptive stomatogastric nervous system, and the lateral chordotonal sense organs), in the Garland cells, the adult midgut precursors, the antenno-maxillary complex, the endoderm, the fat bodies, and the visceral mesoderm. In the larva, alpha-adaptin is localized at the plasma membrane in the synaptic boutons of the neuromuscular junctions. The cells expressing high levels of alpha-adaptin are known or expected to

  16. Alpha-adaptin, a marker for endocytosis, is expressed in complex patterns during Drosophila development.

    PubMed

    Dornan, S; Jackson, A P; Gay, N J

    1997-08-01

    A Drosophila cDNA encoding a structural homologue of the mammalian coated vesicle component alpha-adaptin (AP2 adaptor complex) has been cloned and sequenced. The mammalian and invertebrate sequences are highly conserved, especially within the amino terminal region, a domain that mediates interactions with other components within the AP2 complex and with specific receptors tails. Mammalian alpha-adaptins are encoded by two genes; however, Drosophila alpha-adaptin has a single gene locus, within polytene bands 21C2-C3 on the left arm of the chromosome 2, closely adjacent to the paired homeobox gene aristaless. There seem to be at least two Drosophila alpha-adaptin transcripts expressed, plausibly by alternative splicing. One of the transcripts is more abundant during early embryogenesis and may be of maternal origin. We have studied the distribution of the alpha-adaptin protein throughout embryogenesis and at the neuromuscular junction of the third instar larva. During cellularization of the blastoderm embryo, the protein is seen between and ahead of the elongating nuclei, and then redistributes to the cell surface during gastrulation. These observations suggest a role for endocytosis in cellularization and are consistent with the finding that dynamin (the shibire gene product), another component of the endocytic mechanism, is required for cellularization. At later stages of embryogenesis, alpha-adaptin is expressed in complex and dynamic patterns. It is strongly induced in elements of the central and peripheral nervous system (e.g., in neuroblasts, the presumptive stomatogastric nervous system, and the lateral chordotonal sense organs), in the Garland cells, the adult midgut precursors, the antenno-maxillary complex, the endoderm, the fat bodies, and the visceral mesoderm. In the larva, alpha-adaptin is localized at the plasma membrane in the synaptic boutons of the neuromuscular junctions. The cells expressing high levels of alpha-adaptin are known or expected to

  17. Quantum dots induced interferon beta expression via TRIF-dependent signaling pathways by promoting endocytosis of TLR4.

    PubMed

    Ho, Chia-Chi; Luo, Yueh-Hsia; Chuang, Tsung-Hsien; Lin, Pinpin

    2016-02-17

    Quantum dots (QDs) are nano-sized semiconductors. Previously, intratracheal instillation of QD705s induces persistent inflammation and remodeling in the mouse lung. Expression of interferon beta (IFN-β), involved in tissue remodeling, was induced in the mouse lung. The objective of this study was to understand the mechanism of QD705 induced interferon beta (IFN-β) expression. QD705-COOH and QD705-PEG increased IFN-β and IP-10 mRNA levels during day1 to 90 post-exposure in mouse lungs. QD705-COOH increased IFN-β expression via Toll/interleukin-1 receptor domain-containing adapter protein (TRIF) dependent Toll-like receptor (TLR) signaling pathways in macrophages RAW264.7. Silencing TRIF expression with siRNA or co-treatment with a TRIF inhibitor tremendously abolished QD705s-induced IFN-β expression. Co-treatment with a TLR4 inhibitor completely prevented IFN-β induction by QD705-COOH. QD705-COOH readily entered cells, and co-treatment with either inhibitors of endocytosis or intracellular TLRs prevented IFN-β induction. Thus, activation of the TRIF dependent TLRs pathway by promoting endocytosis of TLR4 is one of the mechanisms for immunomodulatory effects of nanoparticles. PMID:26925925

  18. β-Arrestin-mediated Angiotensin II Signaling Controls the Activation of ARF6 Protein and Endocytosis in Migration of Vascular Smooth Muscle Cells.

    PubMed

    Charles, Ricardo; Namkung, Yoon; Cotton, Mathieu; Laporte, Stéphane A; Claing, Audrey

    2016-02-19

    Angiotensin II (Ang II) is a vasopressive hormone but is also a potent activator of cellular migration. We have previously shown that it can promote the activation of the GTPase ARF6 in a heterologous overexpressing system. The molecular mechanisms by which receptors control the activation of this small G protein remain, however, largely unknown. Furthermore, how ARF6 coordinates the activation of complex cellular responses needs to be further elucidated. In this study, we demonstrate that Ang II receptors engage β-arrestin, but not Gq, to mediate ARF6 activation in HEK 293 cells. To further confirm the key role of β-arrestin proteins, we overexpressed β-arrestin2-(1-320), a dominant negative mutant known to block receptor endocytosis. We show that expression of this truncated construct does not support the activation of the GTPase nor cell migration. Interestingly, β-arrestin2 can interact with the ARF guanine nucleotide exchange factor ARNO, although the C-terminally lacking mutant does not. We finally examined whether receptor endocytosis controlled ARF6 activation and cell migration. Although the clathrin inhibitor PitStop2 did not impact the ability of Ang II to activate ARF6, cell migration was markedly impaired. To further show that ARF activation regulates key signaling events leading to migration, we also examined MAPK activation. We demonstrate that this signaling axis is relevant in smooth muscle cells of the vasculature. Altogether, our findings show for the first time that Ang II receptor signaling to β-arrestin regulates ARF6 activation. These proteins together control receptor endocytosis and ultimately cell migration. PMID:26703465

  19. The Lipid Raft-Associated Protein CD98 Is Required for Vaccinia Virus Endocytosis

    PubMed Central

    Schroeder, Nina; Chung, Che-Sheng; Chen, Chein-Hung; Liao, Chung-Lin

    2012-01-01

    Mature vaccinia virus (vaccinia MV) infects a broad range of animals in vivo and cell cultures in vitro; however, the cellular receptors that determine vaccinia MV tropism and entry pathways are poorly characterized. Here, we performed quantitative proteomic analyses of lipid raft-associated proteins upon vaccinia MV entry into HeLa cells. We found that a type II membrane glycoprotein, CD98, is enriched in lipid rafts upon vaccinia MV infection compared to mock-infected HeLa cells. The knockdown of CD98 expression in HeLa cells significantly reduced vaccinia MV entry. Furthermore, CD98 knockout (KO) mouse embryonic fibroblasts (MEFs) also exhibited reduced vaccinia MV infectivity without affecting MV attachment to cells, suggesting a role for CD98 in the postbinding step of virus entry. Further characterization with inhibitors and dominant negative proteins that block different endocytic pathways revealed that vaccinia MV entry into MEFs occurs through a clathrin-independent, caveolin-independent, dynamin-dependent, fluid-phase endocytic pathway, implying that CD98 plays a specific role in the vaccinia MV endocytic pathway. Infections of wild-type and CD98 KO MEF cells with different strains of vaccinia MV provided further evidence that CD98 plays a specific role in MV endocytosis but not in plasma membrane fusion. Finally, different CD98-C69 chimeric proteins were expressed in CD98 KO MEFs, but none were able to reconstitute MV infectivity, suggesting that the overall structure of the CD98 protein is required for vaccinia MV endocytosis. PMID:22345471

  20. Fast, Temperature-Sensitive and Clathrin-Independent Endocytosis at Central Synapses.

    PubMed

    Delvendahl, Igor; Vyleta, Nicholas P; von Gersdorff, Henrique; Hallermann, Stefan

    2016-05-01

    The fusion of neurotransmitter-filled vesicles during synaptic transmission is balanced by endocytotic membrane retrieval. Despite extensive research, the speed and mechanisms of synaptic vesicle endocytosis have remained controversial. Here, we establish low-noise time-resolved membrane capacitance measurements that allow monitoring changes in surface membrane area elicited by single action potentials and stronger stimuli with high-temporal resolution at physiological temperature in individual bona-fide mature central synapses. We show that single action potentials trigger very rapid endocytosis, retrieving presynaptic membrane with a time constant of 470 ms. This fast endocytosis is independent of clathrin but mediated by dynamin and actin. In contrast, stronger stimuli evoke a slower mode of endocytosis that is clathrin, dynamin, and actin dependent. Furthermore, the speed of endocytosis is highly temperature dependent with a Q10 of ∼3.5. These results demonstrate that distinct molecular modes of endocytosis with markedly different kinetics operate at central synapses. PMID:27146271

  1. Nascent structure-activity relationship study of a diastereomeric series of kappa opioid receptor antagonists derived from CJ-15,208.

    PubMed

    Dolle, Roland E; Michaut, Mathieu; Martinez-Teipel, Blanca; Seida, Pamela R; Ajello, Christopher W; Muller, Alison L; DeHaven, Robert N; Carroll, Patrick J

    2009-07-01

    Cyclic tetrapeptide c[Phe-pro-Phe-trp] 2, a diastereomer of CJ-15,208 (1), was identified as a potent dual kappa/mu opioid receptor antagonist devoid of delta opioid receptor affinity against cloned human receptors: K(i) (2)=3.8nM (kappa), 30nM (mu); IC(50) ([(35)S]GTPgammaS binding)=140nM (kappa), 21nM (mu). The d-tryptophan residue rendered 2 ca. eightfold and fourfold more potent at kappa and mu, respectively, than the corresponding l-configured tryptophan in the natural product 1. Phe analogs 3-10, designed to probe the effect of substituents on receptor affinity and selectivity, possessed K(i) values ranging from 14 to 220nM against the kappa opioid receptor with mu/kappa ratios of 0.45-3.0. An alanine scan of 2 yielded c[Ala-pro-Phe-trp] 12, an analog equipotent to 2. Agents 2 and 12 were pure antagonists in vitro devoid of agonist activity. Ac-pro-Phe-trp-Phe-NH(2)16 and Ac-Phe-trp-Phe-pro-NH(2)17 two of the eight possible acyclic peptides derived from 1 and 2, were selective, modestly potent mu ligands: K(i) (16)=340nM (mu); K(i) (17)=360nM (mu). PMID:19464172

  2. Antidepressant-like Effects of Buprenorphine are Mediated by Kappa Opioid Receptors.

    PubMed

    Falcon, Edgardo; Browne, Caroline A; Leon, Rosa M; Fleites, Vanessa C; Sweeney, Rachel; Kirby, Lynn G; Lucki, Irwin

    2016-08-01

    Previous studies have identified potential antidepressant effects of buprenorphine (BPN), a drug with high affinity for mu opioid receptor (MORs) and kappa opioid receptors (KORs) and some affinity at delta opioid receptor (DOR) and opioid receptor-like 1 (ORL-1) receptors. Therefore, these studies examined which opioid receptors were involved in BPN's effects on animal behavior tests sensitive to antidepressant drugs. The acute effects of BPN were tested in the forced swim test (FST) using mice with genetic deletion of individual opioid receptors or after pharmacological blockade of receptors. For evaluating the effects of BPN on chronic stress, separate groups of mice were exposed to unpredictable chronic mild stress (UCMS) for 3 weeks and treated with BPN for at least 7 days before behavioral assessment and subsequent measurement of Oprk1, Oprm1, and Pdyn mRNA expression in multiple brain regions. BPN did not reduce immobility in mice with KOR deletion or after pretreatment with norbinaltorphimine, even though desipramine remained effective. In contrast, BPN reduced immobility in MOR and DOR knockout mice and in mice pretreated with the ORL-1 antagonist JTC-801. UCMS reduced sucrose preference, decreased time in the light side of the light/dark box, increased immobility in the FST and induced region-specific alterations in Oprk1, Oprm1, and PDYN mRNA expression in the frontal cortex and striatum. All of these changes were normalized following BPN treatment. The KOR was identified as a key player mediating the effects of BPN in tests sensitive to antidepressant drugs in mice. These studies support further development of BPN as a novel antidepressant. PMID:26979295

  3. Regulation by intracellular Ca sup 2+ and cyclic AMP of the growth factor-induced ruffling membrane formation and stimulation of fluid-phase endocytosis and exocytosis

    SciTech Connect

    Miyata, Yoshihiko Tokyo Metropolitan Inst. of Medical Science ); Nishida, Eisuke; Sakai, Hikoichi ); Koyasu, Shigeo; Yahara, Ichiro )

    1989-04-01

    Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) induce formation of ruffling membranes and stimulate the fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells. An increase in intracellular Ca{sup 2+} concentration by treatment with A23187, a calcium ionophore, or an increase in intracellular cAMP level by treatment with dibutyryl cAMP or forskolin almost completely inhibited the insulin-, IGF-I-, or EGF-induced formation of ruffling membranes. Increases in Ca{sup 2+} or cAMP concentration also inhibited almost completely the stimulation of fluid-phase endocytosis and exocytosis elicited by these growth factors. These results suggest that the growth factor-induced ruffling membrane formation and the stimulation of fluid-phase endocytosis and exocytosis have a common regulatory mechanism involving intracellular concentrations of Ca{sup 2+} and cAMP. {sup 125}I-EGF binding assays and immunoprecipitation experiments with anti-phosphotyrosine antibody revealed that treatment of KB cells with A23187, dibutyryl cAMP, or forskolin did not inhibit the EGF binding to the cells nor subsequent tyrosine autophosphorylation of its receptors. These results indicate that Ca{sup 2+}- and/or cAMP-sensitive intracellular reactions exist downstream from the receptor kinase activation in the process of these early cellular responses.

  4. Transferrin Receptor Controls AMPA Receptor Trafficking Efficiency and Synaptic Plasticity

    PubMed Central

    Liu, Ke; Lei, Run; Li, Qiong; Wang, Xin-Xin; Wu, Qian; An, Peng; Zhang, Jianchao; Zhu, Minyan; Xu, Zhiheng; Hong, Yang; Wang, Fudi; Shen, Ying; Li, Hongchang; Li, Huashun

    2016-01-01

    Transferrin receptor (TFR) is an important iron transporter regulating iron homeostasis and has long been used as a marker for clathrin mediated endocytosis. However, little is known about its additional function other than iron transport in the development of central nervous system (CNS). Here we demonstrate that TFR functions as a regulator to control AMPA receptor trafficking efficiency and synaptic plasticity. The conditional knockout (KO) of TFR in neural progenitor cells causes mice to develop progressive epileptic seizure, and dramatically reduces basal synaptic transmission and long-term potentiation (LTP). We further demonstrate that TFR KO remarkably reduces the binding efficiency of GluR2 to AP2 and subsequently decreases AMPA receptor endocytosis and recycling. Thus, our study reveals that TFR functions as a novel regulator to control AMPA trafficking efficiency and synaptic plasticity. PMID:26880306

  5. An Unbiased Screen Identifies the DEP-1 Tumour Suppressor as a Phosphatase Controlling EGFR Endocytosis

    PubMed Central

    Tarcic, Gabi; Boguslavsky, Shlomit K.; Wakim, Jean; Kiuchi, Tai; Liu, Angela; Reinitz, Felicia; Nathanson, David; Takahashi, Takamune; Mischel, Paul S.; Ng, Tony; Yarden, Yosef

    2009-01-01

    Background The epidermal growth factor (EGF) stimulates rapid tyrosine phosphorylation of EGF-receptor (EGFR). This event precedes signalling from both the plasma membrane and from endosomes, and it is essential for recruitment of an ubiquitin ligase, CBL, that sorts activated receptors to endosomes and degradation. Because hyper-phosphorylation of EGFR is involved in oncogenic pathways, we performed an unbiased screen of siRNA oilgonucleotides targeting all human tyrosine phosphatases. Results We report the identification of PTPRK and PTPRJ (DEP-1) as EGFR-targeting phosphatases. DEP-1 is a tumour suppressor that dephosphorylates, thereby stabilizes EGFR by hampering its ability to associate with the CBL-GRB2 ubiquitin ligase complex. DEP-1 silencing enhanced tyrosine phosphorylation of endosomal EGFRs and, accordingly, increased cell proliferation. In line with functional interactions, EGFR and DEP-1 form physical associations, and EGFR phosphorylates a substrtae trapping mutant of DEP-1. Interestingly, the interactions of DEP-1 and EGFR are followed by physical segregation: whereas EGFR undergoes endocytosis, DEP-1 remains confined to the cell surface. Conclusions EGFR and DEP-1 physically interact at the cell surface and maitain bidirectional enzyme-substrate interactions, which are relevant to their respective oncogenic and tumor suppressive functions. These observations highlight the emerging roles of vesicular trafficking in malignant processes. PMID:19836242

  6. Membrane folate-binding proteins are responsible for folate-protein conjugate endocytosis into cultured cells.

    PubMed Central

    Leamon, C P; Low, P S

    1993-01-01

    Folate-protein conjugates have been shown to bind to and enter HeLa and KB cells by receptor-mediated endocytosis [Leamon and Low (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5572-5576]. Although these cells contain a membrane folate-binding protein (FBP) involved in the uptake of free folate, no studies have been conducted to evaluate whether the folate-protein conjugates enter cells via the same protein. To address this issue, HeLa cell monolayers were treated with folate-labelled 125I-RNAase under various conditions characteristic of FBP-mediated folate uptake. Folate-labelled 125I-RNAase was found to bind to cells with high affinity (Kd = 24 nM), and like the free vitamin, its binding could be competitively blocked by excess free folate. Furthermore, binding could be reversed by either washing the cells with acid/saline, pH 3.0, or by treating the cells with phosphatidyl-inositol-specific phospholipase C, an enzyme known to release FBP from cell surfaces. Because cells pretreated with anti-FBP serum were unable to bind folate conjugates, and since the same antiserum identified a single 65 kDa band reminiscent of FBPs found in many other tissues, we conclude that a classical FBP is responsible for the uptake of folate-protein conjugates by receptor-bearing cells. Images Figure 5 PMID:8387781

  7. Abolished thermal and mechanical antinociception but retained visceral chemical antinociception induced by butorphanol in mu-opioid receptor knockout mice.

    PubMed

    Ide, Soichiro; Minami, Masabumi; Ishihara, Kumatoshi; Uhl, George R; Satoh, Masamichi; Sora, Ichiro; Ikeda, Kazutaka

    2008-06-01

    Butorphanol is hypothesized to induce analgesia via opioid pathways, although the precise mechanisms for its effects remain unknown. In this study, we investigated the role of the mu-opioid receptor (MOP) in thermal, mechanical, and visceral chemical antinociception induced by butorphanol using MOP knockout (KO) mice. Butorphanol-induced thermal antinociception, assessed by the hot-plate and tail-flick tests, was significantly reduced in heterozygous and abolished in homozygous MOP-KO mice compared with wildtype mice. The results obtained from our butorphanol-induced mechanical antinociception experiments, assessed by the Randall-Selitto test, were similar to the results obtained from the thermal antinociception experiments in these mice. Interestingly, however, butorphanol retained its ability to induce significant visceral chemical antinociception, assessed by the writhing test, in homozygous MOP-KO mice. The butorphanol-induced visceral chemical antinociception that was retained in homozygous MOP-KO mice was completely blocked by pretreatment with nor-binaltorphimine, a kappa-opioid receptor (KOP) antagonist. In vitro binding and cyclic adenosine monophosphate assays also showed that butorphanol possessed higher affinity for KOPs and MOPs than for delta-opioid receptors. These results molecular pharmacologically confirmed previous studies implicating MOPs, and partially KOPs, in mediating butorphanol-induced analgesia. PMID:18417173

  8. TRIM72 modulates caveolar endocytosis in repair of lung cells.

    PubMed

    Nagre, Nagaraja; Wang, Shaohua; Kellett, Thomas; Kanagasabai, Ragu; Deng, Jing; Nishi, Miyuki; Shilo, Konstantin; Oeckler, Richard A; Yalowich, Jack C; Takeshima, Hiroshi; Christman, John; Hubmayr, Rolf D; Zhao, Xiaoli

    2016-03-01

    Alveolar epithelial and endothelial cell injury is a major feature of the acute respiratory distress syndrome, in particular when in conjunction with ventilation therapies. Previously we showed [Kim SC, Kellett T, Wang S, Nishi M, Nagre N, Zhou B, Flodby P, Shilo K, Ghadiali SN, Takeshima H, Hubmayr RD, Zhao X. Am J Physiol Lung Cell Mol Physiol 307: L449-L459, 2014.] that tripartite motif protein 72 (TRIM72) is essential for amending alveolar epithelial cell injury. Here, we posit that TRIM72 improves cellular integrity through its interaction with caveolin 1 (Cav1). Our data show that, in primary type I alveolar epithelial cells, lack of TRIM72 led to significant reduction of Cav1 at the plasma membrane, accompanied by marked attenuation of caveolar endocytosis. Meanwhile, lentivirus-mediated overexpression of TRIM72 selectively increases caveolar endocytosis in rat lung epithelial cells, suggesting a functional association between these two. Further coimmunoprecipitation assays show that deletion of either functional domain of TRIM72, i.e., RING, B-box, coiled-coil, or PRY-SPRY, abolishes the physical interaction between TRIM72 and Cav1, suggesting that all theoretical domains of TRIM72 are required to forge a strong interaction between these two molecules. Moreover, in vivo studies showed that injurious ventilation-induced lung cell death was significantly increased in knockout (KO) TRIM72(KO) and Cav1(KO) lungs compared with wild-type controls and was particularly pronounced in double KO mutants. Apoptosis was accompanied by accentuation of gross lung injury manifestations in the TRIM72(KO) and Cav1(KO) mice. Our data show that TRIM72 directly and indirectly modulates caveolar endocytosis, an essential process involved in repair of lung epithelial cells through removal of plasma membrane wounds. Given TRIM72's role in endomembrane trafficking and cell repair, we consider this molecule an attractive therapeutic target for patients with injured lungs. PMID

  9. Plasmolipin—a new player in endocytosis and epithelial development

    PubMed Central

    Le Guelte, Armelle; Macara, Ian G

    2015-01-01

    Polarized vesicle sorting is essential not only for epithelial cell function but also for cell polarization and tissue morphogenesis. Endocytosis is a key determinant of the surface abundance of plasma membrane proteins and is highly regulated. In an important recent paper, Rodríguez-Fraticelli et al (4) identify a new player in apical endocytosis—a previously uncharacterized protein called Plasmolipin. They report not only its mechanism of action through binding to an epsin, but also highlight an essential role in regulating Notch signaling, which controls epithelial differentiation. PMID:25825384

  10. Clathrin-mediated endocytosis in budding yeast at a glance.

    PubMed

    Lu, Rebecca; Drubin, David G; Sun, Yidi

    2016-04-15

    Clathrin-mediated endocytosis is an essential cellular process that involves the concerted assembly and disassembly of many different proteins at the plasma membrane. In yeast, live-cell imaging has shown that the spatiotemporal dynamics of these proteins is highly stereotypical. Recent work has focused on determining how the timing and functions of endocytic proteins are regulated. In this Cell Science at a Glance article and accompanying poster, we review our current knowledge of the timeline of endocytic site maturation and discuss recent works focusing on how phosphorylation, ubiquitylation and lipids regulate various aspects of the process. PMID:27084361

  11. Role of preoptic opioid receptors in the body temperature reduction during hypoxia.

    PubMed

    Scarpellini, Carolina da Silveira; Gargaglioni, Luciane H; Branco, Luis G S; Bícego, Kênia C

    2009-08-25

    Evidence indicates that endogenous opioids play a role in body temperature (Tb) regulation in mammals but no data exist about the involvement of the specific opioid receptors, mu, kappa and delta, in the reduction of Tb induced by hypoxia. Thus, we investigated the participation of these opioid receptors in the anteroventral preoptic region (AVPO) in hypoxic decrease of Tb. To this end, Tb of unanesthetized Wistar rats was monitored by temperature data loggers before and after intra-AVPO microinjection of the selective kappa-opioid receptor antagonist nor-binaltorphimine dihydrochloride (nor-BNI; 0.1 and 1.0 microg/100 nL/animal), the selective mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 cyclic (CTAP; 0.1 and 1.0 microg/100 nL/animal), and the selective delta-opioid receptor antagonist Naltrindole (0.06 and 0.6 microg/100 nL/animal) or saline (vehicle, 100 nL/animal), during normoxia and hypoxia (7% inspired O2). Under normoxia, no effect of opioid antagonists on Tb was observed. Hypoxia induced Tb to reduce in vehicle group, a response that was inhibited by the microinjection intra-AVPO of nor-BNI. In contrast, CTAP and Naltrindole did not change Tb during hypoxia but caused a longer latency for the return of Tb to the normoxic values just after low O2 exposure. Our results indicate the kappa-opioid receptor in the AVPO is important for the reduction of Tb during hypoxia while the mu and delta receptors are involved in the increase of Tb during normoxia post-hypoxia. PMID:19545549

  12. Dysferlin is essential for endocytosis in the sea star oocyte

    PubMed Central

    Oulhen, Nathalie; Onorato, Thomas M.; Ramos, Isabela; Wessel, Gary M.

    2014-01-01

    Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function. PMID:24368072

  13. Membrane Mechanics of Endocytosis in Cells with Turgor

    PubMed Central

    Dmitrieff, Serge; Nédélec, François

    2015-01-01

    Endocytosis is an essential process by which cells internalize a piece of plasma membrane and material from the outside. In cells with turgor, pressure opposes membrane deformations, and increases the amount of force that has to be generated by the endocytic machinery. To determine this force, and calculate the shape of the membrane, we used physical theory to model an elastic surface under pressure. Accurate fits of experimental profiles are obtained assuming that the coated membrane is highly rigid and preferentially curved at the endocytic site. The forces required from the actin machinery peaks at the onset of deformation, indicating that once invagination has been initiated, endocytosis is unlikely to stall before completion. Coat proteins do not lower the initiation force but may affect the process by the curvature they induce. In the presence of isotropic curvature inducers, pulling the tip of the invagination can trigger the formation of a neck at the base of the invagination. Hence direct neck constriction by actin may not be required, while its pulling role is essential. Finally, the theory shows that anisotropic curvature effectors stabilize membrane invaginations, and the loss of crescent-shaped BAR domain proteins such as Rvs167 could therefore trigger membrane scission. PMID:26517669

  14. Ricin A chain reaches the endoplasmic reticulum after endocytosis

    SciTech Connect

    Liu Qiong; Zhan Jinbiao . E-mail: jzhan2k@zju.edu.cn; Chen Xinhong; Zheng Shu

    2006-05-12

    Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purified by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum.

  15. Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds.

    PubMed

    Pagnussat, Luciana; Burbach, Christian; Baluška, František; de la Canal, Laura

    2012-03-01

    During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4-64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunolocalization of rhamnogalacturonan-II (RG-II) complexed with boron showed that whereas this pectin is localized only in the cell walls of dry seed embryos, it starts to be intracellular once the imbibition started. Brefeldin A (BFA) exposure resulted in recruitment of the intracellular RG-II pectin complexes into the endocytic BFA-induced compartments, confirming the endocytic origin of the RG-II signal detected intracellularly. Finally, germination was significantly delayed when Arabidopsis seeds were germinated in the presence of inhibitors of endocytic pathways, suggesting that trafficking of extracellular molecules might play an important role in the overcome of germination. This work constitutes the first demonstration of endocytic processes during germination and opens new perspectives about the role of the extracellular matrix and membrane components in seed germination. PMID:22476454

  16. Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds

    PubMed Central

    Pagnussat, Luciana; Burbach, Christian; Baluška, František; de la Canal, Laura

    2012-01-01

    During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4–64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunolocalization of rhamnogalacturonan-II (RG-II) complexed with boron showed that whereas this pectin is localized only in the cell walls of dry seed embryos, it starts to be intracellular once the imbibition started. Brefeldin A (BFA) exposure resulted in recruitment of the intracellular RG-II pectin complexes into the endocytic BFA-induced compartments, confirming the endocytic origin of the RG-II signal detected intracellularly. Finally, germination was significantly delayed when Arabidopsis seeds were germinated in the presence of inhibitors of endocytic pathways, suggesting that trafficking of extracellular molecules might play an important role in the overcome of germination. This work constitutes the first demonstration of endocytic processes during germination and opens new perspectives about the role of the extracellular matrix and membrane components in seed germination. PMID:22476454

  17. Lysosomal Trafficking of TGFBIp via Caveolae-Mediated Endocytosis

    PubMed Central

    Choi, Seung-il; Maeng, Yong-Sun; Kim, Tae-im; Lee, Yangsin; Kim, Yong-Sun; Kim, Eung Kweon

    2015-01-01

    Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin αVβ3. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy. PMID:25853243

  18. Differential Use of the C-Type Lectins L-SIGN and DC-SIGN for Phlebovirus Endocytosis.

    PubMed

    Léger, Psylvia; Tetard, Marilou; Youness, Berthe; Cordes, Nicole; Rouxel, Ronan N; Flamand, Marie; Lozach, Pierre-Yves

    2016-06-01

    Bunyaviruses represent a growing threat to humans and livestock globally. The receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely unidentified and poorly characterized. DC-SIGN is a C-type lectin highly expressed on dermal dendritic cells that has been found to act as an authentic entry receptor for many phleboviruses (Bunyaviridae), including Rift Valley fever virus (RVFV), Toscana virus (TOSV) and Uukuniemi virus (UUKV). We found that these phleboviruses can exploit another C-type lectin, L-SIGN, for infection. L-SIGN shares 77% sequence homology with DC-SIGN and is expressed on liver sinusoidal endothelial cells. L-SIGN is required for UUKV binding but not for virus internalization. An endocytosis-defective mutant of L-SIGN was still able to mediate virus uptake and infection, indicating that L-SIGN acts as an attachment receptor for phleboviruses rather than an endocytic receptor. Our results point out a fundamental difference in the use of the C-type lectins L-SIGN and DC-SIGN by UUKV to enter cells, although both proteins are closely related in terms of molecular structure and biological function. This study sheds new light on the molecular mechanisms by which phleboviruses target the liver and also highlights the added complexity in virus-receptor interactions beyond attachment. PMID:26990254

  19. Endoplasmic Reticulum Chaperone Protein GRP-78 Mediates Endocytosis of Dentin Matrix Protein 1*S⃞

    PubMed Central

    Ravindran, Sriram; Narayanan, Karthikeyan; Eapen, Asha Sarah; Hao, Jianjun; Ramachandran, Amsaveni; Blond, Sylvie; George, Anne

    2008-01-01

    Dentin matrix protein 1 (DMP1), a phosphorylated protein present in the mineral phase of both vertebrates and invertebrates, is a key regulatory protein during biogenic formation of mineral deposits. Previously we showed that DMP1 is localized in the nuclear compartment of preosteoblasts and preodontoblasts. In the nucleus DMP1 might play an important role in the regulation of genes that control osteoblast or odontoblast differentiation. Here, we show that cellular uptake of DMP1 occurs through endocytosis. Interestingly, this process is initiated by DMP1 binding to the glucose-regulated protein-78 (GRP-78) localized on the plasma membrane of preodontoblast cells. Binding of DMP1 to GRP-78 receptor was determined to be specific and saturable with a binding dissociation constant KD = 85 nm. We further depict a road map for the endocytosed DMP1 and demonstrate that the internalization is mediated primarily by caveolae and that the vesicles containing DMP1 are routed to the nucleus along microtubules. Immunohistochemical analysis and binding studies performed with biotin-labeled DMP1 confirm spatial co-localization of DMP1 and GRP-78 in the preodontoblasts of a developing mouse molar. Co-localization of DMP1 with GRP-78 was also observed in T4-4 preodontoblast cells, dental pulp stem cells, and primary preodontoblasts. By small interfering RNA techniques, we demonstrate that the receptor for DMP1 is GRP-78. Therefore, binding of DMP1 with GRP-78 receptor might be an important mechanism by which DMP1 is internalized and transported to the nucleus during bone and tooth development. PMID:18757373

  20. Activity-Regulated Cytoskeleton-Associated Protein Controls AMPAR Endocytosis through a Direct Interaction with Clathrin-Adaptor Protein 2123

    PubMed Central

    Wall, Mark J.; P. de Almeida, Luciana; Wauters, Sandrine C.; Januário, Yunan C.; Müller, Jürgen

    2016-01-01

    Abstract The activity-regulated cytoskeleton-associated (Arc) protein controls synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-EPSCs (mEPSCs). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, an effect that is restored by reintroducing µ2. The Arc–AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. These data provide a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2. PMID:27257628

  1. Expression of mammalian protein kinase C in Schizosaccharomyces pombe: isotype-specific induction of growth arrest, vesicle formation, and endocytosis.

    PubMed Central

    Goode, N T; Hajibagheri, M A; Warren, G; Parker, P J

    1994-01-01

    Mammalian protein kinase C (PKC) isotypes elicit a number of effects on expression in Schizosaccharomyces pombe. A small decrease in growth rate results from PKC-gamma expression, and treatment of these cells with phorbol esters leads to marked growth inhibition and vesicle formation. PKC-delta and -eta expression causes growth inhibition and vesiculation, and the magnitude of both of these effects is increased by phorbol esters. In contrast, PKC-epsilon expression produces growth inhibition but no vesicle accumulation, and this effect is not responsive to phorbol ester. Finally, PKC-zeta has no observable effect. Thus, isotype-specific biological effects are observed. The accumulation of vesicles correlates with phorbol ester-dependent growth inhibition and occurs only with expression of those isotypes that down-regulate in response to phorbol esters in these cells. Antibodies against mammalian clathrin light chain 1a identified clathrin-coated vesicles and up-regulation of clathrin expression in those cells where vesicles accumulate; the increased vesicular traffic includes an element of endocytosis. Thus expression of specific mammalian PKC isotypes up-regulates endocytosis in S. pombe, providing a likely explanation for PKC-mediated receptor internalization in higher eukaryotes. Images PMID:7803858

  2. Integrin alpha5beta1 function is regulated by XGIPC/kermit2 mediated endocytosis during Xenopus laevis gastrulation.

    PubMed

    Spicer, Erin; Suckert, Catherine; Al-Attar, Hyder; Marsden, Mungo

    2010-01-01

    During Xenopus gastrulation alpha5beta1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the alpha5 subunit and regulates the activity of alpha5beta1 integrin. The interaction of kermit2 with alpha5beta1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis. PMID:20498857

  3. The NFL-TBS.40-63 anti-glioblastoma peptide enters selectively in glioma cells by endocytosis.

    PubMed

    Lépinoux-Chambaud, Claire; Eyer, Joël

    2013-10-01

    Glioblastoma are the most frequent and aggressive tumour of the nervous system despite surgical resection associated with chemotherapy and radiotherapy. Recently, we showed that the NFL-TBS.40-63 peptide corresponding to the sequence of a tubulin-binding site of neurofilaments, enters selectively in glioblastoma cells where it blocks microtubule polymerization, inhibits their proliferation, and reduces tumour development in rats bearing glioblastoma (Bocquet et al., 2009; Berges et al., 2012a). Here, we characterized the molecular mechanism responsible for the uptake of NFL-TBS.40-63 peptide by glioblastoma cells. Unlike other cell penetrating peptides (CPPs), which use a balance between endocytosis and direct translocation, the NFL-TBS.40-63 peptide is unable to translocate directly through the membrane when incubated with giant plasma membrane vesicles. Then, using a panel of markers and inhibitors, flow cytometry and confocal microscopy investigations showed that the uptake occurs mainly through endocytosis. Moreover, glycosaminoglycans and αVβ3 integrins are not involved in the NFL-TBS.40-63 peptide recognition and internalization by glioblastoma cells. Finally, the signalling of tyrosine kinase receptors is involved in the peptide uptake, especially via EGFR overexpressed in tumour cells, indicating that the uptake of NFL-TBS.40-63 peptide by glioblastoma cells is related to their abnormally high proliferative activity. PMID:23603097

  4. Integrin α5β1 Function Is Regulated by XGIPC/kermit2 Mediated Endocytosis during Xenopus laevis Gastrulation

    PubMed Central

    Spicer, Erin; Suckert, Catherine; Al-Attar, Hyder; Marsden, Mungo

    2010-01-01

    During Xenopus gastrulation α5β1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the α5 subunit and regulates the activity of α5β1 integrin. The interaction of kermit2 with α5β1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates α5β1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the α5β1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the α5β1 integrin through receptor endocytosis. PMID:20498857

  5. Design, Syntheses, and Pharmacological Characterization of 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3′-carboxamido)morphinan Analogues as Opioid Receptor Ligands

    PubMed Central

    Yuan, Yunyun; Zaidi, Saheem A.; Stevens, David L.; Scoggins, Krista L.; Mosier, Philip D.; Kellogg, Glen E.; Dewey, William L.; Selley, Dana E.; Zhang, Yan

    2015-01-01

    A series of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3′-carboxamido)morphinan (NAQ) analogues were synthesized and pharmacologically characterized to study their structure-activity relationship at the mu opioid receptor (MOR). The competition binding assay showed two-atom spacer and aromatic side chain were optimal for MOR selectivity. Meanwhile, substitutions at the 1′- and/or 4′-position of the isoquinoline ring retained or improved MOR selectivity over the kappa opioid receptor while still possessing above 20-fold MOR selectivity over the delta opioid receptor. In contrast, substitutions at the 6′-and/or 7′-position of the isoquinoline ring reduced MOR selectivity as well as MOR efficacy. Among this series of ligands, compound 11 acted as an antagonist when challenged with morphine in warm-water tail immersion assay and produced less significant withdrawal symptoms compared to naltrexone in morphine-pelleted mice. Compound 11 also antagonized the intracellular Ca2+ increase induced by DAMGO. Molecular dynamics simulation studies of 11 in three opioid receptors indicated orientation of the 6’-nitro group varied significantly in the different “address” domains of the receptors and played a crucial role in the observed binding affinities and selectivity. Collectively, the current findings provide valuable insights for future development of NAQ-based MOR selective ligands. PMID:25783191

  6. Design, syntheses, and pharmacological characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan analogues as opioid receptor ligands.

    PubMed

    Yuan, Yunyun; Zaidi, Saheem A; Stevens, David L; Scoggins, Krista L; Mosier, Philip D; Kellogg, Glen E; Dewey, William L; Selley, Dana E; Zhang, Yan

    2015-04-15

    A series of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan (NAQ) analogues were synthesized and pharmacologically characterized to study their structure-activity relationship at the mu opioid receptor (MOR). The competition binding assay showed two-atom spacer and aromatic side chain were optimal for MOR selectivity. Meanwhile, substitutions at the 1'- and/or 4'-position of the isoquinoline ring retained or improved MOR selectivity over the kappa opioid receptor while still possessing above 20-fold MOR selectivity over the delta opioid receptor. In contrast, substitutions at the 6'- and/or 7'-position of the isoquinoline ring reduced MOR selectivity as well as MOR efficacy. Among this series of ligands, compound 11 acted as an antagonist when challenged with morphine in warm-water tail immersion assay and produced less significant withdrawal symptoms compared to naltrexone in morphine-pelleted mice. Compound 11 also antagonized the intracellular Ca(2+) increase induced by DAMGO. Molecular dynamics simulation studies of 11 in three opioid receptors indicated orientation of the 6'-nitro group varied significantly in the different 'address' domains of the receptors and played a crucial role in the observed binding affinities and selectivity. Collectively, the current findings provide valuable insights for future development of NAQ-based MOR selective ligands. PMID:25783191

  7. Standardizing Scavenger Receptor Nomenclature

    PubMed Central

    PrabhuDas, Mercy; Bowdish, Dawn; Drickamer, Kurt; Febbraio, Maria; Herz, Joachim; Kobzik, Lester; Krieger, Monty; Loike, John; Means, Terry K.; Moestrup, Soren K.; Post, Steven; Sawamura, Tatsuya; Silverstein, Samuel; Wang, Xiang-Yang; El Khoury, Joseph

    2014-01-01

    Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a variety of ligands, including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the U.S. National Institute of Allergy and Infectious Diseases, National Institutes of Health to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of non-self or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. The discussion and nomenclature recommendations described in this report only refer to mammalian scavenger receptors. The purpose of this article is to describe the proposed mammalian nomenclature and classification developed at the workshop and to solicit additional feedback from the broader research community. PMID:24563502

  8. Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

    PubMed

    Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua

    2016-07-01

    At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as

  9. Antiport mediated rounding and endocytosis are enhanced by sulphate.

    PubMed

    Sit, K H; Bay, B H; Wong, K P

    1991-01-01

    Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na+/H+ antiporter via HCO3(-)-dependent H+ pumping, is significantly enhanced by the addition of Na2SO4 (FTR + SO4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive. At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol.wt which is a 455-fold diversity. This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol.wt species that measures up to 5 microns in width. Two million mol.wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion. Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages. However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz. epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202). The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change. However the cell surface reversion is not associated with efflux of FDx which are sorted out into 'granular patches', the later stage endosomes without membrane outlines in AME. FDx-loaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions. Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches. AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an

  10. Identification of an endocytosis motif in an intracellular loop of Wntless protein, essential for its recycling and the control of Wnt protein signaling.

    PubMed

    Gasnereau, Isabelle; Herr, Patrick; Chia, Pei Zhi Cheryl; Basler, Konrad; Gleeson, Paul A

    2011-12-16

    The secretion of Wnt signaling proteins is dependent upon the transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, Caenorhabditis elegans, and the mouse. The sorting signals for the internalization and trafficking of Wls have not been defined. Here, we demonstrate that Wls internalization requires clathrin and dynamin I, components of the clathrin-mediated endocytosis pathway. Moreover, we have identified a conserved YXXϕ endocytosis motif in the third intracellular loop of the multipass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of Wls(AEGL) was rescued by the insertion of a classical YXXϕ motif in the cytoplasmic tail. Significantly, a Drosophila Wls(AEGL) mutant displayed a wing notch phenotype, with reduced Wnt secretion and signaling. These findings demonstrate that YXXϕ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signaling proteins. PMID:22027831

  11. Equine Herpesvirus 1 Entry via Endocytosis Is Facilitated by αV Integrins and an RSD Motif in Glycoprotein D ▿

    PubMed Central

    Van de Walle, Gerlinde R.; Peters, Sarah T.; VanderVen, Brian C.; O'Callaghan, Dennis J.; Osterrieder, Nikolaus

    2008-01-01

    Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as αVβ5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revealed that an RSD motif in the EHV-1 envelope glycoprotein D (gD) is critical for entry via endocytosis. In addition, we show that EHV-1 enters peripheral blood mononuclear cells predominantly via the endocytic pathway, whereas in equine endothelial cells entry occurs mainly via fusion at the plasma membrane. Taken together, the data in this study provide evidence that EHV-1 entry via endocytosis is triggered by the interaction between cellular integrins and the RSD motif present in gD and, moreover, that EHV-1 uses different cellular entry pathways to infect important target cell populations of its natural host. PMID:18815313

  12. Chronic Morphine Reduces Surface Expression of δ-Opioid Receptors in Subregions of Rostral Striatum.

    PubMed

    Leah, Paul M; Heath, Emily M L; Balleine, Bernard W; Christie, Macdonald J

    2016-03-01

    The delta opioid receptor (DOPr), whilst not the primary target of clinically used opioids, is involved in development of opioid tolerance and addiction. There is growing evidence that DOPr trafficking is involved in drug addiction, e.g., a range of studies have shown increased plasma membrane DOPr insertion during chronic treatment with opioids. The present study used a transgenic mouse model in which the C-terminal of the DOPr is tagged with enhanced-green fluorescence protein to examine the effects of chronic morphine treatment on surface membrane expression in striatal cholinergic interneurons that are implicated in motivated learning following both chronic morphine and morphine sensitization treatment schedules in male mice. A sex difference was noted throughout the anterior striatum, which was most prominent in the nucleus accumbens core region. Incontrast with previous studies in other neurons, chronic exposure to a high dose of morphine for 6 days had no effect, or slightly decreased (anterior dorsolateral striatum) surface DOPr expression. A morphine sensitization schedule produced similar results with a significant decrease in surface DOPr expression in nucleus accumbens shell. These results suggest that chronic morphine and morphine sensitisation treatment may have effects on instrumental reward-seeking behaviours and learning processes related to drug addiction, via effects on striatal DOPr function. PMID:26093651

  13. GUCY2C lysosomotropic endocytosis delivers immunotoxin therapy to metastatic colorectal cancer

    PubMed Central

    Marszalowicz, Glen P.; Snook, Adam E.; Magee, Michael S.; Merlino, Dante; Lisa, D. Berman-Booty; Waldman, Scott A.

    2014-01-01

    The emergence of targeted cancer therapy has been limited by the paucity of determinants which are tumor-specific and generally associated with disease, and have cell dynamics which effectively deploy cytotoxic payloads. Guanylyl cyclase C (GUCY2C) may be ideal for targeting because it is normally expressed only in insulated barrier compartments, including intestine and brain, but over-expressed by systemic metastatic colorectal tumors. Here, we reveal that GUCY2C rapidly internalizes from the cell surface to lysosomes in intestinal and colorectal cancer cells. Endocytosis is independent of ligand binding and receptor activation, and is mediated by clathrin. This mechanism suggests a design for immunotoxins comprising a GUCY2C-directed monoclonal antibody conjugated through a reducible disulfide linkage to ricin A chain, which is activated to a potent cytotoxin in lysosomes. Indeed, this immunotoxin specifically killed GUCY2C-expressing colorectal cancer cells in a lysosomal- and clathrin-dependent fashion. Moreover, this immunotoxin reduced pulmonary tumors >80% (p<0.001), and improved survival 25% (p<0.001), in mice with established colorectal cancer metastases. Further, therapeutic efficacy was achieved without histologic evidence of toxicity in normal tissues. These observations support GUCY2C-targeted immunotoxins as novel therapeutics for metastatic tumors originating in the GI tract, including colorectum, stomach, esophagus, and pancreas. PMID:25294806

  14. Membranous intermediates in endocytosis are labile, as shown in a temperature-sensitive mutant.

    PubMed Central

    Kessell, I; Holst, B D; Roth, T F

    1989-01-01

    Membranous tubules, especially prevalent in mammalian absorptive epithelia and insect oocytes, are one of the pleomorphic endocytic compartments that have a role in receptor-mediated endocytosis. To determine whether these tubules are evanescent, and to investigate their temporal relationships with other endocytic intermediates, we studied these tubules during oocyte vitellogenesis in the temperature-sensitive mutant Drosophila melanogaster, shibiretsl. Raising the temperature of shibire oocytes for 1 min from 19 degrees C to 29 degrees C caused a loss of these membranous tubules. The percentage of membrane in tubules decreased from 36% at 19 degrees C to 1.5% after 5 min at 29 degrees C. Concomitantly, the amount of surface membrane increased from 64% at 19 degrees C to 98% after 5 min at 29 degrees C, causing surface membrane invaginations to extend deeper into the cortex. At 29 degrees C the cytoplasmic face of the plasma membrane was studded with coated pits, and the extracellular face was coated with electron-dense material. Return from 29 degrees C to either 19 degrees C or 26 degrees C for 1-2 min produced a rapid reappearance of tubules containing extracellular horseradish peroxidase in the cortex. These data suggest that tubular intermediates are evanescent structures, and that temperature shock (i) rapidly blocks their formation from the plasma membrane, (ii) causes existing tubules to rapidly recycle to the plasma membrane, and (iii) is rapidly reversed, as newly formed tubules derive their membrane and content from the cell surface. Images PMID:2472637

  15. Evolutionary Changes on the Way to Clathrin-Mediated Endocytosis in Animals.

    PubMed

    Dergai, Mykola; Iershov, Anton; Novokhatska, Olga; Pankivskyi, Serhii; Rynditch, Alla

    2016-01-01

    Endocytic pathways constitute an evolutionarily ancient system that significantly contributed to the eukaryotic cell architecture and to the diversity of cell type-specific functions and signaling cascades, in particular of metazoans. Here we used comparative proteomic studies to analyze the universal internalization route in eukaryotes, clathrin-mediated endocytosis (CME), to address the issues of how this system evolved and what are its specific features. Among 35 proteins crucially required for animal CME, we identified a subset of 22 proteins common to major eukaryotic branches and 13 gradually acquired during evolution. Based on exploration of structure-function relationship between conserved homologs in sister, distantly related and early diverged branches, we identified novel features acquired during evolution of endocytic proteins on the way to animals: Elaborated way of cargo recruitment by multiple sorting proteins, structural changes in the core endocytic complex AP2, the emergence of the Fer/Cip4 homology domain-only protein/epidermal growth factor receptor substrate 15/intersectin functional complex as an additional interaction hub and activator of AP2, as well as changes in late endocytic stages due to recruitment of dynamin/sorting nexin 9 complex and involvement of the actin polymerization machinery. The evolutionary reconstruction showed the basis of the CME process and its subsequent step-by-step development. Documented changes imply more precise regulation of the pathway, as well as CME specialization for the uptake of specific cargoes and cell type-specific functions. PMID:26872775

  16. UBE3A Regulates Synaptic Plasticity and Learning and Memory by Controlling SK2 Channel Endocytosis.

    PubMed

    Sun, Jiandong; Zhu, Guoqi; Liu, Yan; Standley, Steve; Ji, Angela; Tunuguntla, Rashmi; Wang, Yubin; Claus, Chad; Luo, Yun; Baudry, Michel; Bi, Xiaoning

    2015-07-21

    Gated solely by activity-induced changes in intracellular calcium, small-conductance potassium channels (SKs) are critical for a variety of functions in the CNS, from learning and memory to rhythmic activity and sleep. While there is a wealth of information on SK2 gating, kinetics, and Ca(2+) sensitivity, little is known regarding the regulation of SK2 subcellular localization. We report here that synaptic SK2 levels are regulated by the E3 ubiquitin ligase UBE3A, whose deficiency results in Angelman syndrome and overexpression in increased risk of autistic spectrum disorder. UBE3A directly ubiquitinates SK2 in the C-terminal domain, which facilitates endocytosis. In UBE3A-deficient mice, increased postsynaptic SK2 levels result in decreased NMDA receptor activation, thereby impairing hippocampal long-term synaptic plasticity. Impairments in both synaptic plasticity and fear conditioning memory in UBE3A-deficient mice are significantly ameliorated by blocking SK2. These results elucidate a mechanism by which UBE3A directly influences cognitive function. PMID:26166566

  17. Evolutionary Changes on the Way to Clathrin-Mediated Endocytosis in Animals

    PubMed Central

    Dergai, Mykola; Iershov, Anton; Novokhatska, Olga; Pankivskyi, Serhii; Rynditch, Alla

    2016-01-01

    Endocytic pathways constitute an evolutionarily ancient system that significantly contributed to the eukaryotic cell architecture and to the diversity of cell type–specific functions and signaling cascades, in particular of metazoans. Here we used comparative proteomic studies to analyze the universal internalization route in eukaryotes, clathrin-mediated endocytosis (CME), to address the issues of how this system evolved and what are its specific features. Among 35 proteins crucially required for animal CME, we identified a subset of 22 proteins common to major eukaryotic branches and 13 gradually acquired during evolution. Based on exploration of structure–function relationship between conserved homologs in sister, distantly related and early diverged branches, we identified novel features acquired during evolution of endocytic proteins on the way to animals: Elaborated way of cargo recruitment by multiple sorting proteins, structural changes in the core endocytic complex AP2, the emergence of the Fer/Cip4 homology domain-only protein/epidermal growth factor receptor substrate 15/intersectin functional complex as an additional interaction hub and activator of AP2, as well as changes in late endocytic stages due to recruitment of dynamin/sorting nexin 9 complex and involvement of the actin polymerization machinery. The evolutionary reconstruction showed the basis of the CME process and its subsequent step-by-step development. Documented changes imply more precise regulation of the pathway, as well as CME specialization for the uptake of specific cargoes and cell type-specific functions. PMID:26872775

  18. Oligomerization and Pore Formation by Equinatoxin II Inhibit Endocytosis and Lead to Plasma Membrane Reorganization*

    PubMed Central

    García-Sáez, Ana J.; Buschhorn, Sabine B.; Keller, Heiko; Anderluh, Gregor; Simons, Kai; Schwille, Petra

    2011-01-01

    Pore-forming toxins have evolved to induce membrane injury by formation of pores in the target cell that alter ion homeostasis and lead to cell death. Many pore-forming toxins use cholesterol, sphingolipids, or other raft components as receptors. However, the role of plasma membrane organization for toxin action is not well understood. In this study, we have investigated cellular dynamics during the attack of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina, by combining time lapse three-dimensional live cell imaging, fluorescence recovery after photobleaching, FRET, and fluorescence cross-correlation spectroscopy. Our results show that membrane binding by equinatoxin II is accompanied by extensive plasma membrane reorganization into microscopic domains that resemble coalesced lipid rafts. Pore formation by the toxin induces Ca2+ entry into the cytosol, which is accompanied by hydrolysis of phosphatidylinositol 4,5-bisphosphate, plasma membrane blebbing, actin cytoskeleton reorganization, and inhibition of endocytosis. We propose that plasma membrane reorganization into stabilized raft domains is part of the killing strategy of equinatoxin II. PMID:21885440

  19. Down-regulation of insulin receptors is related to insulin internalization

    SciTech Connect

    Geiger, D.; Carpentier, J.L.; Gorden, P.; Orci, L. )

    1989-11-01

    In the present study, we have tested the influence of inhibition of endocytosis by hypertonic medium on the regulation of cell surface insulin receptors. We show that active internalization of {sup 125}I-insulin is markedly inhibited by hypertonic media and that, in parallel, cell surface invaginations are significantly diminished. These two events are accompanied by a marked inhibition of cell surface insulin receptor down-regulation. These data provide further strong evidence that receptor-mediated endocytosis is the major mechanism by which insulin receptors are regulated at the surface of target cells.

  20. Endocytosis of simian virus 40 into the endoplasmic reticulum

    SciTech Connect

    Kartenbeck, J.; Stukenbrok, H.; Helenius, A. )

    1989-12-01

    The endocytosis of SV-40 into CV-1 cells we studied using biochemical and ultrastructural techniques. The half-time of binding of ({sup 35}S)methionine-radiolabeled SV-40 to CV-1 cells was 25 min. Most of the incoming virus particles remained undegraded for several hours. Electron microscopy showed that some virus entered the endosomal/lysosomal pathway via coated vesicles, while the majority were endocytosed via small uncoated vesicles. After infection at high multiplicity, one third of total cell-associated virus was observed to enter the ER, starting 1-2 h after virus application. The viruses were present in large, tubular, smooth membrane networks generated as extentions of the ER. The results describe a novel and unique membrane transport pathway that allows endocytosed viral particles to be targeted from the plasma membrane to the ER.

  1. Binding characteristics of [3H]14-methoxymetopon, a high affinity mu-opioid receptor agonist.

    PubMed

    Spetea, Mariana; Tóth, Fanni; Schütz, Johannes; Otvös, Ferenc; Tóth, Géza; Benyhe, Sandor; Borsodi, Anna; Schmidhammer, Helmut

    2003-07-01

    The highly potent micro -opioid receptor agonist 14-methoxymetopon (4,5alpha-epoxy-3-hydroxy-14beta-methoxy-5beta,17-dimethylmorphinan-6-one) was prepared in tritium labelled form by a catalytic dehalogenation method resulting in a specific radioactivity of 15.9 Ci/mmol. Opioid binding characteristics of [3H]14-methoxymetopon were determined using radioligand binding assay in rat brain membranes. [3H]14-Methoxymetopon specifically labelled a single class of opioid sites with affinity in low subnanomolar range (Ki = 0.43 nm) and maximal number of binding sites of 314 fmol/mg protein. Binding of [3H]14-methoxymetopon was inhibited by ligands selective for the micro -opioid receptor with high potency, while selective kappa-opioids and delta-opioids were weaker inhibitors. 14-Methoxymetopon increased guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding with an EC50 of 70.9 nm, thus, providing evidence for the agonist character of this ligand. The increase of [35S]GTPgammaS binding was inhibited by naloxone and selective micro -opioid antagonists, indicating a micro -opioid receptor-mediated action. [3H]14-Methoxymetopon is one of the few nonpeptide mu-opioid receptor agonists available in radiolabelled form up to now. Due to its high affinity and selectivity, high stability and extremely low nonspecific binding (<10%), this radioligand would be an important and useful tool in probing mu-opioid receptor mechanisms, as well as to promote a further understanding of the opioid system at the cellular and molecular level. PMID:12887410

  2. Ezetimibe-sensitive cholesterol uptake by NPC1L1 protein does not require endocytosis

    PubMed Central

    Johnson, Tory A.; Pfeffer, Suzanne R.

    2016-01-01

    Human NPC1L1 protein mediates cholesterol absorption in the intestine and liver and is the target of the drug ezetimibe, which is used to treat hypercholesterolemia. Previous studies concluded that NPC1L1-GFP protein trafficking is regulated by cholesterol binding and that ezetimibe blocks NPC1L1-GFP function by inhibiting its endocytosis. We used cell surface biotinylation to monitor NPC1L1-GFP endocytosis and show that ezetimibe does not alter the rate of NPC1L1-GFP endocytosis in cultured rat hepatocytes grown under normal growth conditions. As expected, NPC1L1-GFP endocytosis depends in part on C-terminal, cytoplasmically oriented sequences, but endocytosis does not require cholesterol binding to NPC1L1’s N-terminal domain. In addition, two small- molecule inhibitors of general (and NPC1L1-GFP) endocytosis failed to inhibit the ezetimibe-sensitive uptake of [3H]cholesterol from taurocholate micelles. These experiments demonstrate that cholesterol uptake by NPC1L1 does not require endocytosis; moreover, ezetimibe interferes with NPC1L1’s cholesterol adsorption activity without blocking NPC1L1 internalization in RH7777 cells. PMID:27075173

  3. Ezetimibe-sensitive cholesterol uptake by NPC1L1 protein does not require endocytosis.

    PubMed

    Johnson, Tory A; Pfeffer, Suzanne R

    2016-06-01

    Human NPC1L1 protein mediates cholesterol absorption in the intestine and liver and is the target of the drug ezetimibe, which is used to treat hypercholesterolemia. Previous studies concluded that NPC1L1-GFP protein trafficking is regulated by cholesterol binding and that ezetimibe blocks NPC1L1-GFP function by inhibiting its endocytosis. We used cell surface biotinylation to monitor NPC1L1-GFP endocytosis and show that ezetimibe does not alter the rate of NPC1L1-GFP endocytosis in cultured rat hepatocytes grown under normal growth conditions. As expected, NPC1L1-GFP endocytosis depends in part on C-terminal, cytoplasmically oriented sequences, but endocytosis does not require cholesterol binding to NPC1L1's N-terminal domain. In addition, two small- molecule inhibitors of general (and NPC1L1-GFP) endocytosis failed to inhibit the ezetimibe-sensitive uptake of [(3)H]cholesterol from taurocholate micelles. These experiments demonstrate that cholesterol uptake by NPC1L1 does not require endocytosis; moreover, ezetimibe interferes with NPC1L1's cholesterol adsorption activity without blocking NPC1L1 internalization in RH7777 cells. PMID:27075173

  4. A role for the dynamin-like protein Vps1 during endocytosis in yeast.

    PubMed

    Smaczynska-de Rooij, Iwona I; Allwood, Ellen G; Aghamohammadzadeh, Soheil; Hettema, Ewald H; Goldberg, Martin W; Ayscough, Kathryn R

    2010-10-15

    Dynamins are a conserved family of proteins involved in membrane fusion and fission. Although mammalian dynamins are known to be involved in several membrane-trafficking events, the role of dynamin-1 in endocytosis is the best-characterised role of this protein family. Despite many similarities between endocytosis in yeast and mammalian cells, a comparable role for dynamins in yeast has not previously been demonstrated. The reported lack of involvement of dynamins in yeast endocytosis has raised questions over the general applicability of the current yeast model of endocytosis, and has also precluded studies using well-developed methods in yeast, to further our understanding of the mechanism of dynamin function during endocytosis. Here, we investigate the yeast dynamin-like protein Vps1 and demonstrate a transient burst of localisation to sites of endocytosis. Using live-cell imaging of endocytic reporters in strains lacking vps1, and also electron microscopy and biochemical approaches, we demonstrate a role for Vps1 in facilitating endocytic invagination. Vps1 mutants were generated, and analysis in several assays reveals a role for the C-terminal self-assembly domain in endocytosis but not in other membrane fission events with which Vps1 has previously been associated. PMID:20841380

  5. A role for the dynamin-like protein Vps1 during endocytosis in yeast

    PubMed Central

    Rooij, Iwona I. Smaczynska-de; Allwood, Ellen G.; Aghamohammadzadeh, Soheil; Hettema, Ewald H.; Goldberg, Martin W.; Ayscough, Kathryn R.

    2010-01-01

    Dynamins are a conserved family of proteins involved in membrane fusion and fission. Although mammalian dynamins are known to be involved in several membrane-trafficking events, the role of dynamin-1 in endocytosis is the best-characterised role of this protein family. Despite many similarities between endocytosis in yeast and mammalian cells, a comparable role for dynamins in yeast has not previously been demonstrated. The reported lack of involvement of dynamins in yeast endocytosis has raised questions over the general applicability of the current yeast model of endocytosis, and has also precluded studies using well-developed methods in yeast, to further our understanding of the mechanism of dynamin function during endocytosis. Here, we investigate the yeast dynamin-like protein Vps1 and demonstrate a transient burst of localisation to sites of endocytosis. Using live-cell imaging of endocytic reporters in strains lacking vps1, and also electron microscopy and biochemical approaches, we demonstrate a role for Vps1 in facilitating endocytic invagination. Vps1 mutants were generated, and analysis in several assays reveals a role for the C-terminal self-assembly domain in endocytosis but not in other membrane fission events with which Vps1 has previously been associated. PMID:20841380

  6. Rabconnectin-3a Regulates Vesicle Endocytosis and Canonical Wnt Signaling in Zebrafish Neural Crest Migration

    PubMed Central

    Tuttle, Adam M.; Hoffman, Trevor L.; Schilling, Thomas F.

    2014-01-01

    Cell migration requires dynamic regulation of cell–cell signaling and cell adhesion. Both of these processes involve endocytosis, lysosomal degradation, and recycling of ligand–receptor complexes and cell adhesion molecules from the plasma membrane. Neural crest (NC) cells in vertebrates are highly migratory cells, which undergo an epithelial–mesenchymal transition (EMT) to leave the neural epithelium and migrate throughout the body to give rise to many different derivatives. Here we show that the v-ATPase interacting protein, Rabconnectin-3a (Rbc3a), controls intracellular trafficking events and Wnt signaling during NC migration. In zebrafish embryos deficient in Rbc3a, or its associated v-ATPase subunit Atp6v0a1, many NC cells fail to migrate and misregulate expression of cadherins. Surprisingly, endosomes in Rbc3a- and Atp6v0a1-deficient NC cells remain immature but still acidify. Rbc3a loss-of-function initially downregulates several canonical Wnt targets involved in EMT, but later Frizzled-7 accumulates at NC cell membranes, and nuclear B-catenin levels increase. Presumably due to this later Wnt signaling increase, Rbc3a-deficient NC cells that fail to migrate become pigment progenitors. We propose that Rbc3a and Atp6v0a1 promote endosomal maturation to coordinate Wnt signaling and intracellular trafficking of Wnt receptors and cadherins required for NC migration and cell fate determination. Our results suggest that different v-ATPases and associated proteins may play cell-type-specific functions in intracellular trafficking in many contexts. PMID:24802872

  7. Visualization of clathrin-mediated endocytosis in live Drosophila egg chambers

    PubMed Central

    Jha, Anupma; Traub, Linton M.

    2015-01-01

    Summary In oviparous animals, clathrin-dependent endocytosis is often critical to stockpile a necessary supply of yolk within the maturing oocyte, which enables subsequent embryonic development. In the physically linked chains of maturing egg chambers within the Drosophila melanogaster ovary, a distinct, morphologically discernable, subset undergoes a massive burst clathrin-mediated endocytosis to accumulate yolk in a process termed vitellogenesis. Here, we describe how to prepare isolated ovaries to follow endocytosis, and detail approaches to follow live uptake of soluble reporters into vitellogenic Drosophila egg chambers. PMID:24947394

  8. Bulk-like endocytosis plays an important role in the recycling of insulin granules in pancreatic beta cells.

    PubMed

    Wen, Du; Xue, Yanhong; Liang, Kuo; Yuan, Tianyi; Lu, Jingze; Zhao, Wei; Xu, Tao; Chen, Liangyi

    2012-08-01

    Although bulk endocytosis has been found in a number of neuronal and endocrine cells, the molecular mechanism and physiological function of bulk endocytosis remain elusive. In pancreatic beta cells, we have observed bulk-like endocytosis evoked both by flash photolysis and trains of depolarization. Bulk-like endocytosis is a clathrin-independent process that is facilitated by enhanced extracellular Ca(2+) entry and suppressed by the inhibition of dynamin function. Moreover, defects in bulk-like endocytosis are accompanied by hyperinsulinemia in primary beta cells dissociated from diabetic KKAy mice, which suggests that bulk-like endocytosis plays an important role in maintaining the exo-endocytosis balance and beta cell secretory capability. PMID:22729398

  9. Phosphorylation of threonine 156 of the mu2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo.

    PubMed

    Olusanya, O; Andrews, P D; Swedlow, J R; Smythe, E

    2001-06-01

    The clathrin-coated pit is the major port of entry for many receptors and pathogens and is the paradigm for membrane-based sorting events in higher cells [1]. Recently, it has been possible to reconstitute in vitro the events leading to assembly, invagination, and budding off of clathrin-coated vesicles, allowing dissection of the machinery required for sequestration of receptors into these structures [2-6]. The AP2 adaptor complex is a key element of this machinery linking receptors to the coat lattice, and it has previously been reported that AP2 can be phosphorylated both in vitro and in vivo [7-10]. However, the physiological significance of this has never been established. Here, we show that phosphorylation of a single threonine residue (Thr156) of the mu2 subunit of the AP2 complex is essential for efficient endocytosis of transferrin both in an in vitro coated-pit budding assay and in living cells. PMID:11516654

  10. The antinociceptive effects of ferulic acid on neuropathic pain: involvement of descending monoaminergic system and opioid receptors.

    PubMed

    Xu, Ying; Lin, Dan; Yu, Xuefeng; Xie, Xupei; Wang, Liqun; Lian, Lejing; Fei, Ning; Chen, Jie; Zhu, Naping; Wang, Gang; Huang, Xianfeng; Pan, Jianchun

    2016-04-12

    Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress-related depression and respond to similar treatment. Ferulic acid (FA) is a major active component of angelica sinensis and has been reported to exert antidepressant-like effects; however, it remains unknown whether FA ameliorate chronic constriction injury (CCI)-induced neuropathic pain and the involvement of descending monoaminergic system and opioid receptors. Chronic treatment with FA (20, 40 and 80 mg/kg) ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal noradrenaline (NA) and serotonin (5-HT) levels. Subsequent study suggested that treatment of CCI animals with 40 and 80 mg/kg FA also inhibited spinal MAO-A levels. FA's effects on mechanical allodynia or thermal hyperalgesiawas blocked by 6-hydroxydopamine (6-OHDA) or p-chlorophenylalanine (PCPA) via pharmacological depletion of spinal noradrenaline or serotonin. Moreover, the anti-allodynic action of FA on mechanical stimuli was prevented by pre-treatment with beta2-adrenoceptor antagonist ICI 118,551, or by the delta-opioid receptor antagonist naltrindole. While the anti-hyperalgesia on thermal stimuli induced by FA was blocked by pre-treatment with 5-HT1A receptor antagonist WAY-100635, or with the irreversible mu-opioid receptor antagonist beta-funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via amelioration of the descending monoaminergic system that coupled with spinal beta2- and 5-HT1A receptors and the downstream delta- and mu-opioid receptors differentially. PMID:26967251

  11. The antinociceptive effects of ferulic acid on neuropathic pain: involvement of descending monoaminergic system and opioid receptors

    PubMed Central

    Xu, Ying; Lin, Dan; Yu, Xuefeng; Xie, Xupei; Wang, Liqun; Lian, Lejing; Fei, Ning; Chen, Jie; Zhu, Naping; Wang, Gang; Huang, Xianfeng; Pan, Jianchun

    2016-01-01

    Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress-related depression and respond to similar treatment. Ferulic acid (FA) is a major active component of angelica sinensis and has been reported to exert antidepressant-like effects; however, it remains unknown whether FA ameliorate chronic constriction injury (CCI)-induced neuropathic pain and the involvement of descending monoaminergic system and opioid receptors. Chronic treatment with FA (20, 40 and 80 mg/kg) ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal noradrenaline (NA) and serotonin (5-HT) levels. Subsequent study suggested that treatment of CCI animals with 40 and 80 mg/kg FA also inhibited spinal MAO-A levels. FA's effects on mechanical allodynia or thermal hyperalgesiawas blocked by 6-hydroxydopamine (6-OHDA) or p-chlorophenylalanine (PCPA) via pharmacological depletion of spinal noradrenaline or serotonin. Moreover, the anti-allodynic action of FA on mechanical stimuli was prevented by pre-treatment with beta2-adrenoceptor antagonist ICI 118,551, or by the delta-opioid receptor antagonist naltrindole. While the anti-hyperalgesia on thermal stimuli induced by FA was blocked by pre-treatment with 5-HT1A receptor antagonist WAY-100635, or with the irreversible mu-opioid receptor antagonist beta-funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via amelioration of the descending monoaminergic system that coupled with spinal beta2- and 5-HT1A receptors and the downstream delta- and mu-opioid receptors differentially. PMID:26967251

  12. Postmortem changes in rat brain: studies on membrane-bound enzymes and receptors.

    PubMed

    Syapin, P J; Ritchie, T; Noble, L; Noble, E P

    1987-04-01

    The relationship between the stability of potential neurochemical markers and autolysis time was studied at 4 degrees C and 25 degrees C using postmortem brain samples from two rat strains. In general, qualitatively similar results were obtained with either N/Nih or Sprague-Dawley rats; however, quantitative differences were often observed, particularly in regard to benzodiazepine receptor changes. For every enzyme activity or binding property examined, no significant change was found when brains were kept at 4 degrees C for up to 72 h prior to freezing at -70 degrees C. Na,K-ATPase and low-affinity Ca-ATPase activities were also stable in brains kept at 25 degrees C for up to 72 h. Mg-ATPase activity was reduced in brains kept at 25 degrees C for 24 and 48 h. [3H]Guanidinoethylmercaptosuccinic acid [( 3H]GEMSA) binding to enkephalin convertase in the cytosol was not significantly changed in brains kept at 25 degrees C; however, a small increase was seen for [3H]GEMSA binding to the membrane fraction at 24, but not 48 and 72 h postmortem. [3H]Quinuclidinyl benzilate [( 3H]QNB) binding to muscarinic cholinergic receptors decreased in brains kept at 25 degrees C for 72 h. Opioid receptor binding also decreased in brains kept at 25 degrees C. Using [3H]2-D-alanine-5-D-leucine enkephalin to label delta opioid receptors, a statistically significant decrease in binding was observed as early as 6 h postmortem, and was completely abolished after 72 h at 25 degrees C. In contrast, [3H]naloxone binding was unchanged after 24 h at 25 degrees C, but was decreased after 48 and 72 h.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3029332

  13. Sensing the delivery and endocytosis of nanoparticles using magneto-photo-acoustic imaging

    PubMed Central

    Qu, M.; Mehrmohammadi, M.; Emelianov, S.Y.

    2015-01-01

    Many biomedical applications necessitate a targeted intracellular delivery of the nanomaterial to specific cells. Therefore, a non-invasive and reliable imaging tool is required to detect both the delivery and cellular endocytosis of the nanoparticles. Herein, we demonstrate that magneto-photo-acoustic (MPA) imaging can be used to monitor the delivery and to identify endocytosis of magnetic and optically absorbing nanoparticles. The relationship between photoacoustic (PA) and magneto-motive ultrasound (MMUS) signals from the in vitro samples were analyzed to identify the delivery and endocytosis of nanoparticles. The results indicated that during the delivery of nanoparticles to the vicinity of the cells, both PA and MMUS signals are almost linearly proportional. However, accumulation of nanoparticles within the cells leads to nonlinear MMUS-PA relationship, due to non-linear MMUS signal amplification. Therefore, through longitudinal MPA imaging, it is possible to monitor the delivery of nanoparticles and identify the endocytosis of the nanoparticles by living cells. PMID:26640773

  14. Tubular endocytosis drives remodelling of the apical surface during epithelial morphogenesis in Drosophila.

    PubMed

    Fabrowski, Piotr; Necakov, Aleksandar S; Mumbauer, Simone; Loeser, Eva; Reversi, Alessandra; Streichan, Sebastian; Briggs, John A G; De Renzis, Stefano

    2013-01-01

    During morphogenesis, remodelling of cell shape requires the expansion or contraction of plasma membrane domains. Here we identify a mechanism underlying the restructuring of the apical surface during epithelial morphogenesis in Drosophila. We show that the retraction of villous protrusions and subsequent apical plasma membrane flattening is an endocytosis-driven morphogenetic process. Quantitation of endogenously tagged GFP::Rab5 dynamics reveals a massive increase in apical endocytosis that correlates with changes in apical morphology. This increase is accompanied by the formation of tubular plasma membrane invaginations that serve as platforms for the de novo generation of Rab5-positive endosomes. We identify the Rab5-effector Rabankyrin-5 as a regulator of this pathway and demonstrate that blocking dynamin activity results in the complete inhibition of tubular endocytosis, in the disappearance of Rab5 endosomes, and in the inhibition of surface flattening. These data collectively demonstrate a requirement for endocytosis in morphogenetic remodelling during epithelial development. PMID:23921440

  15. Recording the dynamic endocytosis of single gold nanoparticles by AFM-based force tracing

    NASA Astrophysics Data System (ADS)

    Ding, Bohua; Tian, Yongmei; Pan, Yangang; Shan, Yuping; Cai, Mingjun; Xu, Haijiao; Sun, Yingchun; Wang, Hongda

    2015-04-01

    We utilized force tracing to directly record the endocytosis of single gold nanoparticles (Au NPs) with different sizes, revealing the size-dependent endocytosis dynamics and the crucial role of membrane cholesterol. The force, duration and velocity of Au NP invagination are accurately determined at the single-particle and microsecond level unprecedentedly.We utilized force tracing to directly record the endocytosis of single gold nanoparticles (Au NPs) with different sizes, revealing the size-dependent endocytosis dynamics and the crucial role of membrane cholesterol. The force, duration and velocity of Au NP invagination are accurately determined at the single-particle and microsecond level unprecedentedly. Electronic supplementary information (ESI) available: Details of the experimental procedures and the results of the control experiments. See DOI: 10.1039/c5nr01020a

  16. Endocytosis is required for synaptic activity-dependent release of amyloid-β in vivo

    PubMed Central

    Cirrito, John R.; Kang, Jae-Eun; Lee, Jiyeon; Stewart, Floy R.; Verges, Deborah K.; Silverio, Luz M.; Bu, Guojun; Mennerick, Steven; Holtzman, David M.

    2008-01-01

    Aggregation of amyloid-β (Aβ) peptide into soluble and insoluble forms within the brain extracellular space is central to the pathogenesis of Alzheimer’s disease. Full length amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce Aβ. Aβ is subsequently released into the brain interstitial fluid (ISF). We hypothesized that synaptic transmission results in more APP endocytosis, thereby increasing Aβ generation and release into the ISF. We found that inhibition of clathrin-mediated endocytosis immediately lowers ISF Aβ levels in vivo. Two distinct methods which increased synaptic transmission resulted in an elevation of ISF Aβ levels. Inhibition of endocytosis, however, prevented the activity-dependent increase in Aβ. We estimate that ~70% of ISF Aβ arises from endocytosis-associated mechanisms with the vast majority of this pool also dependent on synaptic activity. These findings have implications for AD pathogenesis and may provide insights into therapeutic intervention. PMID:18400162

  17. Sugar-induced endocytosis of plant 7TM-RGS proteins

    PubMed Central

    Phan, Nguyen; Urano, Daisuke; Srba, Miroslav; Fischer, Lukas; Jones, Alan M.

    2013-01-01

    Plant cells use sugars mainly as a source or store of energy and carbon skeletons for anabolic reactions and for osmotic regulation. The perception of sugars and their responses are rather complex including the heterotrimeric G protein pathway and a seven-transmembrane RGS molecule. Previously, we found that endocytosis of the 7TM-RGS leads to sustained activation of the G protein pathway in the genetic model Arabidopsis. Here we show that other plants possess similar endocytosis systems of the 7TM-RGS proteins. A phosphorylation site essential for the endocytosis is well conserved in land plant 7TM-RGS proteins. In addition, conifer and tobacco 7TM-RGS proteins are internalized in response to sugar. These results indicate a universal mechanism to activate G signaling by endocytosis in plant cells that have 7TM-RGS proteins. PMID:23154506

  18. Actin-Based Feedback Circuits in Cell Migration and Endocytosis

    NASA Astrophysics Data System (ADS)

    Wang, Xinxin

    In this thesis, we study the switch and pulse functions of actin during two important cellular processes, cell migration and endocytosis. Actin is an abundant protein that can polymerize to form a dendritic network. The actin network can exert force to push or bend the cell membrane. During cell migration, the actin network behaves like a switch, assembling mostly at one end or at the other end. The end with the majority of the actin network is the leading edge, following which the cell can persistently move in the same direction. The other end, with the minority of the actin network, is the trailing edge, which is dragged by the cell as it moves forward. When subjected to large fluctuations or external stimuli, the leading edge and the trailing edge can interchange and change the direction of motion, like a motion switch. Our model of the actin network in a cell reveals that mechanical force is crucial for forming the motion switch. We find a transition from single state symmetric behavior to switch behavior, when tuning parameters such as the force. The model is studied by both stochastic simulations, and a set of rate equations that are consistent with the simulations. Endocytosis is a process by which cells engulf extracellular substances and recycle the cell membrane. In yeast cells, the actin network is transiently needed to overcome the pressure difference across the cell membrane caused by turgor pressure. The actin network behaves like a pulse, which assembles and then disassembles within about 30 seconds. Using a stochastic model, we reproduce the pulse behaviors of the actin network and one of its regulatory proteins, Las17. The model matches green fluorescence protein (GFP) experiments for wild-type cells. The model also predicts some phenotypes that modify or diminish the pulse behavior. The phenotypes are verified with both experiments performed at Washington University and with other groups' experiments. We find that several feedback mechanisms are

  19. Different dynamin blockers interfere with distinct phases of synaptic endocytosis during stimulation in motoneurones

    PubMed Central

    Linares-Clemente, Pedro; Rozas, José L; Mircheski, Josif; García-Junco-Clemente, Pablo; Martínez-López, José A; Nieto-González, José L; Vázquez, M Eugenio; Pintado, C Oscar; Fernández-Chacón, Rafael

    2015-01-01

    Key points Neurotransmitter release requires a tight coupling between synaptic vesicle exocytosis and endocytosis with dynamin being a key protein in that process. We used imaging techniques to examine the time course of endocytosis at mouse motor nerve terminals expressing synaptopHluorin, a genetically encoded reporter of the synaptic vesicle cycle. We separated two sequential phases of endocytosis taking place during the stimulation train: early and late endocytosis. Freshly released synaptic vesicle proteins are preferentially retrieved during the early phase, which is very sensitive to dynasore, an inhibitor of dynamin GTPase activity. Synaptic vesicle proteins pre-existing at the plasma membrane before the stimulation are preferentially retrieved during the late phase, which is very sensitive to myristyl trimethyl ammonium bromide (MitMAB), an inhibitor of the dynamin–phospholipid interaction. Abstract Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by exocytosis. Here, at the neuromuscular junction of synaptopHluorin (spH) transgenic mice, we have used imaging to study exo- and endocytosis occurring simultaneously during nerve stimulation. We observed two endocytosis components, which occur sequentially during stimulation. The early component of endocytosis apparently internalizes spH molecules freshly exocytosed. This component was sensitive to dynasore, a blocker of dynamin 1 GTPase activity. In contrast, this early component was resistant to myristyl trimethyl ammonium bromide (MiTMAB), a competitive agent that blocks dynamin binding to phospholipid membranes. The late component of endocytosis is likely to internalize spH molecules that pre-exist at the plasma membrane before stimulation starts. This component was blocked by MiTMAB, perhaps by impairing the binding of dynamin or other key endocytic proteins to phospholipid membranes. Our study suggests the co-existence of two sequential synaptic endocytosis

  20. Synthesis and evaluation of aryl-naloxamide opiate analgesics targeting truncated exon 11-associated mu opioid receptor (MOR-1) splice variants

    PubMed Central

    Majumdar, Susruta; Subrath, Joan; Le Rouzic, Valerie; Polikar, Lisa; Burgman, Maxim; Nagakura, Kuni; Ocampo, Julie; Haselton, Nathan; Pasternak, Anna R.; Grinnell, Steven; Pan, Ying-Xian; Pasternak, Gavril W.

    2012-01-01

    3-Iodobenzoylnaltrexamide 1 (IBNtxA) is a potent analgesic acting through a novel receptor target that lack many side-effects of traditional opiates composed, in part, of exon 11-associated truncated six transmembrane domain MOR-1 (6TM/E11) splice variants. To better understand the SAR of this drug target, a number of 4,5-epoxymorphinan analogs were synthesized. Results show the importance of a free 3-phenolic group, a phenyl ring at the 6 position, an iodine at the 3′ or 4′ position of the phenyl ring and an N-allyl or c-propylmethyl group to maintain high 6TM/E11 affinity and activity. 3-Iodobenzoylnaloxamide 15 (IBNalA) with a N-allyl group displayed lower delta opioid receptor affinity than its naltrexamine analog, was 10-fold more potent an analgesic than morphine, elicited no respiratory depression or physical dependence and only limited inhibition of gastrointestinal transit. Thus, the aryl-naloxamide scaffold can generate a potent analgesic acting through the 6TM/E11 sites with advantageous side-effect profile and greater selectivity. PMID:22734622

  1. Adaptor Protein Complex 2 (AP-2) Mediated, Clathrin Dependent Endocytosis, And Related Gene Activities, Are A Prominent Feature During Maturation Stage Amelogenesis

    PubMed Central

    LACRUZ, Rodrigo S.; BROOKES, Steven J.; WEN, Xin; JIMENEZ, Jaime M.; VIKMAN, Susanna; HU, Ping; WHITE, Shane N.; LYNGSTADAAS, S. Petter; OKAMOTO, Curtis T.; SMITH, Charles E.; PAINE, Michael L.

    2012-01-01

    Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1), cluster of differentiation 63 and 68 (Cd63 and Cd68), ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2), ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2), chloride channel, voltage-sensitive 7 (Clcn7) and cathepsin K (Ctsk). Immunohistological data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain® showed up-regulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor regulated pathway for the endocytosis of enamel matrix proteins. These data together

  2. CD82 endocytosis and cholesterol-dependent reorganization of tetraspanin webs and lipid rafts

    PubMed Central

    Xu, Congfeng; Zhang, Yanhui H.; Thangavel, Muthusamy; Richardson, Mekel M.; Liu, Li; Zhou, Bin; Zheng, Yi; Ostrom, Rennolds S.; Zhang, Xin A.

    2009-01-01

    Tetraspanin CD82 suppresses cell migration, tumor invasion, and tumor metastasis. To determine the mechanism by which CD82 inhibits motility, most studies have focused on the cell surface CD82, which forms tetraspanin-enriched microdomains (TEMs) with other transmembrane proteins, such as integrins. In this study, we found that CD82 undergoes endocytosis and traffics to endosomes and lysosomes. To determine the endocytic mechanism of CD82, we demonstrated that dynamin and clathrin are not essential for CD82 internalization. Depletion or sequestration of sterol in the plasma membrane markedly inhibited the endocytosis of CD82. Despite the demand on Cdc42 activity, CD82 endocytosis is distinct from macropinocytosis and the documented dynamin-independent pinocytosis. As a TEM component, CD82 reorganizes TEMs and lipid rafts by redistributing cholesterol into these membrane microdomains. CD82-containing TEMs are characterized by the cholesterol-containing microdomains in the extreme light- and intermediate-density fractions. Moreover, the endocytosis of CD82 appears to alleviate CD82-mediated inhibition of cell migration. Taken together, our studies demonstrate that lipid-dependent endocytosis drives CD82 trafficking to late endosomes and lysosomes, and CD82 reorganizes TEMs and lipid rafts through redistribution of cholesterol.—Xu, C., Zhang, Y. H., Thangavel, M., Richardson, M. M., Liu, L., Zhou, B., Zheng, Y., Ostrom, R. S., Zhang, X. A. CD82 endocytosis and cholesterol-dependent reorganization of tetraspanin webs and lipid rafts. PMID:19497983

  3. Quantification of endocytosis using a folate functionalized silica hollow nanoshell platform.

    PubMed

    Sandoval, Sergio; Mendez, Natalie; Alfaro, Jesus G; Yang, Jian; Aschemeyer, Sharraya; Liberman, Alex; Trogler, William C; Kummel, Andrew C

    2015-08-01

    A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N -hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900  μg , respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization. PMID:26315280

  4. ‘Delayed’ endocytosis is regulated by extracellular Ca2+ in snake motor boutons

    PubMed Central

    Teng, Haibing; Wilkinson, Robert S

    2003-01-01

    When cooled below ≈7 °C, recently endocytosed vesicles in the motor terminals of the garter snake fail to shed their clathrin coats. Perhaps as a result, the terminals complete only about one-half of the compensatory endocytosis expected after a given period of stimulation. Upon return to room temperature (RT), endocytosis resumes immediately and is complete within minutes. This ‘delayed’ endocytosis following release from cold block provides an opportunity to study clathrin-dependent endocytotic mechanisms in temporal isolation from those events, such as Ca2+ entry and consequent exocytosis, that are normally associated with the activation of nerve terminals. We have taken advantage of clathrin decoating blockade to examine the rate, temperature dependence and extracellular Ca2+ dependence of endocytosis at the snake nerve-muscle synapse. Endocytosis was fast at RT (complete in < 1 min) and markedly faster still at 35 °C. Moreover, the rate of endocytosis varied significantly with change in [Ca2+]o; the rate at 7.2 mM (single exponential time constant, ≈3 s) was approximately double that at 0 mM (single exponential time constant, ≈7 s). Thus, membrane retrieval via clathrin is rapid and, due to its dependence on [Ca2+]o, potentially regulated by changes in the milieu of the synaptic cleft during neural activity. PMID:12813154

  5. Interferon Gamma Receptor: The Beginning of the Journey

    PubMed Central

    Blouin, Cédric M.; Lamaze, Christophe

    2013-01-01

    Our view of endocytosis and membrane trafficking of transmembrane receptors has dramatically changed over the last 20 years. Several new endocytic routes have been discovered and mechanistically characterized in mammalian cells. Long considered as a passive means to terminate signaling through down-regulation of the number of activated receptors at the plasma membrane, it is now established that receptor endocytosis and endosomal sorting can be directly linked to the regulation of intracellular signaling pathways. The functional links between membrane trafficking of interferon receptors and JAK/STAT signaling have recently begun to be unraveled. These studies raise the exciting possibility that a certain level of signal specificity can be achieved through endocytosis and selective localization of the activated complexes within cellular membranes. The ongoing development of high-resolution cell imaging techniques with better spatial and temporal resolution gives new means of deciphering the inherent complexity of membrane trafficking and signaling. This should help to better comprehend the molecular mechanisms by which endocytosis and endosomal sorting of interferon receptors can orchestrate signaling selectivity within the JAK/STAT pathway that can be activated by as many as 60 different cytokines, growth factors, and hormones. PMID:24027571

  6. Coupling between endocytosis and sphingosine kinase 1 recruitment.

    PubMed

    Shen, Hongying; Giordano, Francesca; Wu, Yumei; Chan, Jason; Zhu, Chen; Milosevic, Ira; Wu, Xudong; Yao, Kai; Chen, Bo; Baumgart, Tobias; Sieburth, Derek; De Camilli, Pietro

    2014-07-01

    Genetic studies have suggested a functional link between cholesterol/sphingolipid metabolism and endocytic membrane traffic. Here we show that perturbing the cholesterol/sphingomyelin balance in the plasma membrane results in the massive formation of clusters of narrow endocytic tubular invaginations positive for N-BAR proteins. These tubules are intensely positive for sphingosine kinase 1 (SPHK1). SPHK1 is also targeted to physiologically occurring early endocytic intermediates, and is highly enriched in nerve terminals, which are cellular compartments specialized for exo/endocytosis. Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme's surface. The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme. Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling. PMID:24929359

  7. Casein Kinase 1 Promotes Initiation of Clathrin-Mediated Endocytosis

    PubMed Central

    Peng, Yutian; Grassart, Alexandre; Lu, Rebecca; Wong, Catherine C. L.; Yates, John; Barnes, Georjana; Drubin, David G.

    2014-01-01

    Summary In budding yeast, over 60 proteins functioning in at least 5 modules are recruited to endocytic sites with predictable order and timing. However, how sites of clathrin-mediated endocytosis are initiated and stabilized is not well understood. Here, the casein kinase 1 (CK1) Hrr25 is shown to be an endocytic protein and to be among the earliest proteins to appear at endocytic sites. Hrr25 absence or overexpression decreases or increases the rate of endocytic site initiation, respectively. Ede1, an early endocytic Eps15-like protein important for endocytic initiation, is an Hrr25 target and is required for Hrr25 recruitment to endocytic sites. Hrr25 phosphorylation of Ede1 is required for Hrr25-Ede1 interaction and promotes efficient initiation of endocytic sites. These observations indicate that Hrr25 kinase and Ede1 cooperate to initiate and stabilize endocytic sites. Analysis of the mammalian homologs CK1δ/ε suggests a conserved role for these protein kinases in endocytic site initiation and stabilization. PMID:25625208

  8. Casein kinase 1 promotes initiation of clathrin-mediated endocytosis.

    PubMed

    Peng, Yutian; Grassart, Alexandre; Lu, Rebecca; Wong, Catherine C L; Yates, John; Barnes, Georjana; Drubin, David G

    2015-01-26

    In budding yeast, over 60 proteins functioning in at least five modules are recruited to endocytic sites with predictable order and timing. However, how sites of clathrin-mediated endocytosis are initiated and stabilized is not well understood. Here, the casein kinase 1 (CK1) Hrr25 is shown to be an endocytic protein and to be among the earliest proteins to appear at endocytic sites. Hrr25 absence or overexpression decreases or increases the rate of endocytic site initiation, respectively. Ede1, an early endocytic Eps15-like protein important for endocytic initiation, is an Hrr25 target and is required for Hrr25 recruitment to endocytic sites. Hrr25 phosphorylation of Ede1 is required for Hrr25-Ede1 interaction and promotes efficient initiation of endocytic sites. These observations indicate that Hrr25 kinase and Ede1 cooperate to initiate and stabilize endocytic sites. Analysis of the mammalian homologs CK1δ/ε suggests a conserved role for these protein kinases in endocytic site initiation and stabilization. PMID:25625208

  9. Quantifying endocytosis in vivo using intravital two-photon microscopy.

    PubMed

    Sandoval, Ruben M; Molitoris, Bruce A

    2008-01-01

    The recent introduction of multiphoton microscopy coupled with advances in optics, computer sciences, designer fluorophores, molecular labeling, and previously developed physiologic approaches have empowered investigators to quantitatively study the cell-specific dynamic events, such as endocytosis, within a functioning organ with subcellular resolution. This rapidly emerging field of investigation, with superior spatial and temporal resolution and high sensitivity, enables investigators to track molecules and determine their mode of cellular uptake, intracellular trafficking, and metabolism in a cell-specific fashion in complex heterogeneous organs such as the kidney with repeated determinations possible over a prolonged period of time. This approach is enhanced by the ability to obtain and quantify volumetric data with using up to three different fluorophores simultaneously. We have utilized this intravital approach to understand and quantify kidney proximal tubule cell uptake and intracellular distribution and metabolism of fluorescently labeled molecules, including folic acid, gentamicin, and small interfering ribonucleic acid (siRNA). Limitations of this technique include tissue penetration, which is the major barrier to successful clinical utilization of this technology. However, its use in preclinical animal models offers new insight into physiologic processes and the pathophysiology and treatment of disease processes. PMID:18369960

  10. Endocytosis and transcytosis of albumin gold through mice peritoneal mesothelium.

    PubMed

    Gotloib, L; Shostak, A

    1995-05-01

    The present transmission electron microscopy (TEM) study was designed to investigate whether mesothelial cells of mice diaphragmatic, parietal and mesenteric peritoneum are actively coupled to the mechanisms involved in the transerosal absorption of albumin gold complexes (Alb-Au). Five albino mice were injected intraperitoneally with 0.5 ml of a suspension of Alb-Au. In three animals, in vivo fixation was done 10 minutes after injection of Alb-Au, whereas in the remaining two, fixation was performed 45 minutes after injection of the tracer. At both time intervals, a substantial part of Alb-Au complexes was observed within plasmalemmal and coated vesicles, mainly attached to the luminal aspect of the internal luminal membranes. The amount of Alb-Au contained in plasmalemmal vesicles was significantly higher than that detected in intermesothelial junctions. Plasmalemmal vesicles were observed discharging Alb-Au complexes in the submesothelial interstitium, showing a significantly higher proportion of the tracer associated with non-junctional areas. Evidence presented in this study supports the idea of local degradation of Alb-Au in mesothelial cells after endocytosis, and that of a continuously transcytotic mechanism transporting polymerized albumin across the mesothelial layer. In this sense, transcytotic vesicles could represent the large pore equivalent. PMID:7637257

  11. Coupling between endocytosis and sphingosine kinase I recruitment

    PubMed Central

    Shen, Hongying; Giordano, Francesca; Wu, Yumei; Chan, Jason; Zhu, Chen; Milosevic, Ira; Wu, Xudong; Yao, Kai; Chen, Bo; Baumgart, Tobias; Sieburth, Derek; De Camilli, Pietro

    2014-01-01

    Genetic studies have suggested a functional link between cholesterol/sphingolipid metabolism and endocytic membrane traffic. Here we show that perturbing the cholesterol/sphingomyelin balance in the plasma membrane results in the massive formation of clusters of narrow endocytic tubular invaginations positive for N-BAR proteins. These tubules are intensely positive for sphingosine kinase 1 (SPHK1). SPHK1 is also targeted to physiologically occurring early endocytic intermediates, and is highly enriched in nerve terminals, cellular compartments specialized for exo-endocytosis. Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme’s surface. The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of C. elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme. Our studies support a role of sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signaling. PMID:24929359

  12. A Yeast t-SNARE Involved in Endocytosis

    PubMed Central

    Séron, Karin; Tieaho, Ville; Prescianotto-Baschong, Cristina; Aust, Thomas; Blondel, Marie-Odile; Guillaud, Philippe; Devilliers, Ginette; Rossanese, Olivia W.; Glick, Benjamin S.; Riezman, Howard; Keränen, Sirkka; Haguenauer-Tsapis, Rosine

    1998-01-01

    The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Δ cells, internalization was normal for two endocytic markers, the pheromone α-factor and the plasma membrane uracil permease. In contrast, degradation of α-factor and uracil permease was delayed in tlg2Δ cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis. PMID:9763449

  13. A yeast t-SNARE involved in endocytosis.

    PubMed

    Séron, K; Tieaho, V; Prescianotto-Baschong, C; Aust, T; Blondel, M O; Guillaud, P; Devilliers, G; Rossanese, O W; Glick, B S; Riezman, H; Keränen, S; Haguenauer-Tsapis, R

    1998-10-01

    The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Delta cells, internalization was normal for two endocytic markers, the pheromone alpha-factor and the plasma membrane uracil permease. In contrast, degradation of alpha-factor and uracil permease was delayed in tlg2Delta cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis. PMID:9763449

  14. Berberine Improves Intestinal Motility and Visceral Pain in the Mouse Models Mimicking Diarrhea-Predominant Irritable Bowel Syndrome (IBS-D) Symptoms in an Opioid-Receptor Dependent Manner

    PubMed Central

    Pan, Qiuhui; Fichna, Jakub; Zheng, Lijun; Wang, Kesheng; Yu, Zhen; Li, Yongyu; Li, Kun; Song, Aihong; Liu, Zhongchen; Song, Zhenshun; Kreis, Martin

    2015-01-01

    Background and Aims Berberine and its derivatives display potent analgesic, anti-inflammatory and anticancer activity. Here we aimed at characterizing the mechanism of action of berberine in the gastrointestinal (GI) tract and cortical neurons using animal models and in vitro tests. Methods The effect of berberine was characterized in murine models mimicking diarrhea-predominant irritable bowel syndrome (IBS-D) symptoms. Then the opioidantagonists were used to identify the receptors involved. Furthermore, the effect of berberineon opioid receptors expression was established in the mouse intestine and rat fetal cortical neurons. Results In mouse models, berberine prolonged GI transit and time to diarrhea in a dose-dependent manner, and significantly reduced visceral pain. In physiological conditions the effects of berberine were mediated by mu- (MOR) and delta- (DOR) opioidreceptors; hypermotility, excessive secretion and nociception were reversed by berberine through MOR and DOR-dependent action. We also found that berberine increased the expression of MOR and DOR in the mouse bowel and rat fetal cortical neurons. Conclusion Berberine significantly improved IBS-D symptoms in animal models, possibly through mu- and delta- opioid receptors. Berberine may become a new drug candidate for the successful treatment of IBS-D in clinical conditions. PMID:26700862

  15. Spatial Organization of EphA2 at the Cell-Cell Interface Modulates Trans-Endocytosis of EphrinA1

    PubMed Central

    Greene, Adrienne C.; Lord, Samuel J.; Tian, Aiwei; Rhodes, Christopher; Kai, Hiroyuki; Groves, Jay T.

    2014-01-01

    EphA2 is a receptor tyrosine kinase (RTK) that is sensitive to spatial and mechanical aspects of the cell’s microenvironment. Misregulation of EphA2 occurs in many aggressive cancers. Although its juxtacrine signaling geometry (EphA2’s cognate ligand ephrinA1 is expressed on the surface of an apposing cell) provides a mechanism by which the receptor may experience extracellular forces, this also renders the system challenging to decode. By depositing living cells on synthetic supported lipid membranes displaying ephrinA1, we have reconstituted key features of the juxtacrine EphA2-ephrinA1 signaling system while maintaining the ability to perturb the spatial and mechanical properties of the membrane-cell interface with precision. In addition, we developed a trans-endocytosis assay to monitor internalization of ephrinA1 from a supported membrane into the apposing cell using a quantitative three-dimensional fluorescence microscopy assay. Using this experimental platform to mimic a cell-cell junction, we found that the signaling complex is not efficiently internalized when lateral reorganization at the membrane-cell contact sites is physically hindered. This suggests that EphA2-ephrinA1 trans-endocytosis is sensitive to the mechanical properties of a cell’s microenvironment and may have implications in physical aspects of tumor biology. PMID:24853748

  16. Aptamer-mediated nanoparticle-based protein labeling platform for intracellular imaging and tracking endocytosis dynamics.

    PubMed

    Chen, Li Qiang; Xiao, Sai Jin; Hu, Ping Ping; Peng, Li; Ma, Jun; Luo, Ling Fei; Li, Yuan Fang; Huang, Cheng Zhi

    2012-04-01

    Although nanoparticles have been widely used as optical contrasts for cell imaging, the complicated prefunctionalized steps and low labeling efficiency of nanoprobes greatly inhibit their applications in cellular protein imaging. In this study, we developed a novel and general strategy that employs an aptamer not only as a recognizer for protein recognition but also as a linker for nanoreporter targeting to specifically label membrane proteins of interest and track their endocytic pathway. With this strategy, three kinds of nanoparticles, including gold nanoparticles, silver nanoparticles, and quantum dots (QDs), have been successfully targeted to the membrane proteins of interest, such as nucleolin or prion protein (PrP(C)). The following investigations on the subcellular distribution with fluorescent immunocolocalization assay indicated that PrP(C)-aptamer-QD complexes most likely internalized into cytoplasm through a classical clathrin-dependent/receptor-mediated pathway. Further single-particle tracking and trajectory analysis demonstrated that PrP(C)-aptamer-QD complexes exhibited a complex dynamic process, which involved three types of movements, including membrane diffusion, vesicle transportation, and confined diffusion, and all types of these movements were associated with distinct phases of PrP(C) endocytosis. Compared with traditional multilayer methods, our proposed aptamer-mediated strategy is simple in procedure, avoiding any complicated probe premodification and purification. In particular, the new double-color labeling strategy is unique and significant due to its superior advantages of targeting two signal reporters simultaneously in a single protein using only one aptamer. What is more important, we have constructed a general and versatile aptamer-mediated protein labeling nanoplatform that has shown great promise for future biomedical labeling and intracellular protein dynamic analysis. PMID:22423600

  17. The AP-2 Adaptor β2 Appendage Scaffolds Alternate Cargo Endocytosis

    PubMed Central

    Keyel, Peter A.; Thieman, James R.; Roth, Robyn; Erkan, Elif; Everett, Eric T.; Watkins, Simon C.; Heuser, John E.

    2008-01-01

    The independently folded appendages of the large α and β2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The β2 subunit appendage contains a common binding site for β-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing β2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 α subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the β2 chain with the closely related endogenous β1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both β1 and β2 subunit transcripts recapitulates the strong α subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive β2-yellow fluorescent protein (YFP) expressed in the β1 + β2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the β appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a β2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a β-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with β2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and β-arrestin binding to a site upon the β2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and β-arrestin depend on a privileged β2 appendage site for proper cargo recruitment to clathrin bud sites. PMID:18843039

  18. Recruitment of endocytosis in sonopermeabilization-mediated drug delivery: a real-time study.

    PubMed

    Derieppe, Marc; Rojek, Katarzyna; Escoffre, Jean-Michel; de Senneville, Baudouin Denis; Moonen, Chrit; Bos, Clemens

    2015-07-01

    Microbubbles (MBs) in combination with ultrasound (US) can enhance cell membrane permeability, and have the potential to facilitate the cellular uptake of hydrophilic molecules. However, the exact mechanism behind US- and MB-mediated intracellular delivery still remains to be fully understood. Among the proposed mechanisms are formation of transient pores and endocytosis stimulation. In our study, we investigated whether endocytosis is involved in US- and MB-mediated delivery of small molecules. Dynamic fluorescence microscopy was used to investigate the effects of endocytosis inhibitors on the pharmacokinetic parameters of US- and MB-mediated uptake of SYTOX Green, a 600 Da hydrophilic model drug. C6 rat glioma cells, together with SonoVue(®) MBs, were exposed to 1.4 MHz US waves at 0.2 MPa peak-negative pressure. Collection of the signal intensity in each individual nucleus was monitored during and after US exposure by a fibered confocal fluorescence microscope designed for real-time imaging. Exposed to US waves, C6 cells pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, showed up to a 2.5-fold significant increase of the uptake time constant, and a 1.1-fold increase with genistein, an inhibitor of caveolae-mediated endocytosis. Both inhibitors slowed down the US-mediated uptake of SYTOX Green. With C6 cells and our experimental settings, these quantitative data indicate that endocytosis plays a role in sonopermeabilization-mediated delivery of small molecules with a more predominant contribution of clathrin-mediated endocytosis. PMID:26118644

  19. Recruitment of endocytosis in sonopermeabilization-mediated drug delivery: a real-time study

    NASA Astrophysics Data System (ADS)

    Derieppe, Marc; Rojek, Katarzyna; Escoffre, Jean-Michel; de Senneville, Baudouin Denis; Moonen, Chrit; Bos, Clemens

    2015-07-01

    Microbubbles (MBs) in combination with ultrasound (US) can enhance cell membrane permeability, and have the potential to facilitate the cellular uptake of hydrophilic molecules. However, the exact mechanism behind US- and MB-mediated intracellular delivery still remains to be fully understood. Among the proposed mechanisms are formation of transient pores and endocytosis stimulation. In our study, we investigated whether endocytosis is involved in US- and MB-mediated delivery of small molecules. Dynamic fluorescence microscopy was used to investigate the effects of endocytosis inhibitors on the pharmacokinetic parameters of US- and MB-mediated uptake of SYTOX Green, a 600 Da hydrophilic model drug. C6 rat glioma cells, together with SonoVue® MBs, were exposed to 1.4 MHz US waves at 0.2 MPa peak-negative pressure. Collection of the signal intensity in each individual nucleus was monitored during and after US exposure by a fibered confocal fluorescence microscope designed for real-time imaging. Exposed to US waves, C6 cells pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, showed up to a 2.5-fold significant increase of the uptake time constant, and a 1.1-fold increase with genistein, an inhibitor of caveolae-mediated endocytosis. Both inhibitors slowed down the US-mediated uptake of SYTOX Green. With C6 cells and our experimental settings, these quantitative data indicate that endocytosis plays a role in sonopermeabilization-mediated delivery of small molecules with a more predominant contribution of clathrin-mediated endocytosis.

  20. β-Hydroxybutyrate supports synaptic vesicle cycling but reduces endocytosis and exocytosis in rat brain synaptosomes.

    PubMed

    Hrynevich, Sviatlana V; Waseem, Tatyana V; Hébert, Audrey; Pellerin, Luc; Fedorovich, Sergei V

    2016-02-01

    The ketogenic diet is used as a prophylactic treatment for different types of brain diseases, such as epilepsy or Alzheimer's disease. In such a diet, carbohydrates are replaced by fats in everyday food, resulting in an elevation of blood-borne ketone bodies levels. Despite clinical applications of this treatment, the molecular mechanisms by which the ketogenic diet exerts its beneficial effects are still uncertain. In this study, we investigated the effect of replacing glucose by the ketone body β-hydroxybutyrate as the main energy substrate on synaptic vesicle recycling in rat brain synaptosomes. First, we observed that exposing presynaptic terminals to nonglycolytic energy substrates instead of glucose did not alter the plasma membrane potential. Next, we found that synaptosomes were able to maintain the synaptic vesicle cycle monitored with the fluorescent dye acridine orange when glucose was replaced by β-hydroxybutyrate. However, in presence of β-hydroxybutyrate, synaptic vesicle recycling was modified with reduced endocytosis. Replacing glucose by pyruvate also led to a reduced endocytosis. Addition of β-hydroxybutyrate to glucose-containing incubation medium was without effect. Reduced endocytosis in presence of β-hydroxybutyrate as sole energy substrate was confirmed using the fluorescent dye FM2-10. Also we found that replacement of glucose by ketone bodies leads to inhibition of exocytosis, monitored by FM2-10. However this reduction was smaller than the effect on endocytosis under the same conditions. Using both acridine orange in synaptosomes and the genetically encoded sensor synaptopHluorin in cortical neurons, we observed that replacing glucose by β-hydroxybutyrate did not modify the pH gradient of synaptic vesicles. In conclusion, the nonglycolytic energy substrates β-hydroxybutyrate and pyruvate are able to support synaptic vesicle recycling. However, they both reduce endocytosis. Reduction of both endocytosis and exocytosis together with

  1. Prolonged Activity of the Pestiviral RNase Erns as an Interferon Antagonist after Uptake by Clathrin-Mediated Endocytosis

    PubMed Central

    Zürcher, Christoph; Sauter, Kay-Sara; Mathys, Veronika; Wyss, Fabienne

    2014-01-01

    ABSTRACT The RNase activity of the envelope glycoprotein Erns of the pestivirus bovine viral diarrhea virus (BVDV) is required to block type I interferon (IFN) synthesis induced by single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in bovine cells. Due to the presence of an unusual membrane anchor at its C terminus, a significant portion of Erns is also secreted. In addition, a binding site for cell surface glycosaminoglycans is located within the C-terminal region of Erns. Here, we show that the activity of soluble Erns as an IFN antagonist is not restricted to bovine cells. Extracellularly applied Erns protein bound to cell surface glycosaminoglycans and was internalized into the cells within 1 h of incubation by an energy-dependent mechanism that could be blocked by inhibitors of clathrin-dependent endocytosis. Erns mutants that lacked the C-terminal membrane anchor retained RNase activity but lost most of their intracellular activity as an IFN antagonist. Surprisingly, once taken up into the cells, Erns remained active and blocked dsRNA-induced IFN synthesis for several days. Thus, we propose that Erns acts as an enzymatically active decoy receptor that degrades extracellularly added viral RNA mainly in endolysosomal compartments that might otherwise activate intracellular pattern recognition receptors (PRRs) in order to maintain a state of innate immunotolerance. IMPORTANCE The pestiviral RNase Erns was previously shown to inhibit viral ssRNA- and dsRNA-induced interferon (IFN) synthesis. However, the localization of Erns at or inside the cells, its species specificity, and its mechanism of interaction with cell membranes in order to block the host's innate immune response are still largely unknown. Here, we provide strong evidence that the pestiviral RNase Erns is taken up within minutes by clathrin-mediated endocytosis and that this uptake is mostly dependent on the glycosaminoglycan binding site located within the C-terminal end of the protein

  2. Cytotoxicity mechanism of α-MMC in normal liver cells through LRP1 mediated endocytosis and JNK activation.

    PubMed

    Wang, Ling; Shen, Fubing; Zhang, Min; He, Qianchuan; Zhao, Hui; Yu, Xiaoping; Yang, Shuxia; Liu, Yang; Deng, Nianhua; Zheng, Juecun; Zhu, Lixia; Liu, Xiaolan

    2016-05-16

    Alpha-momorcharin (α-MMC), a type I ribosome-inactivating protein isolated from Momordica charantia, is a potential drug candidate with strong anti-tumor activity. However, α-MMC has a severe hepatotoxicity when applied in vivo, which may greatly hinders its use in clinic in the future. The biological mechanism of hepatotoxicity induced by α-MMC is largely unknown, especially the mechanism by which α-MMC enters the hepatocytes. In this study, we investigated α-MMC-induced cytotoxicity in normal liver L02 cell line as well as the mechanism underlying it. As expected, α-MMC is more toxic in L02 cells than in various normal cells from other organs. The cytotoxic effect of α-MMC on L02 cells is found to be mediated through cell apoptosis as detected by flow cytometry and fluorescence microscopy. Importantly, α-MMC was shown to bind to a specific receptor on cell membrane, as the density of the cell membrane receptor is closely related to both the amount of α-MMC endocytosed and the cytotoxicity in different cell lines. By using LRP1 competitive inhibitor α2-M or siRNA targeting LRP1, we further identified that LRP1 protein served as the membrane receptor for α-MMC. Both α2-M and siRNA targeting LRP1 can significantly inhibit α-MMC's endocytosis as well as its cytotoxicity in L02 cells. In addition, it was found that α-MMC can activate the JNK signalling pathways via LRP1 in L02 cells. As JNK activation often leads to cell apoptosis, the activation of JNK may play an important role in α-MMC-induced cytotoxicity. To our knowledge, this is the first report showing that LRP1 mediates the cytotoxicity of α-MMC through (1) endocytosis and induced apoptosis and (2) the activation of the JNK pathway. Our findings shed light on the fundamental mechanism of hepatotoxicity of α-MMC and offer reference to understand its mechanism of lymphocytotoxicity and neurotoxicity. PMID:27262837

  3. Increased endocytosis and formation of multivesicular bodies in phorbol-ester-stimulated human monoblastic U-937 cells

    SciTech Connect

    Nilsson, M. ); Nilsson, K.; Forsbeck, K. )

    1989-04-01

    The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to arrest mitotic activity and induce macrophage differentiation in the U-937 monoblastic cell line. The acute effect of TPA on ultrastructural morphology and endocytic activity of U-937 cells was studied. TPA induced within 15 minutes {alpha} marked enlargement of multivesicular bodies (MVBs), comprising both volume and number of inclusion vesicles (other organelles appeared unchanged). At this stage the MVBs frequently showed tubular cytoplasmic extensions. Inclusion vesicles accumulated in MVBs with prolonged incubation (60 minutes). Cellular uptake of {sup 125}I-HRP was increased five times the control values already after 5 minutes of TPA stimulation. The uptake increased further with prolonged incubation (60 minutes), but at a slower rate. Together these indicate a TPA-induced transfer by endocytosis of portions of the plasma membrane to the lysosomal system via MVBs. Consideration of MVBs as part of the receptor-mediated endocytic pathway suggests that this effect of TPA might involve down-regulation of cell-surface receptors. The possibility of MVBs as a proton-sequestrating compartment, responsible for the cytoplasmic alkalinization previously reported for TPA-stimulated U-937 monoblastic cells, is discussed.

  4. Induction of hyperphagia and carbohydrate intake by mu-opioid receptor stimulation in circumscribed regions of frontal cortex

    PubMed Central

    Mena, Jesus D.; Sadeghian, Ken; Baldo, Brian A.

    2011-01-01

    Frontal cortical regions are activated by food-associated stimuli, and this activation appears to be dysregulated in individuals with eating disorders. Nevertheless, frontal control of basic unconditioned feeding responses remains poorly understood. Here we show that hyperphagia can be driven by μ-opioid receptor stimulation in restricted regions of ventral medial prefrontal cortex (vmPFC) and orbitofrontal cortex. In both ad libitum-fed and food-restricted male Sprague-Dawley rats, bilateral infusions of the μ-opioid agonist, DAMGO, markedly increased intake of standard rat chow. When given a choice between palatable fat- versus carbohydrate enriched test diets, intra-vmPFC DAMGO infusions selectively increased carbohydrate intake, even in rats with a baseline fat preference. Rats also exhibited motor hyperactivity characterized by rapid switching between brief bouts of investigatory and ingestive behaviors. Intra-vmPFC DAMGO affected neither water intake nor non-specific oral behavior. Similar DAMGO infusions into neighboring areas of lateral orbital or anterior motor cortex had minimal effects on feeding. Neither stimulation of vmPFC-localized delta-opioid, kappa-opioid, dopaminergic, serotonergic, or noradrenergic receptors, nor antagonism of D1, 5HT1A, or alpha- or beta-adrenoceptors, reproduced the profile of DAMGO effects. Muscimol-mediated inactivation of the vmPFC, and intra-vmPFC stimulation of κ-opioid receptors or blockade of 5HT2A receptors, suppressed motor activity and increased feeding bout duration-a profile opposite to that seen with DAMGO. Hence, μ-opioid-induced hyperphagia and carbohydrate intake can be elicited with remarkable pharmacological and behavioral specificity from discrete subterritories of the frontal cortex. These findings may have implications for understanding affect-driven feeding and loss of restraint in eating disorders. PMID:21368037

  5. Immunomodulatory Glycan Lacto-N-Fucopentaose III Requires Clathrin-Mediated Endocytosis To Induce Alternative Activation of Antigen-Presenting Cells

    PubMed Central

    Srivastava, Leena; Tundup, Smanla; Choi, Beak-San; Norberg, Thomas

    2014-01-01

    The mechanism of alternative activation of antigen-presenting cells (APCs) is largely unknown. Lacto-N-fucopentaose III (LNFPIII) is a biologically conserved pentasaccharide that contains the Lewisx trisaccharide. LNFPIII conjugates and schistosome egg antigens, which contain the Lewisx trisaccharide, drive alternative activation of APCs and induce anti-inflammatory responses in vivo, preventing inflammation-based diseases, including psoriasis, transplant organ rejection, and metabolic disease. In this study, we show that LNFPIII conjugates and schistosome egg antigens interact with APCs via a receptor-mediated process, requiring internalization of these molecules through a clathrin/dynamin-dependent but caveolus-independent endocytic pathway. Using inhibitors/small interfering RNA (siRNA) against dynamin and clathrin, we show for the first time that endocytosis of Lewisx-containing glycans is required to drive alternative maturation of antigen-presenting cells and Th2 immune responses. We identified mouse SIGNR-1 as a cell surface receptor for LNFPIII conjugates. Elimination of SIGNR-1 showed no effect on uptake of LNFPIII conjugates, suggesting that other receptors bind to and facilitate uptake of LNFPIII conjugates. We demonstrate that disruption of actin filaments partially prevented the entry of LNFPIII conjugates into APCs and that LNFPIII colocalizes with both early and late endosomal markers and follows the classical endosomal pathway leading to lysosome maturation. The results of this study show that the ability of LNFPIII to induce alternative activation utilizes a receptor-mediated process that requires a dynamin-dependent endocytosis. Thus, key steps have been defined in the previously unknown mechanism of alternative activation that ultimately leads to induction of anti-inflammatory responses. PMID:24566617

  6. Regulation of clathrin coat assembly by Eps15 homology domain–mediated interactions during endocytosis

    PubMed Central

    Suzuki, Ryohei; Toshima, Junko Y.; Toshima, Jiro

    2012-01-01

    Clathrin-mediated endocytosis involves a coordinated series of molecular events regulated by interactions among a variety of proteins and lipids through specific domains. One such domain is the Eps15 homology (EH) domain, a highly conserved protein–protein interaction domain present in a number of proteins distributed from yeast to mammals. Several lines of evidence suggest that the yeast EH domain–containing proteins Pan1p, End3p, and Ede1p play important roles during endocytosis. Although genetic and cell-biological studies of these proteins suggested a role for the EH domains in clathrin-mediated endocytosis, it was unclear how they regulate clathrin coat assembly. To explore the role of the EH domain in yeast endocytosis, we mutated those of Pan1p, End3p, or Ede1p, respectively, and examined the effects of single, double, or triple mutation on clathrin coat assembly. We found that mutations of the EH domain caused a defect of cargo internalization and a delay of clathrin coat assembly but had no effect on assembly of the actin patch. We also demonstrated functional redundancy among the EH domains of Pan1p, End3p, and Ede1p for endocytosis. Of interest, the dynamics of several endocytic proteins were differentially affected by various EH domain mutations, suggesting functional diversity of each EH domain. PMID:22190739

  7. A Wiskott-Aldrich syndrome protein is involved in endocytosis in Aspergillus nidulans.

    PubMed

    Hoshi, Hiro-Omi; Zheng, Lu; Ohta, Akinori; Horiuchi, Hiroyuki

    2016-09-01

    Endocytosis is vital for hyphal tip growth in filamentous fungi and is involved in the tip localization of various membrane proteins. To investigate the function of a Wiskott-Aldrich syndrome protein (WASP) in endocytosis of filamentous fungi, we identified a WASP ortholog-encoding gene, wspA, in Aspergillus nidulans and characterized it. The wspA product, WspA, localized to the tips of germ tubes during germination and actin rings in the subapical regions of mature hyphae. wspA is essential for the growth and functioned in the polarity establishment and maintenance during germination of conidia. We also investigated its function in endocytosis and revealed that endocytosis of SynA, a synaptobrevin ortholog that is known to be endocytosed at the subapical regions of hyphal tips in A. nidulans, did not occur when wspA expression was repressed. These results suggest that WspA plays roles in endocytosis at hyphal tips and polarity establishment during germination. PMID:26927610

  8. Zinc-regulated ubiquitin conjugation signals endocytosis of the yeast ZRT1 zinc transporter.

    PubMed Central

    Gitan, R S; Eide, D J

    2000-01-01

    The yeast ZRT1 zinc transporter is regulated by zinc at both transcriptional and post-translational levels. At the post-translational level, zinc inactivates ZRT1 by inducing the removal of the protein from the plasma membrane by endocytosis. The zinc transporter is subsequently degraded in the vacuole. This regulatory system allows for the rapid shut off of zinc uptake activity in cells exposed to high zinc concentrations, thereby preventing overaccumulation of this potentially toxic metal. In this report, we examine the role of ubiquitin conjugation in this process. First, we show that ZRT1 is ubiquitinated shortly after zinc treatment and before endocytosis. Secondly, mutations in various components of the ubiquitin conjugation pathway, specifically the RSP5 ubiquitin-protein ligase and the UBC4 and UBC5 ubiquitin conjugating enzymes, inhibit both ubiquitination and endocytosis. Finally, mutation of a specific lysine residue in ZRT1 blocks both ubiquitination and endocytosis. This critical lysine, Lys-195, is located in a cytoplasmic loop region of the protein and may be the residue to which ubiquitin is attached. These results demonstrate that ubiquitin conjugation is a critical step in the signal transduction pathway that controls the rate of ZRT1 endocytosis in response to zinc. PMID:10677350

  9. Pan1 regulates transitions between stages of clathrin-mediated endocytosis

    PubMed Central

    Bradford, Mary Katherine; Whitworth, Karen; Wendland, Beverly

    2015-01-01

    Endocytosis is a well-conserved process by which cells invaginate small portions of the plasma membrane to create vesicles containing extracellular and transmembrane cargo proteins. Dozens of proteins and hundreds of specific binding interactions are needed to coordinate and regulate these events. Saccharomyces cerevisiae is a powerful model system with which to study clathrin-mediated endocytosis (CME). Pan1 is believed to be a scaffolding protein due to its interactions with numerous proteins that act throughout the endocytic process. Previous research characterized many Pan1 binding interactions, but due to Pan1's essential nature, the exact mechanisms of Pan1's function in endocytosis have been difficult to define. We created a novel Pan1-degron allele, Pan1-AID, in which Pan1 can be specifically and efficiently degraded in <1 h upon addition of the plant hormone auxin. The loss of Pan1 caused a delay in endocytic progression and weakened connections between the coat/actin machinery and the membrane, leading to arrest in CME. In addition, we determined a critical role for the central region of Pan1 in endocytosis and viability. The regions important for endocytosis and viability can be separated, suggesting that Pan1 may have a distinct role in the cell that is essential for viability. PMID:25631817

  10. Characterization of endocytosis and exocytosis of cationic nanoparticles in airway epithelium cells

    NASA Astrophysics Data System (ADS)

    Youta Dombu, Christophe; Kroubi, Maya; Zibouche, Rima; Matran, Regis; Betbeder, Didier

    2010-09-01

    A major challenge of drug delivery using colloids via the airway is to understand the mechanism implied in their interactions with epithelial cells. The purpose of this work was to characterize the process of endocytosis and exocytosis of cationic nanoparticles (NPs) made of maltodextrin which were developed as a delivery system for antigens in vaccine applications. Confocal microscopy demonstrated that these NP are rapidly endocytosed after as little as 3 min incubation, and that the endocytosis was also faster than NP binding since most of the NPs were found in the middle of the cells around the nuclei. A saturation limit was observed after a 40 min incubation, probably due to an equilibrium becoming established between endocytosis and exocytosis. Endocytosis was dramatically reduced at 4 °C compared with 37 °C, or by NaN3 treatment, both results suggesting an energy dependent process. Protamine pretreatment of the cells inhibited NPs uptake and we found that clathrin pathway is implied in their endocytosis. Cholesterol depletion increased NP uptake by 300% and this phenomenon was explained by the fact that cholesterol depletion totally blocked NP exocytosis. These results suggest that these cationic NPs interact with anionic sites, are quickly endocytosed via the clathrin pathway and that their exocytosis is cholesterol dependent, and are similar to those obtained in other studies with viruses such as influenza.

  11. ER network homeostasis is critical for plant endosome streaming and endocytosis

    PubMed Central

    Stefano, Giovanni; Renna, Luciana; Lai, YaShiuan; Slabaugh, Erin; Mannino, Nicole; Buono, Rafael A; Otegui, Marisa S; Brandizzi, Federica

    2015-01-01

    Eukaryotic cells internalize cargo at the plasma membrane via endocytosis, a vital process that is accomplished through a complex network of endosomal organelles. In mammalian cells, the ER is in close association with endosomes and regulates their fission. Nonetheless, the physiological role of such interaction on endocytosis is yet unexplored. Here, we probed the existence of ER–endosome association in plant cells and assayed its physiological role in endocytosis. Through live-cell imaging and electron microscopy studies, we established that endosomes are extensively associated with the plant ER, supporting conservation of interaction between heterotypic organelles in evolutionarily distant kingdoms. Furthermore, by analyzing ER–endosome dynamics in genetic backgrounds with defects in ER structure and movement, we also established that the ER network integrity is necessary for homeostasis of the distribution and streaming of various endosome populations as well as for efficient endocytosis. These results support a novel model that endocytosis homeostasis depends on a spatiotemporal control of the endosome dynamics dictated by the ER membrane network. PMID:27462431

  12. HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

    PubMed

    Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M

    2016-01-01

    Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment. PMID:26459807

  13. Endocytosis of Multiwalled Carbon Nanotubes in Bronchial Epithelial and Mesothelial Cells

    PubMed Central

    Maruyama, Kayo; Matsuda, Yoshikazu; Kobayashi, Shinsuke; Tanaka, Manabu; Aoki, Kaoru; Takanashi, Seiji; Okamoto, Masanori; Kato, Hiroyuki

    2015-01-01

    Bronchial epithelial cells and mesothelial cells are crucial targets for the safety assessment of inhalation of carbon nanotubes (CNTs), which resemble asbestos particles in shape. Intrinsic properties of multiwalled CNTs (MWCNTs) are known to cause potentially hazardous effects on intracellular and extracellular pathways. These interactions alter cellular signaling and affect major cell functions, resulting in cell death, lysosome injury, reactive oxygen species production, apoptosis, and cytokine release. Furthermore, CNTs are emerging as a novel class of autophagy inducers. Thus, in this study, we focused on the mechanisms of MWCNT uptake into the human bronchial epithelial cells (HBECs) and human mesothelial cells (HMCs). We verified that MWCNTs are actively internalized into HBECs and HMCs and were accumulated in the lysosomes of the cells after 24-hour treatment. Next, we determined which endocytosis pathways (clathrin-mediated, caveolae-mediated, and macropinocytosis) were associated with MWCNT internalization by using corresponding endocytosis inhibitors, in two nonphagocytic cell lines derived from bronchial epithelial cells and mesothelioma cells. Clathrin-mediated endocytosis inhibitors significantly suppressed MWCNT uptake, whereas caveolae-mediated endocytosis and macropinocytosis were also found to be involved in MWCNT uptake. Thus, MWCNTs were positively taken up by nonphagocytic cells, and their cytotoxicity was closely related to these three endocytosis pathways. PMID:26090445