Quantitative in vivo receptor binding. III. Tracer kinetic modeling of muscarinic cholinergic receptor binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frey, K.A.; Hichwa, R.D.; Ehrenkaufer, R.L.

1985-10-01

A tracer kinetic method is developed for the in vivo estimation of high-affinity radioligand binding to central nervous system receptors. Ligand is considered to exist in three brain pools corresponding to free, nonspecifically bound, and specifically bound tracer. These environments, in addition to that of intravascular tracer, are interrelated by a compartmental model of in vivo ligand distribution. A mathematical description of the model is derived, which allows determination of regional blood-brain barrier permeability, nonspecific binding, the rate of receptor-ligand association, and the rate of dissociation of bound ligand, from the time courses of arterial blood and tissue tracer concentrations.more » The term ''free receptor density'' is introduced to describe the receptor population measured by this method. The technique is applied to the in vivo determination of regional muscarinic acetylcholine receptors in the rat, with the use of (TH)scopolamine. Kinetic estimates of free muscarinic receptor density are in general agreement with binding capacities obtained from previous in vivo and in vitro equilibrium binding studies. In the striatum, however, kinetic estimates of free receptor density are less than those in the neocortex--a reversal of the rank ordering of these regions derived from equilibrium determinations. A simplified model is presented that is applicable to tracers that do not readily dissociate from specific binding sites during the experimental period.« less

Endosomal receptor kinetics determine the stability of intracellular growth factor signalling complexes

PubMed Central

Tzafriri, A. Rami; Edelman, Elazer R.

2006-01-01

There is an emerging paradigm that growth factor signalling continues in the endosome and that cell response to a growth factor is defined by the integration of cell surface and endosomal events. As activated receptors in the endosome are exposed to a different set of binding partners, they probably elicit differential signals compared with when they are at the cell surface. As such, complete appreciation of growth factor signalling requires understanding of growth factor–receptor binding and trafficking kinetics both at the cell surface and in endosomes. Growth factor binding to surface receptors is well characterized, and endosomal binding is assumed to follow surface kinetics if one accounts for changes in pH. Yet, specific binding kinetics within the endosome has not been examined in detail. To parse the factors governing the binding state of endosomal receptors we analysed a whole-cell mathematical model of epidermal growth factor receptor trafficking and binding. We discovered that the stability of growth factor–receptor complexes within endosomes is governed by three primary independent factors: the endosomal dissociation constant, total endosomal volume and the number of endosomal receptors. These factors were combined into a single dimensionless parameter that determines the endosomal binding state of the growth factor–receptor complex and can distinguish different growth factors from each other and different cell states. Our findings indicate that growth factor binding within endosomal compartments cannot be appreciated solely on the basis of the pH-dependence of the dissociation constant and that the concentration of receptors in the endosomal compartment must also be considered. PMID:17117924

Platelet activating factor receptor binding plays a critical role in jet fuel-induced immune suppression.

PubMed

Ramos, Gerardo; Kazimi, Nasser; Nghiem, Dat X; Walterscheid, Jeffrey P; Ullrich, Stephen E

2004-03-15

Applying military jet fuel (JP-8) or commercial jet fuel (Jet-A) to the skin of mice suppresses the immune response in a dose-dependent manner. The release of biological response modifiers, particularly prostaglandin E2 (PGE2), is a critical step in activating immune suppression. Previous studies have shown that injecting selective cyclooxygenase-2 inhibitors into jet fuel-treated mice blocks immune suppression. Because the inflammatory phospholipid mediator, platelet-activating factor (PAF), up-regulates cyclooxygenase-2 production and PGE2 synthesis by keratinocytes, we tested the hypothesis that PAF-receptor binding plays a role in jet fuel-induced immune suppression. Treating keratinocyte cultures with PAF and/or jet fuel (JP-8 and Jet-A) stimulates PGE2 secretion. Jet fuel-induced PGE2 production was suppressed by treating the keratinocytes with specific PAF-receptor antagonists. Injecting mice with PAF, or treating the skin of the mice with JP-8, or Jet-A, induced immune suppression. Jet fuel-induced immune suppression was blocked when the jet fuel-treated mice were injected with PAF-receptor antagonists before treatment. Jet fuel treatment has been reported to activate oxidative stress and treating the mice with anti-oxidants (Vitamins C, or E or beta-hydroxy toluene), before jet fuel application, interfered with immune suppression. These findings confirm previous studies showing that PAF-receptor binding can modulate immune function. Furthermore, they suggest that PAF-receptor binding may be an early event in the induction of immune suppression by immunotoxic environmental agents that target the skin.

The Intracellular Juxtamembrane Domain of the Epidermal Growth Factor (EGF) Receptor Is Responsible for the Allosteric Regulation of EGF Binding*S⃞♦

PubMed Central

Macdonald-Obermann, Jennifer L.; Pike, Linda J.

2009-01-01

We have previously shown that the binding of epidermal growth factor (EGF) to its receptor can best be described by a model that involves negative cooperativity in an aggregating system (Macdonald, J. L., and Pike, L. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 112–117). However, despite the fact that biochemical analyses indicate that EGF induces dimerization of its receptor, the binding data provided no evidence for positive linkage between EGF binding and dimer assembly. By analyzing the binding of EGF to a number of receptor mutants, we now report that in naive, unphosphorylated EGF receptors, ligand binding is positively linked to receptor dimerization but the linkage is abolished upon autophosphorylation of the receptor. Both phosphorylated and unphosphorylated EGF receptors exhibit negative cooperativity, indicating that mechanistically, cooperativity is distinct from the phenomenon of linkage. Nonetheless, both the positive linkage and the negative cooperativity observed in EGF binding require the presence of the intracellular juxtamembrane domain. This indicates the existence of inside-out signaling in the EGF receptor system. The intracellular juxtamembrane domain has previously been shown to be required for the activation of the EGF receptor tyrosine kinase (Thiel, K. W., and Carpenter, G. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 19238–19243). Our experiments expand the role of this domain to include the allosteric control of ligand binding by the extracellular domain. PMID:19336395

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

PubMed Central

Owczarek, C M; Layton, M J; Metcalf, D; Lock, P; Willson, T A; Gough, N M; Nicola, N A

1993-01-01

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins. Images PMID:8253075

Analysis of Ethylene Receptors: Ethylene-Binding Assays.

PubMed

Binder, Brad M; Schaller, G Eric

2017-01-01

Plant ethylene receptors bind ethylene with high affinity. Most of the characterization of ethylene binding to the receptors has been carried out using a radioligand-binding assay on functional receptors expressed in yeast. In this chapter, we describe methods for expressing ethylene receptors in yeast and conducting ethylene-binding assays on intact yeast and yeast membranes. The ethylene-binding assays can be modified to analyze ethylene binding to intact plants and other organisms as well as membranes isolated from any biological source.

DNA binding triggers tetramerization of the glucocorticoid receptor in live cells

PubMed Central

Presman, Diego M.; Ganguly, Sourav; Schiltz, R. Louis; Johnson, Thomas A.; Karpova, Tatiana S.; Hager, Gordon L.

2016-01-01

Transcription factors dynamically bind to chromatin and are essential for the regulation of genes. Although a large percentage of these proteins appear to self-associate to form dimers or higher order oligomers, the stoichiometry of DNA-bound transcription factors has been poorly characterized in vivo. The glucocorticoid receptor (GR) is a ligand-regulated transcription factor widely believed to act as a dimer or a monomer. Using a unique set of imaging techniques coupled with a cell line containing an array of DNA binding elements, we show that GR is predominantly a tetramer when bound to its target DNA. We find that DNA binding triggers an interdomain allosteric regulation within the GR, leading to tetramerization. We therefore propose that dynamic changes in GR stoichiometry represent a previously unidentified level of regulation in steroid receptor activation. Quaternary structure analysis of other members of the steroid receptor family (estrogen, androgen, and progesterone receptors) reveals variation in oligomerization states among this family of transcription factors. Because GR’s oligomerization state has been implicated in therapy outcome, our findings open new doors to the rational design of novel GR ligands and redefine the quaternary structure of steroid receptors. PMID:27382178

The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

NASA Astrophysics Data System (ADS)

Iacob, Roxana E.; Chen, Guodong; Ahn, Joomi; Houel, Stephane; Wei, Hui; Mo, Jingjie; Tao, Li; Cohen, Daniel; Xie, Dianlin; Lin, Zheng; Morin, Paul E.; Doyle, Michael L.; Tymiak, Adrienne A.; Engen, John R.

2014-12-01

The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.

Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation, dimerization and EGF hormone binding.

PubMed

Taylor, Eric S; Pol-Fachin, Laercio; Lins, Roberto D; Lower, Steven K

2017-04-01

The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N-glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF-EGFR binding takes place through a large-scale induced-fitting mechanism. Proteins 2017; 85:561-570. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Digested wheat gluten inhibits binding between leptin and its receptor.

PubMed

Jönsson, Tommy; Memon, Ashfaque A; Sundquist, Kristina; Sundquist, Jan; Olsson, Stefan; Nalla, Amarnadh; Bauer, Mikael; Linse, Sara

2015-01-20

Leptin resistance is considered a primary risk factor for obesity. It has been hypothesized that dietary cereal grain protein could cause leptin resistance by preventing leptin from binding to its receptor. Non-degraded dietary wheat protein has been found in human serum at a mean level of 41 ng/mL. Here, we report our findings from testing whether enzymatically digested gluten from wheat prevents leptin from binding to the leptin receptor in vitro. Gluten from wheat was digested with pepsin and trypsin under physiological conditions. Pepsin and trypsin activity was removed from the gluten digest with a 10 kDa spin-filter or by heat treatment at 100°C for 30 min. Binding to the leptin receptor of leptin mixed with gluten digest at a series of concentrations was measured using surface plasmon resonance technology. Binding of the gluten digest to the leptin receptor was not detected. Spin-filtered gluten digest inhibited binding of leptin to the leptin receptor, with 50% inhibition at a gluten digest concentration of ~10 ng/mL. Heat-treated gluten digest did not inhibit leptin binding. Digested wheat gluten inhibits binding of leptin to the leptin receptor, with half-maximal inhibition at 10 ng/mL. The inhibition is significant at clinically relevant concentrations and could therefore serve as a novel pathway to investigate to understand the molecular basis of leptin resistance, obesity and associated disorders.

NMR study of the transforming growth factor-alpha (TGF-alpha)-epidermal growth factor receptor complex. Visualization of human TGF-alpha binding determinants through nuclear Overhauser enhancement analysis.

PubMed

McInnes, C; Hoyt, D W; Harkins, R N; Pagila, R N; Debanne, M T; O'Connor-McCourt, M; Sykes, B D

1996-12-13

The study of human transforming growth factor-alpha (TGF-alpha) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A- and C-loops of TGF-alpha form the major binding interface, while the B-loop provides a structural scaffold for this site. These results corroborate the conclusions from NMR relaxation studies (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292), which suggest that the C-terminal residues of the polypeptide are immobilized upon receptor binding, while the N terminus of the molecule retains considerable flexibility, and are consistent with structure-function studies of the TGF-alpha/EGF system indicating a multidomain binding model. These results give a visualization, for the first time, of native TGF-alpha in complex with the EGF receptor and generate a picture of the ligand-binding site based upon the intact molecule. This will undoubtedly be of utility in the structure-based design of TGF-alpha/EGF agonists and/or antagonists.

Azadirachtin interacts with the tumor necrosis factor (TNF) binding domain of its receptors and inhibits TNF-induced biological responses.

PubMed

Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A; Manna, Sunil K

2010-02-19

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses*

PubMed Central

Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A.; Manna, Sunil K.

2010-01-01

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy. PMID:20018848

Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding

PubMed Central

Broghammer, Angelique; Krusell, Lene; Blaise, Mickaël; Sauer, Jørgen; Sullivan, John T.; Maolanon, Nicolai; Vinther, Maria; Lorentzen, Andrea; Madsen, Esben B.; Jensen, Knud J.; Roepstorff, Peter; Thirup, Søren; Ronson, Clive W.; Thygesen, Mikkel B.; Stougaard, Jens

2012-01-01

Lipochitin oligosaccharides called Nod factors function as primary rhizobial signal molecules triggering legumes to develop new plant organs: root nodules that host the bacteria as nitrogen-fixing bacteroids. Here, we show that the Lotus japonicus Nod factor receptor 5 (NFR5) and Nod factor receptor 1 (NFR1) bind Nod factor directly at high-affinity binding sites. Both receptor proteins were posttranslationally processed when expressed as fusion proteins and extracted from purified membrane fractions of Nicotiana benthamiana or Arabidopsis thaliana. The N-terminal signal peptides were cleaved, and NFR1 protein retained its in vitro kinase activity. Processing of NFR5 protein was characterized by determining the N-glycosylation patterns of the ectodomain. Two different glycan structures with identical composition, Man3XylFucGlcNAc4, were identified by mass spectrometry and located at amino acid positions N68 and N198. Receptor–ligand interaction was measured by using ligands that were labeled or immobilized by application of chemoselective chemistry at the anomeric center. High-affinity ligand binding was demonstrated with both solid-phase and free solution techniques. The Kd values obtained for Nod factor binding were in the nanomolar range and comparable to the concentration range sufficient for biological activity. Structure-dependent ligand specificity was shown by using chitin oligosaccharides. Taken together, our results suggest that ligand recognition through direct ligand binding is a key step in the receptor-mediated activation mechanism leading to root nodule development in legumes. PMID:22859506

The kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

PubMed

Yandell, C A; Dunbar, A J; Wheldrake, J F; Upton, Z

1999-09-17

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.

Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro.

PubMed

Jonsson, Andreas; Wållberg, Helena; Herne, Nina; Ståhl, Stefan; Frejd, Fredrik Y

2009-08-17

Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For ZTNF-alpha:185, subnanomolar affinity (KD=0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the ZTNF-alpha:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

Dual chain synthetic heparin-binding growth factor analogs

DOEpatents

Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

2012-04-24

The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

Dual chain synthetic heparin-binding growth factor analogs

DOEpatents

Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

2009-10-06

The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baumgold, J.

1986-05-01

Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and themore » remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.« less

Quantification of transcription factor-DNA binding affinity in a living cell

PubMed Central

Belikov, Sergey; Berg, Otto G.; Wrange, Örjan

2016-01-01

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [3H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. PMID:26657626

Aqueous and Ethanolic Valeriana officinalis Extracts Change the Binding of Ligands to Glutamate Receptors.

PubMed

Del Valle-Mojica, Lisa M; Cordero-Hernández, José M; González-Medina, Giselle; Ramos-Vélez, Igmeris; Berríos-Cartagena, Nairimer; Torres-Hernández, Bianca A; Ortíz, José G

2011-01-01

The effects of two valerian extracts (aqueous and hydroalcoholic) were investigated through [(3)H]Glutamate ([(3)H]Glu) and [(3)H]Fluorowillardine ([(3)H]FW) receptor binding assays using rat synaptic membranes in presence of different receptor ligands. In addition, the extract stability was monitored spectrophotometrically. Both extracts demonstrated interaction with ionotropic glutamate receptors (iGluRs). However, the extracts displayed considerable differences in receptor selectivity. The hydroalcoholic extract selectively interacted with quisqualic acid (QA), group I metabotropic glutamate receptor (mGluR) ligand, while the aqueous extract did not alter the binding of QA. The stability of the extracts was examined during several weeks. Freshly prepared extract inhibited 38-60% of [(3)H]FW binding (AMPA). After 10 days, the aqueous extract inhibited 85% of [(3)H]FW binding while the hydroalcoholic extract markedly potentiated (200%) [(3)H]FW binding to AMPA receptors. Thus, our results showed that factors such as extraction solvent and extract stability determine the selectivity for glutamate receptor (GluR) interactions.

IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taketazu, F.; Chiba, S.; Shibuya, K.

1991-02-01

The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of 125I-GM-CSF was incubated. Scatchard analysis of 125I-GM-CSF bindingmore » to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the beta-chain, Mr 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existing specifically on hemopoietic cells.« less

The Growth Factor Progranulin Binds to TNF Receptors and Is Therapeutic Against Inflammatory Arthritis in Mice

PubMed Central

Tang, Wei; Lu, Yi; Tian, Qing-Yun; Zhang, Yan; Guo, Feng-Jin; Liu, Guang-Yi; Syed, Nabeel Muzaffar; Lai, Yongjie; Lin, Edward Alan; Kong, Li; Su, Jeffrey; Yin, Fangfang; Ding, Ai-Hao; Zanin-Zhorov, Alexandra; Dustin, Michael L.; Tao, Jian; Craft, Joseph; Yin, Zhinan; Feng, Jian Q.; Abramson, Steven B.; Yu, Xiu-Ping; Liu, Chuan-ju

2011-01-01

The growth factor progranulin (PGRN) has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation, but its receptors remain unidentified. We report that PGRN bound directly to tumor necrosis factor receptors (TNFR), and disturbed the TNFα/TNFR interaction. PGRN-deficient mice were susceptible to collagen-induced arthritis, and administration of PGRN reversed inflammatory arthritis. Atsttrin, an engineered protein composed of three PGRN fragments, exhibited selective TNFR binding. PGRN and Atsttrin prevented inflammation in multiple arthritis mouse models and inhibited TNFα-activated intracellular signaling. Collectively, these findings demonstrate that PGRN is a ligand of TNFR, an antagonist of TNFα signaling and plays a critical role in the pathogenesis of inflammatory arthritis in mice. They also suggest new potential therapeutic interventions for various TNFα-mediated pathologies and conditions, including rheumatoid arthritis. PMID:21393509

Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.

2010-10-19

Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

PubMed

Hong, Huixiao; Branham, William S; Ng, Hui Wen; Moland, Carrie L; Dial, Stacey L; Fang, Hong; Perkins, Roger; Sheehan, Daniel; Tong, Weida

2015-02-01

One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered. Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.

Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1

NASA Astrophysics Data System (ADS)

Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

2015-01-01

The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr6.63 forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr6.63 to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr3566.63 allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R.

Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1

PubMed Central

Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

2015-01-01

The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr6.63 forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr6.63 to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr3566.63 allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R. PMID:25628267

Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1.

PubMed

Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

2015-01-28

The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr(6.63) forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr(6.63) to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr356(6.63) allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R.

Antitumor activity of a novel anti-vascular endothelial growth factor receptor-1 monoclonal antibody that does not interfere with ligand binding

PubMed Central

Tentori, Lucio; Scimeca, Manuel; Dorio, Annalisa S.; Atzori, Maria Grazia; Failla, Cristina M.; Morea, Veronica; Bonanno, Elena; D'Atri, Stefania; Lacal, Pedro M.

2016-01-01

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase transmembrane receptor that has also a soluble isoform containing most of the extracellular ligand binding domain (sVEGFR-1). VEGF-A binds to both VEGFR-2 and VEGFR-1, whereas placenta growth factor (PlGF) interacts exclusively with VEGFR-1. In this study we generated an anti-VEGFR-1 mAb (D16F7) by immunizing BALB/C mice with a peptide that we had previously reported to inhibit angiogenesis and endothelial cell migration induced by PlGF. D16F7 did not affect binding of VEGF-A or PlGF to VEGFR-1, thus allowing sVEGFR-1 to act as decoy receptor for these growth factors, but it hampered receptor homodimerization and activation. D16F7 inhibited both the chemotactic response of human endothelial, myelomonocytic and melanoma cells to VEGFR-1 ligands and vasculogenic mimicry by tumor cells. Moreover, D16F7 exerted in vivo antiangiogenic effects in a matrigel plug assay. Importantly, D16F7 inhibited tumor growth and was well tolerated by B6D2F1 mice injected with syngeneic B16F10 melanoma cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation. PMID:27655684

Aqueous and Ethanolic Valeriana officinalis Extracts Change the Binding of Ligands to Glutamate Receptors

PubMed Central

Del Valle-Mojica, Lisa M.; Cordero-Hernández, José M.; González-Medina, Giselle; Ramos-Vélez, Igmeris; Berríos-Cartagena, Nairimer; Torres-Hernández, Bianca A.; Ortíz, José G.

2011-01-01

The effects of two valerian extracts (aqueous and hydroalcoholic) were investigated through [3H]Glutamate ([3H]Glu) and [3H]Fluorowillardine ([3H]FW) receptor binding assays using rat synaptic membranes in presence of different receptor ligands. In addition, the extract stability was monitored spectrophotometrically. Both extracts demonstrated interaction with ionotropic glutamate receptors (iGluRs). However, the extracts displayed considerable differences in receptor selectivity. The hydroalcoholic extract selectively interacted with quisqualic acid (QA), group I metabotropic glutamate receptor (mGluR) ligand, while the aqueous extract did not alter the binding of QA. The stability of the extracts was examined during several weeks. Freshly prepared extract inhibited 38–60% of [3H]FW binding (AMPA). After 10 days, the aqueous extract inhibited 85% of [3H]FW binding while the hydroalcoholic extract markedly potentiated (200%) [3H]FW binding to AMPA receptors. Thus, our results showed that factors such as extraction solvent and extract stability determine the selectivity for glutamate receptor (GluR) interactions. PMID:21151614

The pure estrogen receptor antagonist ICI 182,780 promotes a novel interaction of estrogen receptor-alpha with the 3',5'-cyclic adenosine monophosphate response element-binding protein-binding protein/p300 coactivators.

PubMed

Jaber, Basem M; Gao, Tong; Huang, Luping; Karmakar, Sudipan; Smith, Carolyn L

2006-11-01

Estrogen receptor-alpha (ERalpha) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Abundant evidence demonstrates that ERalpha agonists promote, whereas antagonists inhibit, receptor binding to coactivators. In this report we demonstrate that binding of the ICI 182,780 (ICI) pure antiestrogen to ERalpha promotes its interaction with the cAMP response element-binding protein-binding protein (CBP)/p300 but not the p160 family of coactivators, demonstrating the specificity of this interaction. Amino acid mutations within the coactivator binding surface of the ERalpha ligand-binding domain revealed that CBP binds to this region of the ICI-liganded receptor. The carboxy-terminal cysteine-histidine rich domain 3 of CBP, rather than its amino-terminal nuclear interacting domain, shown previously to mediate agonist-dependent interactions of CBP with nuclear receptors, is required for binding to ICI-liganded ERalpha. Chromatin immunoprecipitation assays revealed that ICI but not the partial agonist/antagonist 4-hydroxytamoxifen is able to recruit CBP to the pS2 promoter, and this distinguishes ICI from this class of antiestrogens. Chromatin immunoprecipitation assays for pS2 and cytochrome P450 1B1 promoter regions revealed that ICI-dependent recruitment of CBP, but not receptor, to ERalpha targets is gene specific. ICI treatment did not recruit the steroid receptor coactivator 1 to the pS2 promoter, and it failed to induce the expression of this gene. Taken together, these data indicate that recruitment of the CBP coactivator/cointegrator without steroid receptor coactivator 1 to ERalpha is insufficient to promote transcription of ERalpha target genes.

Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin.

PubMed Central

Schilling, D; Reid IV, J D; Hujer, A; Morgan, D; Demoll, E; Bummer, P; Fenstermaker, R A; Kaetzel, D M

1998-01-01

Site-directed mutagenesis of the platelet-derived growth factor (PDGF) B-chain was conducted to determine the importance of cationic amino acid residues (Arg160-Lys161-Lys162; RKK) located within the loop III region in mediating the biological and cell-association properties of the molecule. Binding to both PDGF alpha-and beta-receptors was inhibited by the conversion of all three cationic residues into anionic glutamates (RKK-->EEE), whereas an RKK-->SSS mutant also exhibited a modest loss in affinity for beta-receptors. Replacements with serine at either Arg160 (RKK-->SKK) or at all three positions (RKK-->SSS) had little effect on binding to alpha-receptors. Replacements with either glutamic or serine residues at any of the three positions also resulted in significant inhibition of heparin-binding activity. Furthermore, the RKK-->EEE mutant exhibited decreased association with the cell surface and accumulated in the culture medium as 29-32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK-->EEE mutants of either the A-chain or the B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor-binding activities, each of the loop III mutants retained full mitogenic activity when applied to cultured Swiss 3T3 cells. CD spectrophotometric analysis of the RKK-->EEE mutant revealed a secondary structure indistinguishable from the wild type, with a high degree of beta-sheet structure and random coil content (50% and 43% respectively). These findings indicate an important role of the Arg160-Lys161-Lys162 sequence in mediating the biological and cell-associative activities of the PDGF-BB homodimer, and reveal that the mitogenic activity of PDGF-BB is insufficient to mediate its full oncogenic properties. PMID:9677323

Diminished hepatic growth hormone receptor binding in sex-linked dwarf broiler and leghorn chickens.

PubMed

Leung, F C; Styles, W J; Rosenblum, C I; Lilburn, M S; Marsh, J A

1987-02-01

Hepatic growth hormone (GH) receptor binding was compared in normal and sex-linked dwarfs (SLD) from both Hubbard and Cornell strain chickens. At 6, 8, and 20 weeks of age, hepatic GH receptor binding in the Hubbard SLD chickens was significantly lower than that of normal fast-growing birds. At 20 weeks of age, only 2 of 22 SLD chickens in the Hubbard broiler strain showed positive binding at a high enough level to allow for Scatchard analysis. The affinity constants and binding capacities of these two SLD chickens were numerically (but not significantly) lower than those of the normal fast-growing birds. We further examined hepatic GH receptor binding in two closely related White Leghorn strains of chickens that have been maintained as closed breeding populations for many years. We observed no detectable hepatic GH binding in the Cornell SLD chickens (N = 20), as compared to the normal-growing control strain (K strain). In both SLD strains, pretreatment with 4 M MgCl2 did not enhance GH binding, suggesting that there was no endogenous GH binding to the receptor. Based on these data, we suggest that the lack, or greatly reduced number, of GH receptors may be a major contributing factor to the dwarfism observed in these strains.

Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D*

PubMed Central

Davydova, Natalia; Harris, Nicole C.; Roufail, Sally; Paquet-Fifield, Sophie; Ishaq, Musarat; Streltsov, Victor A.; Williams, Steven P.; Karnezis, Tara; Stacker, Steven A.; Achen, Marc G.

2016-01-01

VEGF-C and VEGF-D are secreted glycoproteins that induce angiogenesis and lymphangiogenesis in cancer, thereby promoting tumor growth and spread. They exhibit structural homology and activate VEGFR-2 and VEGFR-3, receptors on endothelial cells that signal for growth of blood vessels and lymphatics. VEGF-C and VEGF-D were thought to exhibit similar bioactivities, yet recent studies indicated distinct signaling mechanisms (e.g. tumor-derived VEGF-C promoted expression of the prostaglandin biosynthetic enzyme COX-2 in lymphatics, a response thought to facilitate metastasis via the lymphatic vasculature, whereas VEGF-D did not). Here we explore the basis of the distinct bioactivities of VEGF-D using a neutralizing antibody, peptide mapping, and mutagenesis to demonstrate that the N-terminal α-helix of mature VEGF-D (Phe93–Arg108) is critical for binding VEGFR-2 and VEGFR-3. Importantly, the N-terminal part of this α-helix, from Phe93 to Thr98, is required for binding VEGFR-3 but not VEGFR-2. Surprisingly, the corresponding part of the α-helix in mature VEGF-C did not influence binding to either VEGFR-2 or VEGFR-3, indicating distinct determinants of receptor binding by these growth factors. A variant of mature VEGF-D harboring a mutation in the N-terminal α-helix, D103A, exhibited enhanced potency for activating VEGFR-3, was able to promote increased COX-2 mRNA levels in lymphatic endothelial cells, and had enhanced capacity to induce lymphatic sprouting in vivo. This mutant may be useful for developing protein-based therapeutics to drive lymphangiogenesis in clinical settings, such as lymphedema. Our studies shed light on the VEGF-D structure/function relationship and provide a basis for understanding functional differences compared with VEGF-C. PMID:27852824

Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

PubMed Central

Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

2016-01-01

Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface. PMID:27353000

Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

NASA Astrophysics Data System (ADS)

Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

2016-06-01

Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor*

PubMed Central

Lawrence, Callum F.; Margetts, Mai B.; Menting, John G.; Smith, Nicholas A.; Smith, Brian J.; Ward, Colin W.; Lawrence, Michael C.

2016-01-01

Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucine-rich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor α-chain (termed αCT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective αCT residues Phe701 and Phe705. The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor. PMID:27281820

Identification of heparin-binding EGF-like growth factor as a target in intercellular regulation of epidermal basal cell growth by suprabasal retinoic acid receptors.

PubMed Central

Xiao, J H; Feng, X; Di, W; Peng, Z H; Li, L A; Chambon, P; Voorhees, J J

1999-01-01

The role of retinoic acid receptors (RARs) in intercellular regulation of cell growth was assessed by targeting a dominant-negative RARalpha mutant (dnRARalpha) to differentiated suprabasal cells of mouse epidermis. dnRARalpha lacks transcriptional activation but not DNA-binding and receptor dimerization functions. Analysis of transgenic mice revealed that dnRARalpha dose-dependently impaired induction of basal cell proliferation and epidermal hyperplasia by all-trans RA (tRA). dnRARalpha formed heterodimers with endogenous retinoid X receptor-alpha (RXRalpha) over RA response elements in competition with remaining endogenous RARgamma-RXRalpha heterodimers, and dose-dependently impaired retinoid-dependent gene transcription. To identify genes regulated by retinoid receptors and involved in cell growth control, we analyzed the retinoid effects on expression of the epidermal growth factor (EGF) receptor, EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin genes. In normal epidermis, tRA rapidly and selectively induced expression of HB-EGF but not the others. This induction occurred exclusively in suprabasal cells. In transgenic epidermis, dnRARalpha dose-dependently inhibited tRA induction of suprabasal HB-EGF and subsequent basal cell hyperproliferation. Together, our observations suggest that retinoid receptor heterodimers located in differentiated suprabasal cells mediate retinoid induction of HB-EGF, which in turn stimulates basal cell growth via intercellular signaling. These events may underlie retinoid action in epidermal regeneration during wound healing. PMID:10075925

Quantitative in vivo receptor binding. I. Theory and application to the muscarinic cholinergic receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frey, K.A.; Ehrenkaufer, R.L.; Beaucage, S.

1985-02-01

A novel approach to in vivo receptor binding experiments is presented which allows direct quantitation of binding site densities. The method is based on an equilibrium model of tracer uptake and is designed to produce a static distribution proportional to receptor density and to minimize possible confounding influences of regional blood flow, blood-brain barrier permeability, and nonspecific binding. This technique was applied to the measurement of regional muscarinic cholinergic receptor densities in rat brain using (/sup 3/H)scopolamine. Specific in vivo binding of scopolamine demonstrated saturability, a pharmacologic profile, and regional densities which are consistent with interaction of the tracer withmore » the muscarinic receptor. Estimates of receptor density obtained with the in vivo method and in vitro measurements in homogenates were highly correlated. Furthermore, reduction in striatal muscarinic receptors following ibotenic acid lesions resulted in a significant decrease in tracer uptake in vivo, indicating that the correlation between scopolamine distribution and receptor density may be used to demonstrate pathologic conditions. We propose that the general method presented here is directly applicable to investigation of high affinity binding sites for a variety of radioligands.« less

A Major Binding Protein for Leukemia Inhibitory Factor in Normal Mouse Serum: Identification as a Soluble Form of the Cellular Receptor

NASA Astrophysics Data System (ADS)

Layton, Meredith J.; Cross, Bronwyn A.; Metcalf, Donald; Ward, Larry D.; Simpson, Richard J.; Nicola, Nicos A.

1992-09-01

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (K_d = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the α chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 μg/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

Phosphorylation and Intramolecular Stabilization of the Ligand Binding Domain in the Nuclear Receptor Steroidogenic Factor 1

PubMed Central

Desclozeaux, Marion; Krylova, Irina N.; Horn, Florence; Fletterick, Robert J.; Ingraham, Holly A.

2002-01-01

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor with no known ligand. We showed previously that phosphorylation at serine 203 located N′-terminal to the ligand binding domain (LBD) enhanced cofactor recruitment, analogous to the ligand-mediated recruitment in ligand-dependent receptors. In this study, results of biochemical analyses and an LBD helix assembly assay suggest that the SF-1 LBD adopts an active conformation, with helices 1 and 12 packed against the predicted alpha-helical bundle, in the apparent absence of ligand. Fine mapping of the previously defined proximal activation function in SF-1 showed that the activation function mapped fully to helix 1 of the LBD. Limited proteolyses demonstrate that phosphorylation of S203 in the hinge region mimics the stabilizing effects of ligand on the LBD. Moreover, similar effects were observed in an SF-1/thyroid hormone LBD chimera receptor, illustrating that the S203 phosphorylation effects are transferable to a heterologous ligand-dependent receptor. Our collective data suggest that the hinge together with helix 1 is an individualized specific motif, which is tightly associated with its cognate LBD. For SF-1, we find that this intramolecular association and hence receptor activity are further enhanced by mitogen-activated protein kinase phosphorylation, thus mimicking many of the ligand-induced changes observed for ligand-dependent receptors. PMID:12242296

Ligand binding was acquired during evolution of nuclear receptors

PubMed Central

Escriva, Hector; Safi, Rachid; Hänni, Catherine; Langlois, Marie-Claire; Saumitou-Laprade, Pierre; Stehelin, Dominique; Capron, André; Pierce, Raymond; Laudet, Vincent

1997-01-01

The nuclear receptor (NR) superfamily comprises, in addition to ligand-activated transcription factors, members for which no ligand has been identified to date. We demonstrate that orphan receptors are randomly distributed in the evolutionary tree and that there is no relationship between the position of a given liganded receptor in the tree and the chemical nature of its ligand. NRs are specific to metazoans, as revealed by a screen of NR-related sequences in early- and non-metazoan organisms. The analysis of the NR gene duplication pattern during the evolution of metazoans shows that the present NR diversity arose from two waves of gene duplications. Strikingly, our results suggest that the ancestral NR was an orphan receptor that acquired ligand-binding ability during subsequent evolution. PMID:9192646

The Evolving Field of Human Papillomavirus Receptor Research: a Review of Binding and Entry

PubMed Central

Raff, Adam B.; Woodham, Andrew W.; Raff, Laura M.; Skeate, Joseph G.; Yan, Lisa; Da Silva, Diane M.; Schelhaas, Mario

2013-01-01

Human papillomaviruses (HPVs) infect epithelia and can lead to the development of lesions, some of which have malignant potential. HPV type 16 (HPV16) is the most oncogenic genotype and causes various types of cancer, including cervical, anal, and head and neck cancers. However, despite significant research, our understanding of the mechanism by which HPV16 binds to and enters host cells remains fragmented. Over several decades, many HPV receptors and entry pathways have been described. This review puts those studies into context and offers a model of HPV16 binding and entry as a framework for future research. Our model suggests that HPV16 binds to heparin sulfate proteoglycans (HSPGs) on either the epithelial cell surface or basement membrane through interactions with the L1 major capsid protein. Growth factor receptors may also become activated through HSPG/growth factor/HPV16 complexes that initiate signaling cascades during early virion-host cell interactions. After binding to HSPGs, the virion undergoes conformational changes, leading to isomerization by cyclophilin B and proprotein convertase-mediated L2 minor capsid protein cleavage that increases L2 N terminus exposure. Along with binding to HSPGs, HPV16 binds to α6 integrins, which initiate further intracellular signaling events. Following these primary binding events, HPV16 binds to a newly identified L2-specific receptor, the annexin A2 heterotetramer. Subsequently, clathrin-, caveolin-, lipid raft-, flotillin-, cholesterol-, and dynamin-independent endocytosis of HPV16 occurs. PMID:23536685

Structure of dual receptor binding to botulinum neurotoxin B.

PubMed

Berntsson, Ronnie P-A; Peng, Lisheng; Dong, Min; Stenmark, Pål

2013-01-01

Botulinum neurotoxins are highly toxic, and bind two receptors to achieve their high affinity and specificity for neurons. Here we present the first structure of a botulinum neurotoxin bound to both its receptors. We determine the 2.3-Å structure of a ternary complex of botulinum neurotoxin type B bound to both its protein receptor synaptotagmin II and its ganglioside receptor GD1a. We show that there is no direct contact between the two receptors, and that the binding affinity towards synaptotagmin II is not influenced by the presence of GD1a. The interactions of botulinum neurotoxin type B with the sialic acid 5 moiety of GD1a are important for the ganglioside selectivity. The structure demonstrates that the protein receptor and the ganglioside receptor occupy nearby but separate binding sites, thus providing two independent anchoring points.

The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Varnum, Susan M.

2012-03-01

Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was foundmore » to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.« less

Fibroblast growth factor receptor inhibitors.

PubMed

Kumar, Suneel B V S; Narasu, Lakshmi; Gundla, Rambabu; Dayam, Raveendra; J A R P, Sarma

2013-01-01

Fibroblast growth factor receptors (FGFRs) play an important role in embryonic development, angiogenesis, wound healing, cell proliferation and differentiation. The fibroblast growth factor receptor (FGFR) isoforms have been under intense scrutiny for effective anticancer drug candidates. The fibroblast growth factor (FGF) and its receptor (FGFR) provide another pathway that seems critical to monitoring angiogenesis. Recent findings suggest that FGFR mediates signaling, regulates the PKM2 activity, and plays a crucial role in cancer metabolism. The current review also covers the recent findings on the role of FGFR1 in cancer metabolism. This paper reviews the progress, mechanism, and binding modes of recently known kinase inhibitors such as PD173074, SU series and other inhibitors still under clinical development. Some of the structural classes that will be highlighted in this review include Pyrido[2,3-d]pyrimidines, Indolin- 2-one, Pyrrolo[2,1-f][1,2,4]triazine, Pyrido[2,3-d]pyrimidin-7(8H)-one, and 1,6- Naphthyridin-2(1H)-ones.

Role of G protein-coupled estrogen receptor-1, matrix metalloproteinases 2 and 9, and heparin binding epidermal growth factor-like growth factor in estradiol-17β-stimulated bovine satellite cell proliferation.

PubMed

Kamanga-Sollo, E; Thornton, K J; White, M E; Dayton, W R

2014-10-01

In feedlot steers, estradiol-17β (E2) and combined E2 and trenbolone acetate (a testosterone analog) implants enhance rate and efficiency of muscle growth; and, consequently, these compounds are widely used as growth promoters. Although the positive effects of E2 on rate and efficiency of bovine muscle growth are well established, the mechanisms involved in these effects are not well understood. Combined E2 and trenbolone acetate implants result in significantly increased muscle satellite cell number in feedlot steers. Additionally, E2 treatment stimulates proliferation of cultured bovine satellite cells (BSC). Studies in nonmuscle cells have shown that binding of E2 to G protein-coupled estrogen receptor (GPER)-1 results in activation of matrix metalloproteinases 2 and 9 (MMP2/9) resulting in proteolytic release of heparin binding epidermal growth factor-like growth factor (hbEGF) from the cell surface. Released hbEGF binds to and activates the epidermal growth factor receptor resulting in increased proliferation. To assess if GPER-1, MMP2/9, and/or hbEGF are involved in the mechanism of E2-stimulated BSC proliferation, we have examined the effects of G36 (a specific inhibitor of GPER-1), CRM197 (a specific inhibitor of hbEGF), and MMP-2/MMP-9 Inhibitor II (an inhibitor of MMP2/9 activity) on E2-stimulated BSC proliferation. Inhibition of GPER-1, MMP2/9, or hbEGF suppresses E2-stimulated BSC proliferation (P < 0.001) suggesting that all these are required in order for E2 to stimulate BSC proliferation. These results strongly suggest that E2 may stimulate BSC proliferation by binding to GPER-1 resulting in MMP2/9-catalyzed release of cell membrane-bound hbEGF and subsequent activation of epidermal growth factor receptor by binding of released hbEGF. Copyright © 2014 Elsevier Inc. All rights reserved.

Tomography of epidermal growth factor receptor binding to fluorescent Affibody in vivo studied with magnetic resonance guided fluorescence recovery in varying orthotopic glioma sizes

NASA Astrophysics Data System (ADS)

Holt, Robert W.; Demers, Jennifer-Lynn H.; Sexton, Kristian J.; Gunn, Jason R.; Davis, Scott C.; Samkoe, Kimberley S.; Pogue, Brian W.

2015-02-01

The ability to image targeted tracer binding to epidermal growth factor receptor (EGFR) was studied in vivo in orthotopically grown glioma tumors of different sizes. The binding potential was quantified using a dual-tracer approach, which employs a fluorescently labeled peptide targeted to EGFR and a reference tracer with similar pharmacokinetic properties but no specific binding, to estimate the relative bound fraction from kinetic compartment modeling. The recovered values of binding potential did not vary significantly as a function of tumor size (1 to 33 mm3), suggesting that binding potential may be consistent in the U251 tumors regardless of size or stage after implantation. However, the fluorescence yield of the targeted fluorescent tracers in the tumor was affected significantly by tumor size, suggesting that dual-tracer imaging helps account for variations in absolute uptake, which plague single-tracer imaging techniques. Ex vivo analysis showed relatively high spatial heterogeneity in each tumor that cannot be resolved by tomographic techniques. Nonetheless, the dual-tracer tomographic technique is a powerful tool for longitudinal bulk estimation of receptor binding.

Targeting the heparin-binding domain of fibroblast growth factor receptor 1 as a potential cancer therapy.

PubMed

Ling, Ling; Tan, Si Kee; Goh, Ting Hwee; Cheung, Edwin; Nurcombe, Victor; van Wijnen, Andre J; Cool, Simon M

2015-07-23

Aberrant activation of fibroblast growth factor receptors (FGFRs) deregulates cell proliferation and promotes cell survival, and may predispose to tumorigenesis. Therefore, selective inactivation of FGFRs is an important strategy for cancer therapy. Here as a proof-of-concept study, we developed a FGFR1 neutralizing antisera, IMB-R1, employing a novel strategy aimed at preventing the access of essential heparan sulfate (HS) co-receptors to the heparin-binding domain on FGFR1. The mRNA and protein expression level of FGFR1 and other FGFRs were examined in several lines of breast cancer and osteosarcoma cells and corresponding normal cells using Taqman real-time quantitative PCR and Western blot analysis. The specificity of IMB-R1 against FGFR1 was assessed with various ELISA-based approaches and Receptor Tyrosine Kinase array. Proliferation assay and apoptosis analysis were performed to assess the effect of IMB-R1 on cancer cell growth and apoptosis, respectively, in comparison with known FGFR1 inhibitors. The IMB-R1 induced alteration of intracellular signaling and gene expression were analysed using Western blot and microarray approaches. Immunohistochemical staining of FGFR1 using IMB-R1 were carried out in different cancer tissues from clinical patients. Throughout the study, statistical differences were determined by Student's t test where appropriate and reported when a p value was less than 0.05. We demonstrate that IMB-R1 is minimally cross-reactive for other FGFRs, and that it potently and specifically inhibits binding of heparin to FGFR1. Furthermore, IMB-R1 blocks the interaction of FGF2 with FGFR1, the kinase activity of FGFR1 and activation of intracellular FGFR signaling. Cancer cells treated with IMB-R1 displayed impaired FGF2 signaling, were unable to grow and instead underwent apoptosis. IMB-R1-induced cell death correlated with a disruption of antioxidative defense networks and increased expression of several tumor suppressors and apoptotic

Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells

PubMed Central

2013-01-01

Background The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. Results To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Conclusions Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases. PMID:23731667

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to "Biased Opioids"?

PubMed

Root-Bernstein, Robert; Turke, Miah; Subhramanyam, Udaya K Tiruttani; Churchill, Beth; Labahn, Joerg

2018-01-17

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

Energetics of Glutamate Binding to an Ionotropic Glutamate Receptor.

PubMed

Yu, Alvin; Lau, Albert Y

2017-11-22

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that are responsible for the majority of excitatory transmission at the synaptic cleft. Mechanically speaking, agonist binding to the ligand binding domain (LBD) activates the receptor by triggering a conformational change that is transmitted to the transmembrane region, opening the ion channel pore. We use fully atomistic molecular dynamics simulations to investigate the binding process in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, an iGluR subtype. The string method with swarms of trajectories was applied to calculate the possible pathways glutamate traverses during ligand binding. Residues peripheral to the binding cleft are found to metastably bind the ligand prior to ligand entry into the binding pocket. Umbrella sampling simulations were performed to compute the free energy barriers along the binding pathways. The calculated free energy profiles demonstrate that metastable interactions contribute substantially to the energetics of ligand binding and form local minima in the overall free energy landscape. Protein-ligand interactions at sites outside of the orthosteric agonist-binding site may serve to lower the transition barriers of the binding process.

Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor.

PubMed Central

Glickman, J N; Conibear, E; Pearse, B M

1989-01-01

Adaptors mediate the interaction of clathrin with select groups of receptors. Two distinct types of adaptors, the HA-II adaptors (found in plasma membrane coated pits) and the HA-I adaptors (localized to Golgi coated pits) bind to the cytoplasmic portion of the 270 kd mannose 6-phosphate (M6P) receptor-a receptor which is concentrated in coated pits on both the plasma membrane and in the trans-Golgi network. Neither type of adaptor appears to compete with the other for binding, suggesting that each type recognizes a distinct site on the M6P receptor tail. Mutation of the two tyrosines in the tail essentially eliminates the interaction with the HA-II plasma membrane adaptor, which recognizes a 'tyrosine' signal on other endocytosed receptors (for example, the LDL receptor and the poly Ig receptor). In contrast, the wild type and the mutant M6P receptor tail (lacking tyrosines) are equally effective at binding HA-I adaptors. This suggests that there is an HA-I recognition signal in another region of the M6P receptor tail, C-terminal to the tyrosine residues, which remains intact in the mutant. This signal is presumably responsible for the concentration of the M6P receptor, with bound lysosomal enzymes, into coated pits which bud from the trans-Golgi network, thus mediating efficient transfer of these enzymes to lysosomes. Images PMID:2545438

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

PubMed

Roskoski, Robert

2005-11-11

Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of

Tactics for preclinical validation of receptor-binding radiotracers

PubMed Central

Lever, Susan Z.; Fan, Kuo-Hsien; Lever, John R.

2016-01-01

Introduction Aspects of radiopharmaceutical development are illustrated through preclinical studies of [125I]-(E)-1-(2-(2,3-dihydrobenzofuran-5-yl)ethyl)-4-(iodoallyl)piperazine ([125I]-E-IA- BF-PE-PIPZE), a radioligand for sigma-1 (σ1) receptors, coupled with examples from the recent literature. Findings are compared to those previously observed for [125I]-(E)-1-(2-(2,3-dimethoxy-5-yl)ethyl)-4-(iodoallyl)piperazine ([125I]-E-IA-DM-PE-PIPZE). Methods Syntheses of E-IA-BF-PE-PIPZE and [125I]-E-IA-BF-PE-PIPZE were accomplished by standard methods. In vitro receptor binding studies and autoradiography were performed, and binding potential was predicted. Measurements of lipophilicity and protein binding were obtained. In vivo studies were conducted in mice to evaluate radioligand stability, as well as specific binding to σ1 sites in brain, brain regions and peripheral organs in the presence and absence of potential blockers. Results E-IA-BF-PE-PIPZE exhibited high affinity and selectivity for σ1 receptors (Ki = 0.43 ± 0.03 nM, σ2 / σ1 = 173). [125I]-E-IA-BF-PE-PIPZE was prepared in good yield and purity, with high specific activity. Radioligand binding provided dissociation (koff) and association (kon) rate constants, along with a measured Kd of 0.24 ± 0.01 nM and Bmax of 472 ± 13 fmol / mg protein. The radioligand proved suitable for quantitative autoradiography in vitro using brain sections. Moderate lipophilicity, Log D7.4 2.69 ± 0.28, was determined, and protein binding was 71 ± 0.3%. In vivo, high initial whole brain uptake, > 6% injected dose / g, cleared slowly over 24 h. Specific binding represented 75% to 93% of total binding from 15 min to 24 h. Findings were confirmed and extended by regional brain biodistribution. Radiometabolites were not observed in brain (1%). Conclusions Substitution of dihydrobenzofuranylethyl for dimethoxyphenethyl increased radioligand affinity for σ1 receptors by 16-fold. While high specific binding to σ1 receptors was

Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.

PubMed

Disatnik, M H; Hernandez-Sotomayor, S M; Jones, G; Carpenter, G; Mochly-Rosen, D

1994-01-18

Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.

Docking simulations suggest that all-trans retinoic acid could bind to retinoid X receptors.

PubMed

Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

2015-10-01

Retinoid X receptors (RXRs) are ligand-controlled transcription factors which heterodimerize with other nuclear receptors to regulate gene transcriptions associated with crucial biological events. 9-cis retinoic acid (9cRA), which transactivates RXRs, is believed to be an endogenous RXR ligand. All-trans retinoic acid (ATRA) is a natural ligand for retinoic acid receptors (RARs), which heterodimerize with RXRs. Although the concentration of 9cRA in tissues is very low, ATRA is relatively abundant and some reports show that ATRA activates RXRs. We computationally studied the possibility of ATRA binding to RXRs using two different docking methods with our developed programs to assess the binding affinities of naturally occurring retinoids. The simulations showed good correlations to the reported binding affinities of these molecules for RXRs and RARs.

Molecular Modeling, de novo Design and Synthesis of a Novel, Extracellular Binding Fibroblast Growth Factor Receptor 2 Inhibitor Alofanib (RPT835).

PubMed

Tsimafeyeu, Ilya; Daeyaert, Frits; Joos, Jean-Baptiste; Aken, Koen V; Ludes-Meyers, John; Byakhov, Mikhail; Tjulandin, Sergei

2016-01-01

Fibroblast growth factor (FGF) receptors (FGFRs) play a key role in tumor growth and angiogenesis. The present report describes our search for an extracellularly binding FGFR inhibitor using a combined molecular modeling and de novo design strategy. Based upon crystal structures of the receptor with its native ligand and knowledge of inhibiting peptides, we have developed a computational protocol that predicts the putative binding of a molecule to the extracellular domains of the receptor. This protocol, or scoring function, was used in combination with the de novo synthesis program 'SYNOPSIS' to generate high scoring and synthetically accessible compounds. Eight compounds belonging to 3 separate chemical classes were synthesized. One of these compounds, alofanib (RPT835), was found to be an effective inhibitor of the FGF/FGFR2 pathway. The preclinical in vitro data support an allosteric inhibition mechanism of RPT835. RPT835 potently inhibited growth of KATO III gastric cancer cells expressing FGFR2, with GI50 value of 10 nmol/L. These results provide strong rationale for the evaluation of compound in advanced cancers.

Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization

NASA Technical Reports Server (NTRS)

Barmina, O. Y.; Walling, H. W.; Fiacco, G. J.; Freije, J. M.; Lopez-Otin, C.; Jeffrey, J. J.; Partridge, N. C.

1999-01-01

We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

PubMed

Philips, Brian J; Ansell, Pete J; Newton, Leslie G; Harada, Nobuhiro; Honda, Shin-Ichiro; Ganjam, Venkataseshu K; Rottinghaus, George E; Welshons, Wade V; Lubahn, Dennis B

2004-06-01

Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.

2011-09-28

Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusivemore » protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.« less

Structure-function relationship in the binding of snake neurotoxins to the torpedo membrane receptor.

PubMed

Chicheportiche, R; Vincent, J P; Kopeyan, C; Schweitz, H; Lazdunski, M

1975-05-20

The Cys30-Cus34 bridge present in all long neutotoxins (71-74 amino acids, 5 disulfide bridges), but not in short toxins (60-63 amino acids, 4 disulfide bridges), is exposed at the surface since it can be reduced rapidly and selectively by sodium borohydride. Reduction and alkylation of the Cys30-Cys34 bridge of Naja haje neurotoxin III hardly alter the conformational properties of this model long toxin. Although alkylation by iodoacetic acid of th -SH groups liberated by reduction abolishes the toxicity, alkylation by iodoacetamide or ethylenimine does not affect the curarizing efficacy of the toxin. The Cys30-Cys34 bridge is not very important for the toxic activity of long neurotoxins. Reduction of the Cys30-Cys34 bridge followed by alkylation with radioactive iodoacetamide gave a labeled and active toxin which is a convenient derivative for binding experiments to the toxin receptor in membranes of the Torpedo electric organ. The binding capacity of these membrane is 1200 pmol of toxin/mg of membrane protein. The dissociation constant of the modified toxin-receptor complex at pH 7.4, 20 degrees is 10 minus 8m. Reduction with carbroxamidomethylation of the Cys30-Cys34 bridge decreases the affinity of the native Naja haje toxin only by a factor of 15. Carboxymethylation after reduction prevents binding to the membrane receptor. The binding properties of the derivative obtained by reduction and aminoethylation of Cys30-Cys34 are very similar to those of native neurotoxin III; the affinity is decreased only by a factor of 5. Binding properties to Toredo membrane of long neurotoxins (Naja haje neurotoxin III) and short neurotoxins (Naje haje toxin I and Naja mossambica toxin I) have been compared. Dissociation constants of receptor-long neurotoxin and receptor-short neurotoxin complexes are very similar (5.7 minus 8.2 times 10(-10) M at pH 7.4, 20degrees. However, the kinetics of complex formation and complex dissociation are quite different. Short neurotoxins

Enantiomeric cannabidiol derivatives: synthesis and binding to cannabinoid receptors.

PubMed

Hanus, Lumír O; Tchilibon, Susanna; Ponde, Datta E; Breuer, Aviva; Fride, Ester; Mechoulam, Raphael

2005-03-21

(-)-Cannabidiol (CBD) is a major, non psychotropic constituent of cannabis. It has been shown to cause numerous physiological effects of therapeutic importance. We have reported that CBD derivatives in both enantiomeric series are of pharmaceutical interest. Here we describe the syntheses of the major CBD metabolites, (-)-7-hydroxy-CBD and (-)-CBD-7-oic acid and their dimethylheptyl (DMH) homologs, as well as of the corresponding compounds in the enantiomeric (+)-CBD series. The starting materials were the respective CBD enantiomers and their DMH homologs. The binding of these compounds to the CB(1) and CB(2) cannabinoid receptors are compared. Surprisingly, contrary to the compounds in the (-) series, which do not bind to the receptors, most of the derivatives in the (+) series bind to the CB(1) receptor in the low nanomole range. Some of these compounds also bind weakly to the CB(2) receptor.

Solution structure of the chick TGFbeta type II receptor ligand-binding domain.

PubMed

Marlow, Michael S; Brown, Christopher B; Barnett, Joey V; Krezel, Andrzej M

2003-02-28

The transforming growth factor beta (TGFbeta) signaling pathway influences cell proliferation, immune responses, and extracellular matrix reorganization throughout the vertebrate life cycle. The signaling cascade is initiated by ligand-binding to its cognate type II receptor. Here, we present the structure of the chick type II TGFbeta receptor determined by solution NMR methods. Distance and angular constraints were derived from 15N and 13C edited NMR experiments. Torsion angle dynamics was used throughout the structure calculations and refinement. The 20 final structures were energy minimized using the generalized Born solvent model. For these 20 structures, the average backbone root-mean-square distance from the average structure is below 0.6A. The overall fold of this 109-residue domain is conserved within the superfamily of these receptors. Chick receptors fully recognize and respond to human TGFbeta ligands despite only 60% identity at the sequence level. Comparison with the human TGFbeta receptor determined by X-ray crystallography reveals different conformations in several regions. Sequence divergence and crystal packing interactions under low pH conditions are likely causes. This solution structure identifies regions were structural changes, however subtle, may occur upon ligand-binding. We also identified two very well conserved molecular surfaces. One was found to bind ligand in the crystallized human TGFbeta3:TGFbeta type II receptor complex. The other, newly identified area can be the interaction site with type I and/or type III receptors of the TGFbeta signaling complex.

Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bielmann, Regula; Habann, Matthias; Eugster, Marcel R.

Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wallmore » teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.« less

Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J.H.; el-Fakahany, E.E.

1985-06-01

The binding properties of (-)-(/sup 3/H)quinuclidinyl benzilate and (/sup 3/H) N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-(/sup 3/H)quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of (/sup 3/H)N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when (/supmore » 3/H)N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-(/sup 3/H) quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.« less

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to “Biased Opioids”?

PubMed Central

Turke, Miah; Subhramanyam, Udaya K. Tiruttani; Churchill, Beth; Labahn, Joerg

2018-01-01

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists. PMID:29342106

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-02-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

Modeling Conformational Transitions and Energetics of Ligand Binding with the Glutamate Receptor Ligand Binding Domain

NASA Astrophysics Data System (ADS)

Kurnikova, Maria

2009-03-01

Understanding of protein motion and energetics of conformational transitions is crucial to understanding protein function. The glutamate receptor ligand binding domain (GluR2 S1S2) is a two lobe protein, which binds ligand at the interface of two lobes and undergoes conformational transition. The cleft closure conformational transition of S1S2 has been implicated in gating of the ion channel formed by the transmembrane domain of the receptor. In this study we present a composite multi-faceted theoretical analysis of the detailed mechanism of this conformational transition based on rigid cluster decomposition of the protein structure [1] and identifying hydrogen bonds that are responsible for stabilizing the closed conformation [2]. Free energy of the protein reorganization upon ligand binding was calculated using combined Thermodynamic Integration (TI) and Umbrella Sampling (US) simulations [3]. Ligand -- protein interactions in the binding cleft were analyzed using Molecular Dynamics, continuum electrostatics and QM/MM models [4]. All model calculations compare well with corresponding experimental measurements. [4pt] [1] Protein Flexibility using Constraints from Molecular Dynamics Simulations T. Mamonova, B. Hespenheide, R. Straub, M. F. Thorpe, M. G. Kurnikova , Phys. Biol., 2, S137 (2005)[0pt] [2] Theoretical Study of the Glutamate Receptor Ligand Binding Domain Flexibility and Conformational Reorganization T. Mamonova, K. Speranskiy, and M. Kurnikova , Prot.: Struct., Func., Bioinf., 73,656 (2008)[0pt] [3] Energetics of the cleft closing transition and glutamate binding in the Glutamate Receptor ligand Binding Domain T. Mamonova, M. Yonkunas, and M. Kurnikova Biochemistry 47, 11077 (2008)[0pt] [4] On the Binding Determinants of the Glutamate Agonist with the Glutamate Receptor Ligand Binding Domain K. Speranskiy and M. Kurnikova Biochemistry 44, 11208 (2005)

Ligand recognition by RAR and RXR receptors: binding and selectivity.

PubMed

Sussman, Fredy; de Lera, Angel R

2005-10-06

Fundamental biological functions, most notably embriogenesis, cell growth, cell differentiation, and cell apoptosis, are in part regulated by a complex genomic network that starts with the binding (and activation) of retinoids to their cognate receptors, members of the superfamily of nuclear receptors. We have studied ligand recognition of retinoic receptors (RXRalpha and RARgamma) using a molecular-mechanics-based docking method. The protocol used in this work is able to rank the affinity of pairs of ligands for a single retinoid receptor, the highest values corresponding to those that adapt better to the shape of the binding site and generate the optimal set of electrostatic and apolar interactions with the receptor. Moreover, our studies shed light onto some of the energetic contributions to retinoid receptor ligand selectivity. In this regard we show that there is a difference in polarity between the binding site regions that anchor the carboxylate in RAR and RXR, which translates itself into large differences in the energy of interaction of both receptors with the same ligand. We observe that the latter energy change is canceled off by the solvation energy penalty upon binding. This energy compensation is borne out as well by experiments that address the effect of site-directed mutagenesis on ligand binding to RARgamma. The hypothesis that the difference in binding site polarity might be exploited to build RXR-selective ligands is tested with some compounds having a thiazolidinedione anchoring group.

Sex Differences in Serotonin 1 Receptor Binding in Rat Brain

NASA Astrophysics Data System (ADS)

Fischette, Christine T.; Biegon, Anat; McEwen, Bruce S.

1983-10-01

Male and female rats exhibit sex differences in binding by serotonin 1 receptors in discrete areas of the brain, some of which have been implicated in the control of ovulation and of gonadotropin release. The sex-specific changes in binding, which occur in response to the same hormonal (estrogenic) stimulus, are due to changes in the number of binding sites. Castration alone also affects the number of binding sites in certain areas. The results lead to the conclusion that peripheral hormones modulate binding by serotonin 1 receptors. The status of the serotonin receptor system may affect the reproductive capacity of an organism and may be related to sex-linked emotional disturbances in humans.

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

NASA Astrophysics Data System (ADS)

Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, Esam E.; Doležal, Vladimír

2017-01-01

Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

Neurosteroid binding to the amino terminal and glutamate binding domains of ionotropic glutamate receptors.

PubMed

Cameron, Krasnodara; Bartle, Emily; Roark, Ryan; Fanelli, David; Pham, Melissa; Pollard, Beth; Borkowski, Brian; Rhoads, Sarah; Kim, Joon; Rocha, Monica; Kahlson, Martha; Kangala, Melinda; Gentile, Lisa

2012-06-01

The endogenous neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5β-pregnan-20-one sulfate (PREGAS), have been shown to differentially regulate the ionotropic glutamate receptor (iGluR) family of ligand-gated ion channels. Upon binding to these receptors, PREGAS decreases current flow through the channels. Upon binding to non-NMDA or NMDA receptors containing an GluN2C or GluN2D subunit, PS also decreases current flow through the channels, however, upon binding to NMDA receptors containing an GluN2A or GluN2B subunit, flow through the channels increases. To begin to understand this differential regulation, we have cloned the S1S2 and amino terminal domains (ATD) of the NMDA GluN2B and GluN2D and AMPA GluA2 subunits. Here we present results that show that PS and PREGAS bind to different sites in the ATD of the GluA2 subunit, which when combined with previous results from our lab, now identifies two binding domains for each neurosteroid. We also show both neurosteroids bind only to the ATD of the GluN2D subunit, suggesting that this binding is distinct from that of the AMPA GluA2 subunit, with both leading to iGluR inhibition. Finally, we provide evidence that both PS and PREGAS bind to the S1S2 domain of the NMDA GluN2B subunit. Neurosteroid binding to the S1S2 domain of NMDA subunits responsible for potentiation of iGluRs and to the ATD of NMDA subunits responsible for inhibition of iGluRs, provides an interesting option for therapeutic design. Copyright © 2012 Elsevier Inc. All rights reserved.

The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

PubMed

Cui, Meng; Jiang, Peihua; Maillet, Emeline; Max, Marianna; Margolskee, Robert F; Osman, Roman

2006-01-01

The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

PubMed

Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

2017-05-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. © FASEB.

Expression of human peroxisome proliferator-activated receptors ligand binding domain-maltose binding protein fusion protein in Escherichia coli: a convenient and reliable method for preparing receptor for screening ligands.

PubMed

Li, Changqing; Tian, Mi; Yuan, Ye; Zhou, Qinxin

2008-12-01

Human peroxisome proliferator-activated receptors (hPPARs) are ligand-activated transcription factors and are the target for the treatment of many diseases. Screening of their ligands is mainly based on assays of ligand binding to the ligand binding domain (LBD) of hPPARs.However, such assays are difficult because of the preparation of hPPARs LBD. In order to yield functional hPPARs LBD for screening ligands, hPPARs LBD was fused with maltose-binding protein(MBP) using the pMAL-p2x expression system through the gene engineering technique. The radioligand binding assay showed that MBP did not affect ligand binding with hPPARs LBD in the fusion proteins, which means that MBP-hPPARs LBD can be used instead of hPPARs LBD in ligand screening work. The results show that the new strategy using MBP as a fusion tag for preparing hPPARs LBD for screening ligands is a convenient and reliable method. It may be used to easily obtain the other nuclear receptors.

Benzodiazepine and kainate receptor binding sites in the RCS rat retina.

PubMed

Stasi, Kalliopi; Naskar, Rita; Thanos, Solon; Kouvelas, Elias D; Mitsacos, Ada

2003-02-01

The effect of age and photoreceptor degeneration on the kainate subtype of glutamate receptors and on the benzodiazepine-sensitive gamma-aminobutyric acid-A receptors (GABA(A)) in normal and RCS (Royal College of Surgeons) rats were investigated. [(3)H]Kainate and [(3)H]flunitrazepam were used as radioligands for kainate and GABA(A)/benzodiazepine()receptors, respectively, using the quantitative receptor autoradiography technique. In both normal and RCS rat retina we observed that [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were several times higher in inner plexiform layer (IPL) than in outer plexiform layer (OPL) at all four ages studied (P17, P35, P60 and P180). Age-related changes in receptor binding were observed in normal rat retina: [(3)Eta]flunitrazepam binding showed a significant decrease of 25% between P17 and P60 in IPL,and [(3)Eta]kainate binding showed significant decreases between P17 and P35 in both synaptic layers (71% in IPL and 63% in OPL). Degeneration-related changes in benzodiazepine and kainate receptor binding were observed in RCS rat retina. In IPL, [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were higher than in normal retina at P35 (by 24% and 86%, respectively). In OPL, [(3)Eta]flunitrazepam binding was higher in RCS than in normal retina on P35 (74%) and also on P60 (62%). The results indicate that postnatal changes occur in kainate and benzodiazepine receptor binding sites in OPL and IPL of the rat retina up to 6 months of age. The data also suggest that the receptor binding changes observed in the RCS retina could be a consequence of the primary photoreceptor degeneration.

GRB2 Interaction with the Ecotropic Murine Leukemia Virus Receptor, mCAT-1, Controls Virus Entry and Is Stimulated by Virus Binding

PubMed Central

Chen, Zeming; Kolokoltsov, Andrey A.; Wang, Jia; Adhikary, Shramika; Lorinczi, Marta; Elferink, Lisa A.

2012-01-01

For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2–mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2–mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by

GRB2 interaction with the ecotropic murine leukemia virus receptor, mCAT-1, controls virus entry and is stimulated by virus binding.

PubMed

Chen, Zeming; Kolokoltsov, Andrey A; Wang, Jia; Adhikary, Shramika; Lorinczi, Marta; Elferink, Lisa A; Davey, Robert A

2012-02-01

For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2-mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2-mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating

Effect of receptor binding specificity on the immunogenicity and protective efficacy of influenza virus A H1 vaccines

PubMed Central

Sun, Xiangjie; Cao, Weiping; Pappas, Claudia; Liu, Feng; Katz, Jacqueline M.; Tumpey, Terrence M.

2018-01-01

The biological basis for the poor immunogenicity of unadjuvanted avian influenza A virus vaccines in mammals is not well understood. Here, we mutated the hemagglutinin (HA) of two H1N1 virus vaccines to determine whether virus receptor binding specificity contributes to the low immunogenicity of avian influenza virus vaccines. Mutations were introduced into the HA of an avian influenza virus, A/Duck/New York/15024–21/96 (Dk/96) which switched the binding preference from α2,3- to α2,6-linked sialic acid (SA). A switch in receptor specificity of the human A/South Carolina/1/18 (SC/18) virus generated a mutant virus with α2,3 SA (avian) binding preference. Inactivated vaccines were generated and administered to mice and ferrets intramuscularly. We found that the vaccines with human receptor binding preference induced slightly higher antibody titers and cell-mediated immune responses compared to their isogenic viruses with avian receptor binding specificity. Upon challenge with DK/96 or SC18 virus, differences in lung virus titers between the vaccine groups with different receptor-binding specificities were minimal. Overall, our data suggest that receptor binding specificity contributes only marginally to the immunogenicity of avian influenza vaccines and that other factors may also be involved. PMID:25078114

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed Central

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-01-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation. PMID:9927419

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

PubMed Central

Gaviglio, Angela L.; Knelson, Erik H.; Blobe, Gerard C.

2017-01-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor–like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.—Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. PMID:28174207

Macrophage Migration Inhibitory Factor-CXCR4 Receptor Interactions

PubMed Central

Rajasekaran, Deepa; Gröning, Sabine; Schmitz, Corinna; Zierow, Swen; Drucker, Natalie; Bakou, Maria; Kohl, Kristian; Mertens, André; Lue, Hongqi; Weber, Christian; Xiao, Annie; Luker, Gary; Kapurniotu, Aphrodite; Lolis, Elias; Bernhagen, Jürgen

2016-01-01

An emerging number of non-chemokine mediators are found to bind to classical chemokine receptors and to elicit critical biological responses. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that exhibits chemokine-like activities through non-cognate interactions with the chemokine receptors CXCR2 and CXCR4, in addition to activating the type II receptor CD74. Activation of the MIF-CXCR2 and -CXCR4 axes promotes leukocyte recruitment, mediating the exacerbating role of MIF in atherosclerosis and contributing to the wealth of other MIF biological activities. Although the structural basis of the MIF-CXCR2 interaction has been well studied and was found to engage a pseudo-ELR and an N-like loop motif, nothing is known about the regions of CXCR4 and MIF that are involved in binding to each other. Using a genetic strain of Saccharomyces cerevisiae that expresses a functional CXCR4 receptor, site-specific mutagenesis, hybrid CXCR3/CXCR4 receptors, pharmacological reagents, peptide array analysis, chemotaxis, fluorescence spectroscopy, and circular dichroism, we provide novel molecular information about the structural elements that govern the interaction between MIF and CXCR4. The data identify similarities with classical chemokine-receptor interactions but also provide evidence for a partial allosteric agonist compared with CXCL12 that is possible due to the two binding sites of CXCR4. PMID:27226569

Alginate Sulfates Mitigate Binding Kinetics of Proangiogenic Growth Factors with Receptors toward Revascularization.

PubMed

Schmidt, John; Lee, Min Kyung; Ko, Eunkyung; Jeong, Jae Hyun; DiPietro, Luisa A; Kong, Hyunjoon

2016-07-05

Ever since proangiogenic growth factors have been used as a vascular medicine to treat tissue ischemia, efforts have been increasingly made to develop a method to enhance efficacy of growth factors in recreating microvascular networks, especially at low dose. To this end, we hypothesized that polysaccharides substituted with sulfate groups would amplify growth factor receptor activation and stimulate phenotypic activities of endothelial cells involved in neovascularization. We examined this hypothesis by modifying alginate with a controlled number of sulfates and using it to derive a complex with vascular endothelial growth factor (VEGF), as confirmed with fluorescence resonance energy transfer (FRET) assay. Compared with the bare VEGF and with a mixture of VEGF and unmodified alginates, the VEGF complexed with alginate sulfates significantly reduced the dissociation rate with the VEGFR-2, elevated VEGFR-2 phosphorylation level, and increased the number of endothelial sprouts in vitro. Furthermore, the VEGF-alginate sulfate complex improved recovery of perfusion in an ischemic hindlimb of a mouse due to the increase of the capillary density. Overall, this study not only demonstrates an important cofactor of VEGF but also uncovers an underlying mechanism by which the cofactor mitigates the VEGF-induced signaling involved in the binding kinetics and activation of VEGFR. We therefore believe that the results of this study will be highly useful in improving the therapeutic efficacy of various growth factors and expediting their uses in clinical treatments of wounds and tissue defects.

Design of an Insulin Analog with Enhanced Receptor Binding Selectivity

PubMed Central

Zhao, Ming; Wan, Zhu-li; Whittaker, Linda; Xu, Bin; Phillips, Nelson B.; Katsoyannis, Panayotis G.; Ismail-Beigi, Faramarz; Whittaker, Jonathan; Weiss, Michael A.

2009-01-01

Insulin binds with high affinity to the insulin receptor (IR) and with low affinity to the type 1 insulin-like growth factor (IGF) receptor (IGFR). Such cross-binding, which reflects homologies within the insulin-IGF signaling system, is of clinical interest in relation to the association between hyperinsulinemia and colorectal cancer. Here, we employ nonstandard mutagenesis to design an insulin analog with enhanced affinity for the IR but reduced affinity for the IGFR. Unnatural amino acids were introduced by chemical synthesis at the N- and C-capping positions of a recognition α-helix (residues A1 and A8). These sites adjoin the hormone-receptor interface as indicated by photocross-linking studies. Specificity is enhanced more than 3-fold on the following: (i) substitution of GlyA1 by d-Ala or d-Leu, and (ii) substitution of ThrA8 by diaminobutyric acid (Dab). The crystal structure of [d-AlaA1,DabA8]insulin, as determined within a T6 zinc hexamer to a resolution of 1.35 Å, is essentially identical to that of human insulin. The nonstandard side chains project into solvent at the edge of a conserved receptor-binding surface shared by insulin and IGF-I. Our results demonstrate that modifications at this edge discriminate between IR and IGFR. Because hyperinsulinemia is typically characterized by a 3-fold increase in integrated postprandial insulin concentrations, we envisage that such insulin analogs may facilitate studies of the initiation and progression of cancer in animal models. Future development of clinical analogs lacking significant IGFR cross-binding may enhance the safety of insulin replacement therapy in patients with type 2 diabetes mellitus at increased risk of colorectal cancer. PMID:19773552

The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

PubMed Central

Klippel, A; Escobedo, J A; Fantl, W J; Williams, L T

1992-01-01

Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor. Images PMID:1312663

Characterization of the Igf-II Binding Site of the IGF-II/MAN-6-P Receptor Extracellular Domain.

NASA Astrophysics Data System (ADS)

Garmroudi, Farideh

1995-01-01

In mammals, insulin-like growth factor II (IGF -II) and glycoproteins bearing the mannose 6-phosphate (Man -6-P) recognition marker bind with high affinity to the same receptor. The functional consequences of IGF-II binding to the receptor at the cell surface are not clear. In these studies, we sought to broaden our understanding of the functional regions of the receptor regarding its IGF -II binding site. The IGF-II binding/cross-linking domain of the IGF-II/Man-6-P receptor was mapped by sequencing receptor fragments covalently attached to IGF-II. Purified rat placental or bovine liver receptors were affinity-labeled, with ^{125}I-IGF-II and digested with endoproteinase Glu-C. Analysis of digests by gel electrophoresis revealed a major radiolabeled band of 18 kDa, which was purified by gel filtration chromatography followed by reverse-phase HPLC and electroblotting. Sequence analysis revealed that, the peptide S(H)VNSXPMF, located within extracellular repeat 10 and beginning with serine 1488 of the bovine receptor, was the best candidate for the IGF-II cross-linked peptide. These data indicated that residues within repeats 10-11 were important for IGF -II binding. To define the location of the IGF-II binding site further, a nested set of six human receptor cDNA constructs was designed to produce epitope-tagged fusion proteins encompassing the region between repeats 8 and 11 of the human IGF-II/Man-6-P receptor extracellular domain. These truncated receptors were transiently expressed in COS-7 cells, immunoprecipitated and analyzed for their abilities to bind and cross-link to IGF-II. All of the constructs were capable of binding/cross-linking to IGF-II, except for the 9.0-11 construct. Displacement curve analysis indicated that the truncated receptors were approximately equivalent in IGF-II binding affinity, but were of 5- to 10-fold lower affinity than full-length receptors. Sequencing of the 9.0-11 construct indicated the presence of a point mutation

Takifugu rubripes cation independent mannose 6-phosphate receptor: Cloning, expression and functional characterization of the IGF-II binding domain.

PubMed

A, Ajith Kumar; Nadimpalli, Siva Kumar

2018-07-01

Mannose 6-phosphate/IGF-II receptor mediated lysosomal clearance of insulin-like growth factor-II is significantly associated with the evolution of placental mammals. The protein is also referred to as the IGF-II receptor. Earlier studies suggested relatively low binding affinity between the receptor and ligand in prototherian and metatherian mammals. In the present study, we cloned the IGF-II binding domain of the early vertebrate fugu fish and expressed it in bacteria. A 72000Da truncated receptor containing the IGF-II binding domain was obtained. Analysis of this protein (covering domains 11-13 of the CIMPR) for its affinity to fish and human IGF-II by ligand blot assays and ELISA showed that the expressed receptor can specifically bind to both fish and human IGF-II. Additionally, a peptide-specific antibody raised against the region of the IGF-II binding domain also was able to recognize the IGF-II binding regions of mammalian and non-mammalian cation independent MPR protein. These interactions were further characterized by Surface Plasma resonance support that the receptor binds to fish IGF-II, with a dissociation constant of 548nM. Preliminary analysis suggests that the binding mechanism as well as the affinity of the fish and human receptor for IGF-II may have varied according to different evolutionary pressures. Copyright © 2018. Published by Elsevier B.V.

High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

PubMed

Verrier, C S; Roodi, N; Yee, C J; Bailey, L R; Jensen, R A; Bustin, M; Parl, F F

1997-07-01

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

Effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro.

PubMed

Lee, J S; Kim, J M; Hong, E K; Kim, S-O; Yoo, Y-J; Cha, J-H

2009-02-01

A growing amount of attention has been placed on periodontal regeneration and wound healing for periodontal therapy. This study was conducted in an effort to determine the effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro. Human periodontal ligament cells were acquired from explant tissue of human healthy periodontal ligament. After the wounding of periodontal ligament cells, the change in expression of heparin-binding epidermal growth factor-like growth factor and epidermal growth factor receptors 1-4 mRNA was assessed. The effects of heparin-binding epidermal growth factor-like growth factor on periodontal ligament cell proliferation and repopulation were assessed in vitro via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and by photographing the injuries, respectively. Extracellular signal-regulated kinase (Erk)1/2, p38 and Akt phosphorylation was characterized via western blotting. Scratch wounding resulted in a significant up-regulation of heparin-binding epidermal growth factor-like growth factor mRNA expression, whereas wounding had no effect on the expression levels of epidermal growth factor receptors 1-4. Interestingly, no expression of epidermal growth factor receptors 2 and 4 was detectable prior to or after wounding. Heparin-binding epidermal growth factor-like growth factor treatment promoted the proliferation and repopulation of periodontal ligament cells. The scratch wounding also stimulated the phosphorylation of Erk1/2 and p38, but not of Akt, in periodontal ligament cells, and heparin-binding epidermal growth factor-like growth factor treatment applied after wounding amplified and extended the activations of Erk1/2 and p38, but not of Akt. Furthermore, Erk1/2 inhibition blocked the process of cell repopulation induced by heparin-binding epidermal growth factor-like growth factor, whereas the

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

PubMed Central

Shi, Yuan; Chen, Xiang; Elsasser, Suzanne; Stocks, Bradley B.; Tian, Geng; Lee, Byung-Hoon; Shi, Yanhong; Zhang, Naixia; de Poot, Stefanie A. H.; Tuebing, Fabian; Sun, Shuangwu; Vannoy, Jacob; Tarasov, Sergey G.; Engen, John R.; Finley, Daniel; Walters, Kylie J.

2016-01-01

Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced

Gonadotropin-Releasing Hormone (GnRH) Receptor Structure and GnRH Binding

PubMed Central

Flanagan, Colleen A.; Manilall, Ashmeetha

2017-01-01

Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues

Regulation of insulin-like growth factor I receptors on vascular smooth muscle cells by growth factors and phorbol esters.

PubMed

Ververis, J J; Ku, L; Delafontaine, P

1993-06-01

Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells. To characterize regulation of vascular IGF I receptors, we performed radioligand displacement experiments using rat aortic smooth muscle cells (RASMs). Serum deprivation for 48 hours caused a 40% decrease in IGF I receptor number. Exposure of quiescent RASMs to platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or angiotensin II (Ang II) caused a 1.5-2.0-fold increase in IGF I receptors per cell. After FGF exposure, there was a marked increase in the mitogenic response to IGF I. IGF I downregulated its receptors in the presence of platelet-poor plasma. Stimulation of protein kinase C (PKC) by exposure of quiescent RASMs to phorbol 12-myristate 13-acetate caused a biphasic response in IGF I binding; there was a 42% decrease in receptor number at 45 minutes and a 238% increase at 24 hours. To determine the role of PKC in growth factor-induced regulation of IGF I receptors, we downregulated PKC by exposing RASMs to phorbol 12,13-dibutyrate (PDBu) for 48 hours. PDGF- and FGF- but not Ang II-mediated upregulation of IGF I receptors was completely inhibited in PDBu-treated cells. Thus, acute PKC activation by phorbol esters inhibits IGF I binding, whereas chronic PKC activation increases IGF I binding. PDGF and FGF but not Ang II regulate vascular IGF I receptors through a PKC-dependent pathway. These data provide new insights into the regulation of vascular smooth muscle cell IGF I receptors in vitro and are of potential importance in characterizing vascular proliferative responses in vivo.

Synthesis and binding affinity of neuropeptide Y at opiate receptors.

PubMed

Kiddle, James J; McCreery, Heather J; Soles, Sonia

2003-03-24

Neuropeptide Y and several metabolic fragments were synthesized and evaluated for binding affinity at non-selective opiate receptors. Neuropeptide Y and several C-terminal fragments were shown to bind to non-selective opiate receptors with an affinity similar to that of Leu-enkephalin.

Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor*

PubMed Central

An, Bo; Abbonante, Vittorio; Xu, Huifang; Gavriilidou, Despoina; Yoshizumi, Ayumi; Bihan, Dominique; Farndale, Richard W.; Kaplan, David L.; Balduini, Alessandra; Leitinger, Birgit; Brodsky, Barbara

2016-01-01

A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities. PMID:26702058

Receptor-binding, biodistribution, and metabolism studies of 64Cu-DOTA-cetuximab, a PET-imaging agent for epidermal growth-factor receptor-positive tumors.

PubMed

Ping Li, Wen; Meyer, Laura A; Capretto, David A; Sherman, Christopher D; Anderson, Carolyn J

2008-04-01

The epidermal growth-factor receptor (EGFR) and its ligands have been recognized as critical factors in the pathophysiology of tumorigenesis. Overexpression of the EGFR plays a significant role in the tumor progression of a wide variety of solid human cancers. Therefore, the EGFR represents an attractive target for the design of novel diagnostic and therapeutic agents for cancer. Cetuximab (C225, Erbitux) was the first monoclonal antibody targeted against the ligand-binding site of EGFR approved by the Food and Drug Administration for the treatment of patients with EGFR-expressing, metastatic colorectal carcinoma, although clinical trials showed variability in the response to this treatment. The aim of this study involved using cetuximab to design a positron emission tomography (PET) agent to image the overexpression of EGFR in tumors. Cetuximab was conjugated with the chelator, DOTA, for radiolabeling with the positron-emitter, 64Cu (T(1/2) = 12.7 hours). 64Cu-DOTA-cetuximab showed high binding affinity to EGFR-positive A431 cells (K(D) of 0.28 nM). Both biodistribution and microPET imaging studies with 64Cu-DOTA-cetuximab demonstrated greater uptake at 24 hours postinjection in EGFR-positive A431 tumors (18.49% +/- 6.50% injected dose per gram [ID/g]), compared to EGFR-negative MDA-MB-435 tumors (2.60% +/- 0.35% ID/g). A431 tumor uptake at 24 hours was blocked with unlabeled cetuximab (10.69% +/- 2.72% ID/g), suggesting that the tumor uptake was receptor mediated. Metabolism experiments in vivo showed that 64Cu-DOTA-cetuximab was relatively stable in the blood of tumor-bearing mice; however, there was significant metabolism in the liver and tumors. 64Cu-DOTA-cetuximab is a potential agent for imaging EGFR-positive tumors in humans.

Characterization of ( sup 3 H)alprazolam binding to central benzodiazepine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCabe, R.T.; Mahan, D.R.; Smith, R.B.

1990-10-01

The binding of the triazolobenzodiazepine ({sup 3}H)alprazolam was studied to characterize the in vitro interactions with benzodiazepine receptors in membrane preparations of rat brain. Studies using nonequilibrium and equilibrium binding conditions for ({sup 3}H)alprazolam resulted in high specific to nonspecific (signal to noise) binding ratios. The binding of ({sup 3}H)alprazolam was saturable and specific with a low nanomolar affinity for benzodiazepine receptors in the rat brain. The Kd was 4.6 nM and the Bmax was 2.6 pmol/mg protein. GABA enhanced ({sup 3}H)alprazolam binding while several benzodiazepine receptor ligands were competitive inhibitors of this drug. Compounds that bind to other receptormore » sites had a very weak or negligible effect on ({sup 3}H)alprazolam binding. Alprazolam, an agent used as an anxiolytic and in the treatment of depression, acts in vitro as a selective and specific ligand for benzodiazepine receptors in the rat brain. The biochemical binding profile does not appear to account for the unique therapeutic properties which distinguish this compound from the other benzodiazepines in its class.« less

Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

PubMed Central

Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

2013-01-01

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

A novel substance P binding site in rat brain regions modulates TRH receptor binding.

PubMed

Sharif, N A

1990-10-01

Binding sites for thyrotropin-releasing hormone (TRH) were labelled with [3H](2-Me-His3)TRH ([3H]MeTRH) on membranes from rat brain regions at 0 degrees C for 5 h. Amygdaloid membranes bound [3H]MeTRH with high-affinity (Kd = 3.1 +/- 0.5 nM (n = 4)). Five TRH analogs competed for this binding with the same rank order and with affinities that matched the pharmacological specificity of pituitary TRH receptors. Substance P (SP) and its C-terminal fragments reduced amygdaloid TRH receptor binding in a concentration dependent manner (IC50 for SP = 65 microM). The rank order of potency of SP analogs at inhibiting TRH receptor binding was: SP greater than nonapeptide (3-11) greater than hexapeptide (6-11) greater than heptapeptide (5-11) greater than pentapeptide (7-11). However, other tachykinins were inactive in this system. SP was a potent inhibitor of [3H]MeTRH binding in hippocampus greater than spinal cord greater than retina greater than n. accumbens greater than hypothalamus greater than amygdaloid greater than olfactory bulb greater than or equal to pituitary greater than pons/medulla in parallel assays. In amygdaloid membranes SP (50 microM) reduced the apparent maximum receptor density by 39% (p less than 0.01) without altering the binding affinity, and 100 microM SP induced a biphasic dissociation of [3H]MeTRH with kinetics faster than those induced by both TRH (10 microM) and serotonin (100 microM). In contrast, other neuropeptides such as neurotensin, proctolin, angiotensin II, bombesin and luteinizing hormone releasing hormone did not significantly inhibit [3H]MeTRH binding to amygdaloid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Competitive Binding Assay for the G-Protein-Coupled Receptor 30 (GPR30) or G-Protein-Coupled Estrogen Receptor (GPER).

PubMed

Thekkumkara, Thomas; Snyder, Russell; Karamyan, Vardan T

2016-01-01

The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate the underlying effects of this novel estrogen metabolite and to validate its targets; therefore, this competitive receptor-binding assay protocol was developed in order to assess the membrane receptor binding and affinity of 2-methyoxyestradiol.

Characterization of binding affinity of CJ-023,423 for human prostanoid EP4 receptor.

PubMed

Murase, Akio; Nakao, Kazunari; Takada, Junji

2008-01-01

In order to characterize the receptor binding pharmacology of CJ-023,423, a potent and selective EP4 antagonist, we performed a radioligand receptor binding assay under various assay conditions. An acidic (pH 6) and hypotonic buffer is a conventional, well-known buffer for prostaglandin E2 receptor binding assays. CJ-023,423 showed moderate binding affinity for human EP4 receptor under conventional buffer conditions. However, its binding affinity was greatly increased under neutral (pH 7.4) and isotonic buffer conditions. In this report, the binding mechanism between CJ-023,423 and human EP4 receptor is discussed based on the binding affinities determined under various assay conditions. Copyright 2008 S. Karger AG, Basel.

Glycosylation of Cblns attenuates their receptor binding.

PubMed

Rong, Yongqi; Bansal, Parmil K; Wei, Peng; Guo, Hong; Correia, Kristen; Parris, Jennifer; Morgan, James I

2018-05-18

Cbln1 is the prototype of a family (Cbln1-Cbln4) of secreted glycoproteins and is essential for normal synapse structure and function in cerebellum by bridging presynaptic Nrxn to postsynaptic Grid2. Here we report the effects of glycosylation on the in vitro receptor binding properties of Cblns. Cbln1, 2 and 4 harbor two N-linked glycosylation sites, one at the N-terminus is in a region implicated in Nrxn binding and the second is in the C1q domain, a region involved in Grid2 binding. Mutation (asparagine to glutamine) of the N-terminal site, increased neurexin binding whereas mutation of the C1q site markedly increased Grid2 binding. These mutations did not influence subunit composition of Cbln trimeric complexes (mediated through the C1q domain) nor their assembly into hexamers (mediated by the N-terminal region). Therefore, glycosylation likely masks the receptor binding interfaces of Cblns. As Cbln4 has undetectable Grid2 binding in vitro we assessed whether transgenic expression of wild type Cbln4 or its glycosylation mutants rescued the Cbln1-null phenotype in vivo. Cbln4 partially rescued and both glycosylation mutants completely rescued ataxia in cbln1-null mice. Thus Cbln4 has intrinsic Grid2 binding that is attenuated by glycosylation, and glycosylation mutants exhibit gain of function in vivo. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Steroid hormone and epidermal growth factor receptors in meningiomas.

PubMed

Horsfall, D J; Goldsmith, K G; Ricciardelli, C; Skinner, J M; Tilley, W D; Marshall, V R

1989-11-01

A prospective study of steroid hormone and epidermal growth factor receptor expression in 57 meningiomas is presented. Scatchard analysis of radioligand binding identified 20% of meningiomas as expressing classical oestrogen receptors (ER) at levels below that normally accepted for positivity, the remainder being negative. ER could not be visualized in any meningioma using immunocytochemistry. Alternatively, 74% of meningiomas demonstrated the presence of progesterone receptors (PR) by Scatchard analysis, the specificity of which could not be attributed to glucocorticoid or androgen receptors. Confirmation of classical PR presence was determined by immunocytochemical staining. The presence of epidermal growth factor receptor (EGFR) was demonstrated in 100% of meningiomas using immunocytochemical staining. These data are reviewed in the context of previously reported results and are discussed in relation to the potential for medical therapy as an adjunct to surgery.

Fibroblast growth factor receptors, developmental corruption and malignant disease.

PubMed

Kelleher, Fergal C; O'Sullivan, Hazel; Smyth, Elizabeth; McDermott, Ray; Viterbo, Antonella

2013-10-01

Fibroblast growth factors (FGF) are a family of ligands that bind to four different types of cell surface receptor entitled, FGFR1, FGFR2, FGFR3 and FGFR4. These receptors differ in their ligand binding affinity and tissue distribution. The prototypical receptor structure is that of an extracellular region comprising three immunoglobulin (Ig)-like domains, a hydrophobic transmembrane segment and a split intracellular tyrosine kinase domain. Alternative gene splicing affecting the extracellular third Ig loop also creates different receptor isoforms entitled FGFRIIIb and FGFRIIIc. Somatic fibroblast growth factor receptor (FGFR) mutations are implicated in different types of cancer and germline FGFR mutations occur in developmental syndromes particularly those in which craniosynostosis is a feature. The mutations found in both conditions are often identical. Many somatic FGFR mutations in cancer are gain-of-function mutations of established preclinical oncogenic potential. Gene amplification can also occur with 19-22% of squamous cell lung cancers for example having amplification of FGFR1. Ontologic comparators can be informative such as aberrant spermatogenesis being implicated in both spermatocytic seminomas and Apert syndrome. The former arises from somatic FGFR3 mutations and Apert syndrome arises from germline FGFR2 mutations. Finally, therapeutics directed at inhibiting the FGF/FGFR interaction are a promising subject for clinical trials.

Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site

PubMed Central

Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J.

2016-01-01

ABSTRACT Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. IMPORTANCE We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. PMID:26764003

Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

PubMed

Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

2016-01-13

Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Design and structure of stapled peptides binding to estrogen receptors.

PubMed

Phillips, Chris; Roberts, Lee R; Schade, Markus; Bazin, Richard; Bent, Andrew; Davies, Nichola L; Moore, Rob; Pannifer, Andrew D; Pickford, Andrew R; Prior, Stephen H; Read, Christopher M; Scott, Andrew; Brown, David G; Xu, Bin; Irving, Stephen L

2011-06-29

Synthetic peptides that specifically bind nuclear hormone receptors offer an alternative approach to small molecules for the modulation of receptor signaling and subsequent gene expression. Here we describe the design of a series of novel stapled peptides that bind the coactivator peptide site of estrogen receptors. Using a number of biophysical techniques, including crystal structure analysis of receptor-stapled peptide complexes, we describe in detail the molecular interactions and demonstrate that all-hydrocarbon staples modulate molecular recognition events. The findings have implications for the design of stapled peptides in general.

Receptor binding properties and antinociceptive effects of chimeric peptides consisting of a micro-opioid receptor agonist and an ORL1 receptor antagonist.

PubMed

Kawano, Susumu; Ito, Risa; Nishiyama, Miharu; Kubo, Mai; Matsushima, Tomoko; Minamisawa, Motoko; Ambo, Akihiro; Sasaki, Yusuke

2007-07-01

Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100< pmol/mouse) than when administered i.t. (ED(50): 1.34-4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.

Reduced post-synaptic serotonin type 1A receptor binding in bipolar depression

PubMed Central

Nugent, Allison C.; Bain, Earle E.; Carlson, Paul J.; Neumeister, Alexander; Bonne, Omer; Carson, Richard E.; Eckelman, William; Herscovitch, Peter; Zarate, Carlos A.; Charney, Dennis S.; Drevets, Wayne C.

2013-01-01

Multiple lines of evidence suggest that serotonin type 1A (5-HT1A) receptor dysfunction is involved in the pathophysiology of mood disorders, and that alterations in 5-HT1A receptor function play a role in the mechanisms of antidepressant and mood stabilizer treatment. The literature is in disagreement, however, as to whether 5-HT1A receptor binding abnormalities exist in bipolar disorder (BD). We acquired PET images of 5-HT1A receptor binding in 26 unmedicated BD subjects and 37 healthy controls using [18F]FCWAY, a highly selective 5-HT1A receptor radio-ligand. The mean 5-HT1A receptor binding potential (BPP) was significantly lower in BD subjects compared to controls in cortical regions where 5-HT1A receptors are expressed post-synaptically, most prominently in the mesiotemporal cortex. Post-hoc assessments involving other receptor specific binding parameters suggested that this difference particularly affected the females with BD. The mean BPP did not differ between groups in the raphe nucleus, however, where 5-HT1A receptors are predominantly expressed pre-synaptically. Across subjects the BPP in the mesiotemporal cortex was inversely correlated with trough plasma cortisol levels, consistent with preclinical literature indicating that hippocampal 5-HT1A receptor expression is inhibited by glucocorticoid receptor stimulation. These findings suggest that 5-HT1A receptor binding is abnormally reduced in BD, and this abnormality may particularly involve the postsynaptic 5-HT1A receptor system of individuals with a tendency toward cortisol hypersecretion. PMID:23434290

Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

PubMed Central

Ramsey, Simeon J; Attkins, Neil J; Fish, Rebecca; van der Graaf, Piet H

2011-01-01

BACKGROUND AND PURPOSE A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF1) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan. EXPERIMENTAL APPROACH A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF1 antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic–pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF1 receptor binding of novel compounds can be predicted on the basis of in vitro assays. KEY RESULTS The non-competitive antagonist behaviour appeared to be correlated to the CRF1 receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations. CONCLUSIONS AND IMPLICATIONS This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF1 receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF1 receptor antagonists in an efficient manner in a drug discovery setting. PMID:21449919

Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor {alpha}/retinoid X receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belanger, Adam J.; Luo Zhengyu; Vincent, Karen A.

2007-12-21

In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid {beta}-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid {beta}-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1{alpha}/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferasemore » I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for {beta}-oxidation. Furthermore, adenovirus-mediated expression of HIF-1{alpha}/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})/retinoid X receptor (RXR), in the presence or absence of a PPAR{alpha} ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPAR{alpha}/RXR.« less

Molecular Determinants of Epidermal Growth Factor Binding: A Molecular Dynamics Study

PubMed Central

Sanders, Jeffrey M.; Wampole, Matthew E.; Thakur, Mathew L.; Wickstrom, Eric

2013-01-01

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF

Nuclear actions of insulin-like growth factor binding protein-3.

PubMed

Baxter, Robert C

2015-09-10

In addition to its actions outside the cell, cellular uptake and nuclear import of insulin-like growth factor binding protein-3 (IGFBP-3) has been recognized for almost two decades, but knowledge of its nuclear actions has been slow to emerge. IGFBP-3 has a functional nuclear localization signal and interacts with the nuclear transport protein importin-β. Within the nucleus IGFBP-3 appears to have a role in transcriptional regulation. It can bind to the nuclear receptor, retinoid X receptor-α and several of its dimerization partners, including retinoic acid receptor, vitamin D receptor (VDR), and peroxisome proliferator-activated receptor-γ (PPARγ). These interactions modulate the functions of these receptors, for example inhibiting VDR-dependent transcription in osteoblasts and PPARγ-dependent transcription in adipocytes. Nuclear IGFBP-3 can be detected by immunohistochemistry in cancer and other tissues, and its presence in the nucleus has been shown in many cell culture studies to be necessary for its pro-apoptotic effect, which may also involve interaction with the nuclear receptor Nur77, and export from the nucleus. IGFBP-3 is p53-inducible and in response to DNA damage, forms a complex with the epidermal growth factor receptor (EGFR), translocating to the nucleus to interact with DNA-dependent protein kinase. Inhibition of EGFR kinase activity or downregulation of IGFBP-3 can inhibit DNA double strand-break repair by nonhomologous end joining. IGFBP-3 thus has the ability to influence many cell functions through its interactions with intranuclear pathways, but the importance of these interactions in vivo, and their potential to be targeted for therapeutic benefit, require further investigation. Copyright © 2015 Elsevier B.V. All rights reserved.

The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemoto, Takayuki; Ohara-Nemoto, Yuko; Denis, M.

1990-02-20

High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, the authors decided to investigate the possible relationship between these two phenomena. The binding of ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas ({sup 3}H)TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR showed the presence of twomore » types of binding sites. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low ({sup 3}H)TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. These results indicate that the transformed GR has a reduced ({sup 3}H)TA-binding affinity as compared to the nontransformed GR. The steroid-binding domain (amino acids 477-777) and the DNA- and steroid-binding domains (amino acids 415-777) of the human GR were expressed in Escherichia coli as protein A fused proteins. Taken together, these results suggest that the component(s) associating with the nontransformed GR, possibly the heat shock protein hsp 90, play(s) an important role in stabilizing the GR in a high-affinity state for steroids.« less

In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2

PubMed Central

Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R

2018-01-01

Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V1) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys8]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg8]-vasopressin (AVP) at V1 and vasopressin-2 (V2) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V1 and V2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [3H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V1) and cyclic adenosine monophosphate (V2). Binding potency at V1 and V2 was AVP>LVP>>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V1 than for V2. Cellular activity potency was also AVP>LVP>>terlipressin. Terlipressin was a partial agonist at V1 and a full agonist at V2; LVP was a full agonist at both V1 and V2. The in vivo response to terlipressin is likely due to the partial V1 agonist activity of terlipressin and full V1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors. PMID:29302194

In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2.

PubMed

Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R

2018-01-01

Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V 1 ) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys 8 ]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg 8 ]-vasopressin (AVP) at V 1 and vasopressin-2 (V 2 ) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V 1 and V 2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [ 3 H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V 1 ) and cyclic adenosine monophosphate (V 2 ). Binding potency at V 1 and V 2 was AVP>LVP>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V 1 than for V 2 . Cellular activity potency was also AVP>LVP>terlipressin. Terlipressin was a partial agonist at V 1 and a full agonist at V 2 ; LVP was a full agonist at both V 1 and V 2 . The in vivo response to terlipressin is likely due to the partial V 1 agonist activity of terlipressin and full V 1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors.

Structural analysis of the receptor binding domain of botulinum neurotoxin serotype D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Buchko, Garry W.; Qin, Lin

2010-10-28

Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65 Å resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences aremore » located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10 Å relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.« less

Structural Analysis of the Receptor Binding Domain of Botulinum Neurotoxin Serotype D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y Zhang; G Buchko; L Qin

2011-12-31

Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65{angstrom} resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are locatedmore » at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10{angstrom} relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.« less

Alternative dimerization interfaces in the glucocorticoid receptor-α ligand binding domain.

PubMed

Bianchetti, Laurent; Wassmer, Bianca; Defosset, Audrey; Smertina, Anna; Tiberti, Marion L; Stote, Roland H; Dejaegere, Annick

2018-04-30

Nuclear hormone receptors (NRs) constitute a large family of multi-domain ligand-activated transcription factors. Dimerization is essential for their regulation, and both DNA binding domain (DBD) and ligand binding domain (LBD) are implicated in dimerization. Intriguingly, the glucocorticoid receptor-α (GRα) presents a DBD dimeric architecture similar to that of the homologous estrogen receptor-α (ERα), but an atypical dimeric architecture for the LBD. The physiological relevance of the proposed GRα LBD dimer is a subject of debate. We analyzed all GRα LBD homodimers observed in crystals using an energetic analysis based on the PISA and on the MM/PBSA methods and a sequence conservation analysis, using the ERα LBD dimer as a reference point. Several dimeric assemblies were observed for GRα LBD. The assembly generally taken to be physiologically relevant showed weak binding free energy and no significant residue conservation at the contact interface, while an alternative homodimer mediated by both helix 9 and C-terminal residues showed significant binding free energy and residue conservation. However, none of the GRα LBD assemblies found in crystals are as stable or conserved as the canonical ERα LBD dimer. GRα C-terminal sequence (F-domain) forms a steric obstacle to the canonical dimer assembly in all available structures. Our analysis calls for a re-examination of the currently accepted GRα homodimer structure and experimental investigations of the alternative architectures. This work questions the validity of the currently accepted architecture. This has implications for interpreting physiological data and for therapeutic design pertaining to glucocorticoid research. Copyright © 2018. Published by Elsevier B.V.

Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

PubMed

Carbone, V; Kim, H; Huang, J X; Baker, M A; Ong, C; Cooper, M A; Li, J; Rockman, S; Velkov, T

2013-01-01

Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

Accelerated molecular dynamics simulations of ligand binding to a muscarinic G-protein-coupled receptor.

PubMed

Kappel, Kalli; Miao, Yinglong; McCammon, J Andrew

2015-11-01

Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein-coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein-ligand binding, especially the drug recognition of GPCRs.

Radioiodination of chicken luteinizing hormone without affecting receptor binding potency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kikuchi, M.; Ishii, S.

1989-12-01

By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of (1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; (2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reactionmore » time and low temperature; and (3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final {sup 125}I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. {sup 125}I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.« less

Glycosylation of immunoglobulin A influences its receptor binding.

PubMed

Basset, C; Devauchelle, V; Durand, V; Jamin, C; Pennec, Y L; Youinou, P; Dueymes, M

1999-12-01

Immunoglobulin A (IgA), which is heavily glycosylated, interacts with a variety of receptors, e.g. the asialoglycoprotein receptor (ASGP-R), which binds terminal galactose residues, and the Fcalpha receptor (FcalphaRI). It has thus been proposed that elevated serum levels of IgA in primary Sjögren's syndrome (pSS) are caused by its defective clearance. To test this hypothesis, we developed a method (based on sialyl transferases eluted from a hepatoma cell line) to increase the amount of sialic acid (SA) on IgA, and used a battery of IgA1- and IgA2-specific glycosidases to reduce this amount. Binding of IgA1 and IgA2 to ASGP-R and FcalphaRI was found to be sugar dependent because oversialylated IgA bound less than native or desialylated IgA. However, individual sugars did not play a direct role in this binding. Given that IgA are oversialylated in pSS, defective clearance of IgA may indeed be ascribed to an excess of SA in IgA1 and IgA2.

Adaptation of avian influenza A (H6N1) virus from avian to human receptor-binding preference

PubMed Central

Wang, Fei; Qi, Jianxun; Bi, Yuhai; Zhang, Wei; Wang, Min; Zhang, Baorong; Wang, Ming; Liu, Jinhua; Yan, Jinghua; Shi, Yi; Gao, George F

2015-01-01

The receptor-binding specificity of influenza A viruses is a major determinant for the host tropism of the virus, which enables interspecies transmission. In 2013, the first human case of infection with avian influenza A (H6N1) virus was reported in Taiwan. To gather evidence concerning the epidemic potential of H6 subtype viruses, we performed comprehensive analysis of receptor-binding properties of Taiwan-isolated H6 HAs from 1972 to 2013. We propose that the receptor-binding properties of Taiwan-isolated H6 HAs have undergone three major stages: initially avian receptor-binding preference, secondarily obtaining human receptor-binding capacity, and recently human receptor-binding preference, which has been confirmed by receptor-binding assessment of three representative virus isolates. Mutagenesis work revealed that E190V and G228S substitutions are important to acquire the human receptor-binding capacity, and the P186L substitution could reduce the binding to avian receptor. Further structural analysis revealed how the P186L substitution in the receptor-binding site of HA determines the receptor-binding preference change. We conclude that the human-infecting H6N1 evolved into a human receptor preference. PMID:25940072

Molecular Modeling of Structures and Interaction of Human Corticotropin-Releasing Factor (CRF) Binding Protein and CRF Type-2 Receptor

PubMed Central

Slater, Paula G.; Gutierrez-Maldonado, Sebastian E.; Gysling, Katia; Lagos, Carlos F.

2018-01-01

The corticotropin-releasing factor (CRF) system is a key mediator of the stress response and addictive behavior. The CRF system includes four peptides: The CRF system includes four peptides: CRF, urocortins I–III, CRF binding protein (CRF-BP) that binds CRF with high affinity, and two class B G-protein coupled receptors CRF1R and CRF2R. CRF-BP is a secreted protein without significant sequence homology to CRF receptors or to any other known class of protein. Recently, it has been described a potentiation role of CRF-BP over CRF signaling through CRF2R in addictive-related neuronal plasticity and behavior. In addition, it has been described that CRF-BP is capable to physically interact specifically with the α isoform of CRF2R and acts like an escort protein increasing the amount of the receptor in the plasma membrane. At present, there are no available structures for CRF-BP or for full-length CRFR. Knowing and studying the structure of these proteins could be beneficial in order to characterize the CRF-BP/CRF2αR interaction. In this work, we report the modeling of CRF-BP and of full-length CRF2αR and CRF2βR based on the recently solved crystal structures of the transmembrane domains of the human glucagon receptor and human CRF1R, in addition with the resolved N-terminal extracellular domain of CRFRs. These models were further studied using molecular dynamics simulations and protein–protein docking. The results predicted a higher possibility of interaction of CRF-BP with CRF2αR than CRF2βR and yielded the possible residues conforming the interacting interface. Thus, the present study provides a framework for further investigation of the CRF-BP/CRF2αR interaction. PMID:29515519

Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivkees, S.A.; Carlson, L.L.; Reppert, S.M.

Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using {sup 125}I-labeled melatonin ({sup 125}I-Mel), a potent melatonin agonist. {sup 125}I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K{sub d} of 2.3 {plus minus} 1.0 {times} 10{sup {minus}11} M and 2.06 {plus minus} 0.43 {times} 10{sup {minus}10} M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)), significantly reduced the number of high-affinity receptors and increasedmore » the dissociation rate of {sup 125}I-Mel from its receptor. Furthermore, GTP({gamma}S) treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of {sup 125}I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M{sub r} > 400,000 and M{sub r} ca. 110,000. This elution profile was markedly altered by pretreatment with GTP({gamma}S) before solubilization; only the M{sub r} 110,000 peak was present in GTP({gamma}S)-pretreated membranes. The results strongly suggest that {sup 125}I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.« less

Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor.

PubMed

Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

2014-12-12

The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50° kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface.

Cholinergic Receptor Binding in Alzheimer Disease and Healthy Aging: Assessment In Vivo with Positron Emission Tomography Imaging.

PubMed

Sultzer, David L; Melrose, Rebecca J; Riskin-Jones, Hannah; Narvaez, Theresa A; Veliz, Joseph; Ando, Timothy K; Juarez, Kevin O; Harwood, Dylan G; Brody, Arthur L; Mandelkern, Mark A

2017-04-01

To compare regional nicotinic cholinergic receptor binding in older adults with Alzheimer disease (AD) and healthy older adults in vivo and to assess relationships between receptor binding and clinical symptoms. Using cross-sectional positron emission tomography (PET) neuroimaging and structured clinical assessment, outpatients with mild to moderate AD (N = 24) and healthy older adults without cognitive complaints (C group; N = 22) were studied. PET imaging of α4β2* nicotinic cholinergic receptor binding using 2-[ 18 F]fluoro-3-(2(S)azetidinylmethoxy)pyridine (2FA) and clinical measures of global cognition, attention/processing speed, verbal memory, visuospatial memory, and neuropsychiatric symptoms were used. 2FA binding was lower in the AD group compared with the C group in the medial thalamus, medial temporal cortex, anterior cingulate, insula/opercula, inferior caudate, and brainstem (p < 0.05, corrected cluster), but binding was not associated with cognition. The C group had significant inverse correlations between 2FA binding in the thalamus (left: r s  = -0.55, p = 0.008; right: r s  = -0.50, p = 0.02; N = 22) and hippocampus (left: r s  = -0.65, p = 0.001; right: r s  = -0.55, p = 0.009; N = 22) and the Trails A score. The AD group had inverse correlation between 2FA binding in anterior cingulate (left: r s  = -0.50, p = 0.01; right: r s  = -0.50, p = 0.01; N = 24) and Neurobehavioral Rating Scale agitation/disinhibition factor score. Cholinergic receptor binding is reduced in specific brain regions in mild to moderate AD and is related to neuropsychiatric symptoms. Among healthy older adults, lower receptor binding may be associated with slower processing speed. Cholinergic receptor binding in vivo may reveal links to other key brain changes associated with aging and AD and may provide a potential molecular treatment target. Published by Elsevier Inc.

Muscarinic Receptor Binding in Rat Bladder Urothelium and Detrusor Muscle by Intravesical Solifenacin.

PubMed

Ito, Yoshihiko; Kashiwabara, Michishi; Yoshida, Akira; Hikiyama, Eriko; Onoue, Satomi; Yamada, Shizuo

2016-01-01

Solifenacin is an antimuscarinic agent used to treat symptoms of overactive bladder. Pharmacologically significant amounts of solifenacin were excreted in the urine of humans taking a clinical dose of this drug. The aim of this study is to measure muscarinic receptor binding in the bladder urothelium and detrusor muscles of rats following the intravesical instillation of solifenacin. Muscarinic receptors were measured by radioreceptor assay using [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS), a selective radioligand of muscarinic receptors. Solifenacin showed concentration-dependent inhibition of specific [(3)H]NMS binding in the bladder urothelium and detrusor muscle of rats, with no significant difference in Ki values or Hill coefficients between these tissues. Following the intravesical instillation of solifenacin, there was significant muscarinic receptor binding (increase in Kd for specific [(3)H]NMS binding) in the bladder urothelium and detrusor muscle of rats. Similar bladder muscarinic receptor binding was observed by the intravesical instillation of oxybutynin, but not with trospium. In conclusion, the present study has demonstrated that solifenacin binds muscarinic receptors not only in the detrusor muscle but also in the bladder urothelium with high affinity. These bladder muscarinic receptors may be significantly affected by solifenacin excreted in the urine.

Structure of nerve growth factor complexed with the shared neurotrophin receptor p75.

PubMed

He, Xiao-Lin; Garcia, K Christopher

2004-05-07

Neurotrophins are secreted growth factors critical for the development and maintenance of the vertebrate nervous system. Neurotrophins activate two types of cell surface receptors, the Trk receptor tyrosine kinases and the shared p75 neurotrophin receptor. We have determined the 2.4 A crystal structure of the prototypic neurotrophin, nerve growth factor (NGF), complexed with the extracellular domain of p75. Surprisingly, the complex is composed of an NGF homodimer asymmetrically bound to a single p75. p75 binds along the homodimeric interface of NGF, which disables NGF's symmetry-related second p75 binding site through an allosteric conformational change. Thus, neurotrophin signaling through p75 may occur by disassembly of p75 dimers and assembly of asymmetric 2:1 neurotrophin/p75 complexes, which could potentially engage a Trk receptor to form a trimolecular signaling complex.

Differences in the binding mechanism of RU486 and progesterone to the progesterone receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Skafar, D.F.

1991-11-12

The binding mechanism of the antagonist RU486 to the progesterone receptor was compared with that of the agonists progesterone and R5020. Both progesterone and RU486 bound to the receptor with a Hill coefficient of 1.2, indicating the binding of each ligand is positive cooperative. However, when each ligand was used to compete with ({sup 3}H)progesterone for binding to the receptor at receptor concentrations near 8 nM, at which the receptor is likely a dimer, the competition curve for RU486 was significantly steeper than the curves for progesterone and R5020. This indicated that a difference in the binding mechanism of RU486more » and progesterone can be detected when both ligands are present. In contrast, at receptor concentrations near 1 nM, at which the receptor is likely a monomer, the competition curves for all three ligands were indistinguishable. These results indicate that RU486 and agonists have different binding mechanisms for the receptor and further suggest that this difference may be related to site-site interactions within the receptor.« less

Synthesis and receptor binding studies of (+/-)1-iodo-MK-801

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, D.J.; Ciliax, B.J.; Van Dort, M.E.

1989-06-01

The glutamate analogue N-methyl-D-aspartate (NMDA) binds to a subset of glutamate receptors that are coupled to a voltage-sensitive cation channel. This NMDA-linked channel is the likely binding locus of the potent anticonvulsant MK-801. To develop single-photon emission computed tomography (SPECT) probes of this brain channel, we synthesized (+/)1-iodo-MK-801 and (+/-)1-({sup 125}I)iodo-MK-801. The effect of (+/-)1-iodo-MK-801 on ligand binding to the NMDA-linked glutamate receptor site was assessed using a rat brain homogenate assay. (+/-)1-Iodo-MK-801 displaced the dissociative anesthetic ligand ({sup 3}H)N-(1-(2-thienyl)cyclohexyl)piperidine (({sup 3}H)TCP) binding with an IC50 of 1 microM, which is a 10-fold lower binding affinity than that of (+/-)MK-801.more » In in vivo autoradiographic studies, (+/-)MK-801 failed to block selective uptake of (+/-)1-iodo-MK-801 in rat brain. These results suggest that (+/-)1-iodo-MK-801 may not be a suitable ligand for mapping NMDA-linked glutamate receptor channels.« less

Specific receptors for epidermal growth factor in rat intestinal microvillus membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, J.F.

Epidermal growth factor (EGF) is present in high concentrations in milk, salivary, and pancreaticobiliary secretions. EGF, delivered to the intestinal lumen by these fluids, appears to influence intestinal proliferation. Because EGF exerts its mitogenic effect through binding to specific membrane-bound receptors, binding studies of {sup 125}I-labeled EGF to purified microvillus membrane (MVM) preparations fetal, newborn, and adult rat small intestine were performed. Using the membrane filter technique, binding of {sup 125}I-EGF to adult MVM was specific, saturable, and reversible. Adult and fetal MVM binding was rapid and reached a plateau after 30 min at both 20 and 37{degree}C. No bindingmore » was detected at 4{degree}C. Specific binding increased linearly from 0 to 75 {mu}g MVM protein. Scatchard analysis revealed a single class of receptors in fetal and adult MVM with an association constant of 1.0 {+-} 0.35 {times} 10{sup 9} and 2.3 {+-} 1.6 {times} 10{sup 9} M{sup {minus}1}, respectively. Binding capacity was 435.0 {+-} 89 and 97.7 {+-} 41.3 fmol {sup 125}I-EGF bound/mg MVM protein for fetal and adult MVM, respectively. Newborn MVM binding was negligible. After binding, cross-linking utilizing disuccinimidyl suberate, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography revealed a 170-kDa receptor. These data demonstrate specific receptors for EGF on MVM of rat small intestine and, thus, suggest a mechanism for the intraluminal regulation of enterocyte proliferation by EGF.« less

Bio-nanocapsule-based scaffold improves the sensitivity and ligand-binding capacity of mammalian receptors on the sensor chip.

PubMed

Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Maturana, Andrés D; Kuroda, Shun'ichi

2016-06-01

Mammalian receptors are recognized as target molecules for drug discovery, and chemical libraries have been screened for both potential antagonists and agonists mainly by ligand-binding assays using immobilized receptors. A bio-nanocapsule (BNC) of approximately 30 nm that displays a tandem form of the protein A-derived immunoglobulin G (IgG) Fc-binding Z domains (denoted as ZZ-BNC) has been developed for both clustering and oriented immobilization of IgGs on the solid phase of immunosensors. In this study, human IgG1 Fc-fused vascular endothelial growth factor (VEGF) receptor was immobilized through ZZ-BNC on the sensor chip of quartz crystal microbalance (ZZ-BNC-coating). When compared with direct adsorption and protein A-coating, the sensor chip showed higher sensitivity (∽46- and ∽165-fold, respectively) and larger ligand-binding capacity (∽4- and ∽18-fold, respectively). Furthermore, the number of VEGF molecules bound to its receptor increased from 0.20 (direct adsorption) to 2.06 by ZZ-BNC-coating, strongly suggesting that ZZ-BNC reduced the steric hindrance near ligand recognition sites through oriented immobilization. Similarly, the sensitivity and ligand-binding capacity of leptin and prolactin receptors were both enhanced at a level comparable to that observed for the VEGF receptor. Thus, the combination of ZZ-BNC and Fc-fused receptors could significantly improve the function of ligand-binding assays. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transforming Growth Factor β Suppresses Peroxisome Proliferator–Activated Receptor γ Expression via Both SMAD Binding and Novel TGF-β Inhibitory Elements

PubMed Central

Lakshmi, Sowmya P.; Reddy, Aravind T.; Reddy, Raju C.

2017-01-01

Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other’s expression, and such PPARγ downregulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here we show that TGF-β induces association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive corepressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. PMID:28100650

Binding modes of dihydroquinoxalinones in a homology model of bradykinin receptor 1.

PubMed

Ha, Sookhee N; Hey, Pat J; Ransom, Rick W; Harrell, C Meacham; Murphy, Kathryn L; Chang, Ray; Chen, Tsing-Bau; Su, Dai-Shi; Markowitz, M Kristine; Bock, Mark G; Freidinger, Roger M; Hess, Fred J

2005-05-27

We report the first homology model of human bradykinin receptor B1 generated from the crystal structure of bovine rhodopsin as a template. Using an automated docking procedure, two B1 receptor antagonists of the dihydroquinoxalinone structural class were docked into the receptor model. Site-directed mutagenesis data of the amino acid residues in TM1, TM3, TM6, and TM7 were incorporated to place the compounds in the binding site of the homology model of the human B1 bradykinin receptor. The best pose in agreement with the mutation data was selected for detailed study of the receptor-antagonist interaction. To test the model, the calculated antagonist-receptor binding energy was correlated with the experimentally measured binding affinity (K(i)) for nine dihydroquinoxalinone analogs. The model was used to gain insight into the molecular mechanism for receptor function and to optimize the dihydroquinoxalinone analogs.

Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suino-Powell, Kelly; Xu, Yong; Zhang, Chenghai

A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GRmore » LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 {angstrom}{sup 3}, effectively doubling the size of the GR dexamethasone-binding pocket of 540 {angstrom}{sup 3} and yet leaving the structure of the coactivator binding site intact. DAC occupies only {approx}50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.« less

Rationalizing 5000-Fold Differences in Receptor-Binding Rate Constants of Four Cytokines

PubMed Central

Pang, Xiaodong; Qin, Sanbo; Zhou, Huan-Xiang

2011-01-01

The four cytokines erythropoietin (EPO), interleukin-4 (IL4), human growth hormone (hGH), and prolactin (PRL) all form four-helix bundles and bind to type I cytokine receptors. However, their receptor-binding rate constants span a 5000-fold range. Here, we quantitatively rationalize these vast differences in rate constants by our transient-complex theory for protein-protein association. In the transient complex, the two proteins have near-native separation and relative orientation, but have yet to form the short-range specific interactions of the native complex. The theory predicts the association rate constant as ka=ka0exp(−ΔGel∗/kBT) where ka0 is the basal rate constant for reaching the transient complex by random diffusion, and the Boltzmann factor captures the rate enhancement due to electrostatic attraction. We found that the vast differences in receptor-binding rate constants of the four cytokines arise mostly from the differences in charge complementarity among the four cytokine-receptor complexes. The basal rate constants (ka0) of EPO, IL4, hGH, and PRL were similar (5.2 × 105 M−1s−1, 2.4 × 105 M−1s−1, 1.7 × 105 M−1s−1, and 1.7 × 105 M−1s−1, respectively). However, the average electrostatic free energies (ΔGe1∗) were very different (−4.2 kcal/mol, −2.4 kcal/mol, −0.1 kcal/mol, and −0.5 kcal/mol, respectively, at ionic strength = 160 mM). The receptor-binding rate constants predicted without adjusting any parameters, 6.2 × 108 M−1s−1, 1.3 × 107 M−1s−1, 2.0 × 105 M−1s−1, and 7.6 × 104 M−1s−1, respectively, for EPO, IL4, hGH, and PRL agree well with experimental results. We uncover that these diverse rate constants are anticorrelated with the circulation concentrations of the cytokines, with the resulting cytokine-receptor binding rates very close to the limits set by the half-lives of the receptors, suggesting that these binding rates are functionally relevant and perhaps evolutionarily

Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

PubMed

Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M

1996-11-01

It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

Understanding Cytokine and Growth Factor Receptor Activation Mechanisms

PubMed Central

Atanasova, Mariya; Whitty, Adrian

2012-01-01

Our understanding of the detailed mechanism of action of cytokine and growth factor receptors – and particularly our quantitative understanding of the link between structure, mechanism and function – lags significantly behind our knowledge of comparable functional protein classes such as enzymes, G protein-coupled receptors, and ion channels. In particular, it remains controversial whether such receptors are activated by a mechanism of ligand-induced oligomerization, versus a mechanism in which the ligand binds to a pre-associated receptor dimer or oligomer that becomes activated through subsequent conformational rearrangement. A major limitation to progress has been the relative paucity of methods for performing quantitative mechanistic experiments on unmodified receptors expressed at endogenous levels on live cells. In this article we review the current state of knowledge on the activation mechanisms of cytokine and growth factor receptors, critically evaluate the evidence for and against the different proposed mechanisms, and highlight other key questions that remain unanswered. New approaches and techniques have led to rapid recent progress in this area, and the field is poised for major advances in the coming years, which promises to revolutionize our understanding of this large and biologically and medically important class of receptors. PMID:23046381

Nicotinic Cholinergic Receptor Binding Sites in the Brain: Regulation in vivo

NASA Astrophysics Data System (ADS)

Schwartz, Rochelle D.; Kellar, Kenneth J.

1983-04-01

Tritiated acetylcholine was used to measure binding sites with characteristics of nicotinic cholinergic receptors in rat brain. Regulation of the binding sites in vivo was examined by administering two drugs that stimulate nicotinic receptors directly or indirectly. After 10 days of exposure to the cholinesterase inhibitor diisopropyl fluorophosphate, binding of tritiated acetylcholine in the cerebral cortex was decreased. However, after repeated administration of nicotine for 10 days, binding of tritiated acetylcholine in the cortex was increased. Saturation analysis of tritiated acetylcholine binding in the cortices of rats treated with diisopropyl fluorophosphate or nicotine indicated that the number of binding sites decreased and increased, respectively, while the affinity of the sites was unaltered.

A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling through CREB-Binding Protein

PubMed Central

Mahajan, Muktar A.; Samuels, Herbert H.

2000-01-01

We describe the cloning and characterization of a new family of nuclear receptor coregulators (NRCs) which modulate the function of nuclear hormone receptors in a ligand-dependent manner. NRCs are expressed as alternatively spliced isoforms which may exhibit different intrinsic activities and receptor specificities. The NRCs are organized into several modular structures and contain a single functional LXXLL motif which associates with members of the steroid hormone and thyroid hormone/retinoid receptor subfamilies with high affinity. Human NRC (hNRC) harbors a potent N-terminal activation domain (AD1), which is as active as the herpesvirus VP16 activation domain, and a second activation domain (AD2) which overlaps with the receptor-interacting LXXLL region. The C-terminal region of hNRC appears to function as an inhibitory domain which influences the overall transcriptional activity of the protein. Our results suggest that NRC binds to liganded receptors as a dimer and this association leads to a structural change in NRC resulting in activation. hNRC binds CREB-binding protein (CBP) with high affinity in vivo, suggesting that hNRC may be an important functional component of a CBP complex involved in mediating the transcriptional effects of nuclear hormone receptors. PMID:10866662

Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity.

PubMed

Adu-Gyamfi, Emmanuel; Kim, Lori S; Jardetzky, Theodore S; Lamb, Robert A

2016-09-01

Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the

Phosphorylation of a conserved serine in the deoxyribonucleic acid binding domain of nuclear receptors alters intracellular localization.

PubMed

Sun, Kai; Montana, Vedrana; Chellappa, Karthikeyani; Brelivet, Yann; Moras, Dino; Maeda, Yutaka; Parpura, Vladimir; Paschal, Bryce M; Sladek, Frances M

2007-06-01

Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4alpha (HNF4alpha). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4alpha and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4alpha (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4alpha as well as decreased endogenous HNF4alpha protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor alpha, retinoid X receptor alpha, and thyroid hormone receptor beta) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor alpha and peroxisome proliferator-activated receptor alpha. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.

Viral receptor-binding site antibodies with diverse germline origins.

PubMed

Schmidt, Aaron G; Therkelsen, Matthew D; Stewart, Shaun; Kepler, Thomas B; Liao, Hua-Xin; Moody, M Anthony; Haynes, Barton F; Harrison, Stephen C

2015-05-21

Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by 11 different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B cell targets. Copyright © 2015 Elsevier Inc. All rights reserved.

A Pro-Nerve Growth Factor (proNGF) and NGF Binding Protein, α2-Macroglobulin, Differentially Regulates p75 and TrkA Receptors and Is Relevant to Neurodegeneration Ex Vivo and In Vivo

PubMed Central

Barcelona, Pablo F.

2015-01-01

Nerve growth factor (NGF) is generated from a precursor, proNGF, that is proteolytically processed. NGF preferentially binds a trophic tyrosine kinase receptor, TrkA, while proNGF binds a neurotrophin receptor (NTR), p75NTR, that can have neurotoxic activity. Previously, we along with others showed that the soluble protein α2-macroglobulin (α2M) is neurotoxic. Toxicity is due in part to α2M binding to NGF and inhibiting trophic activity, presumably by preventing NGF binding to TrkA. However, the mechanisms remained unclear. Here, we show ex vivo and in vivo three mechanisms for α2M neurotoxicity. First, unexpectedly the α2M-NGF complexes do bind TrkA receptors but do not induce TrkA dimerization or activation, resulting in deficient trophic support. Second, α2M makes stable complexes with proNGF, conveying resistance to proteolysis that results in more proNGF and less NGF. Third, α2M-proNGF complexes bind p75NTR and are more potent agonists than free proNGF, inducing tumor necrosis factor alpha (TNF-α) production. Hence, α2M regulates proNGF/p75NTR positively and mature NGF/TrkA negatively, causing neuronal death ex vivo. These three mechanisms are operative in vivo, and α2M causes neurodegeneration in a p75NTR- and proNGF-dependent manner. α2M could be exploited as a therapeutic target, or as a modifier of neurotrophin signals. PMID:26217017

The alpha-fetoprotein third domain receptor binding fragment: in search of scavenger and associated receptor targets.

PubMed

Mizejewski, G J

2015-01-01

Recent studies have demonstrated that the carboxyterminal third domain of alpha-fetoprotein (AFP-CD) binds with various ligands and receptors. Reports within the last decade have established that AFP-CD contains a large fragment of amino acids that interact with several different receptor types. Using computer software specifically designed to identify protein-to-protein interaction at amino acid sequence docking sites, the computer searches identified several types of scavenger-associated receptors and their amino acid sequence locations on the AFP-CD polypeptide chain. The scavenger receptors (SRs) identified were CD36, CD163, Stabilin, SSC5D, SRB1 and SREC; the SR-associated receptors included the mannose, low-density lipoprotein receptors, the asialoglycoprotein receptor, and the receptor for advanced glycation endproducts (RAGE). Interestingly, some SR interaction sites were localized on the AFP-derived Growth Inhibitory Peptide (GIP) segment at amino acids #480-500. Following the detection studies, a structural subdomain analysis of both the receptor and the AFP-CD revealed the presence of epidermal growth factor (EGF) repeats, extracellular matrix-like protein regions, amino acid-rich motifs and dimerization subdomains. For the first time, it was reported that EGF-like sequence repeats were identified on each of the three domains of AFP. Thereafter, the localization of receptors on specific cell types were reviewed and their functions were discussed.

A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

PubMed

Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

2015-05-21

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. Copyright © 2015 Elsevier Inc. All rights reserved.

Proposed Mode of Binding and Action of Positive Allosteric Modulators at Opioid Receptors

PubMed Central

2016-01-01

Available crystal structures of opioid receptors provide a high-resolution picture of ligand binding at the primary (“orthosteric”) site, that is, the site targeted by endogenous ligands. Recently, positive allosteric modulators of opioid receptors have also been discovered, but their modes of binding and action remain unknown. Here, we use a metadynamics-based strategy to efficiently sample the binding process of a recently discovered positive allosteric modulator of the δ-opioid receptor, BMS-986187, in the presence of the orthosteric agonist SNC-80, and with the receptor embedded in an explicit lipid–water environment. The dynamics of BMS-986187 were enhanced by biasing the potential acting on the ligand–receptor distance and ligand–receptor interaction contacts. Representative lowest-energy structures from the reconstructed free-energy landscape revealed two alternative ligand binding poses at an allosteric site delineated by transmembrane (TM) helices TM1, TM2, and TM7, with some participation of TM6. Mutations of amino acid residues at these proposed allosteric sites were found to either affect the binding of BMS-986187 or its ability to modulate the affinity and/or efficacy of SNC-80. Taken together, these combined experimental and computational studies provide the first atomic-level insight into the modulation of opioid receptor binding and signaling by allosteric modulators. PMID:26841170

Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system.

PubMed

Rovira, X; Vivó, M; Serra, J; Roche, D; Strange, P G; Giraldo, J

2009-01-01

Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.

Binding Mode and Structure-Activity Relationships of ITE as an Aryl Hydrocarbon Receptor (AhR) Agonist.

PubMed

Dolciami, Daniela; Gargaro, Marco; Cerra, Bruno; Scalisi, Giulia; Bagnoli, Luana; Servillo, Giuseppe; Fazia, Maria Agnese Della; Puccetti, Paolo; Quintana, Francisco J; Fallarino, Francesca; Macchiarulo, Antonio

2018-02-06

Discovered as a modulator of the toxic response to environmental pollutants, aryl hydrocarbon receptor (AhR) has recently gained attention for its involvement in various physiological and pathological pathways. AhR is a ligand-dependent transcription factor activated by a large array of chemical compounds, which include metabolites of l-tryptophan (l-Trp) catabolism as endogenous ligands of the receptor. Among these, 2-(1'H-indole-3'-carbonyl)thiazole-4-carboxylic acid methyl ester (ITE) has attracted interest in the scientific community, being endowed with nontoxic, immunomodulatory, and anticancer AhR-mediated functions. So far, no information about the binding mode and interactions of ITE with AhR is available. In this study, we used docking and molecular dynamics to propose a putative binding mode of ITE into the ligand binding pocket of AhR. Mutagenesis studies were then instrumental in validating the proposed binding mode, identifying His 285 and Tyr 316 as important key residues for ligand-dependent receptor activation. Finally, a set of ITE analogues was synthesized and tested to further probe molecular interactions of ITE to AhR and characterize the relevance of specific functional groups in the chemical structure for receptor activity. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of a novel virtual screening strategy using receptor decoy binding sites.

PubMed

Patel, Hershna; Kukol, Andreas

2016-08-23

Virtual screening is used in biomedical research to predict the binding affinity of a large set of small organic molecules to protein receptor targets. This report shows the development and evaluation of a novel yet straightforward attempt to improve this ranking in receptor-based molecular docking using a receptor-decoy strategy. This strategy includes defining a decoy binding site on the receptor and adjusting the ranking of the true binding-site virtual screen based on the decoy-site screen. The results show that by docking against a receptor-decoy site with Autodock Vina, improved Receiver Operator Characteristic Enrichment (ROCE) was achieved for 5 out of fifteen receptor targets investigated, when up to 15 % of a decoy site rank list was considered. No improved enrichment was seen for 7 targets, while for 3 targets the ROCE was reduced. The extent to which this strategy can effectively improve ligand prediction is dependent on the target receptor investigated.

Identification of key residues involved in adrenomedullin binding to the AM1 receptor

PubMed Central

Watkins, HA; Au, M; Bobby, R; Archbold, JK; Abdul-Manan, N; Moore, JM; Middleditch, MJ; Williams, GM; Brimble, MA; Dingley, AJ; Hay, DL

2013-01-01

Background and Purpose Adrenomedullin (AM) is a peptide hormone whose receptors are members of the class B GPCR family. They comprise a heteromer between the GPCR, the calcitonin receptor-like receptor and one of the receptor activity-modifying proteins 1–3. AM plays a significant role in angiogenesis and its antagonist fragment AM22–52 can inhibit blood vessel and tumour growth. The mechanism by which AM interacts with its receptors is unknown. Experimental Approach We determined the AM22–52 binding epitope for the AM1 receptor extracellular domain using biophysical techniques, heteronuclear magnetic resonance spectroscopy and alanine scanning. Key Results Chemical shift perturbation experiments located the main binding epitope for AM22–52 at the AM1 receptor to the C-terminal 8 amino acids. Isothermal titration calorimetry of AM22–52 alanine-substituted peptides indicated that Y52, G51 and I47 are essential for AM1 receptor binding and that K46 and P49 and R44 have a smaller role to play. Characterization of these peptides at the full-length AM receptors was assessed in Cos7 cells by cAMP assay. This confirmed the essential role of Y52, G51 and I47 in binding to the AM1 receptor, with their substitution resulting in ≥100-fold reduction in antagonist potency compared with AM22–52. R44A, K46A, S48A and P49A AM22–52 decreased antagonist potency by approximately 10-fold. Conclusions and Implications This study localizes the main binding epitope of AM22–52 to its C-terminal amino acids and distinguishes essential residues involved in this binding. This will inform the development of improved AM receptor antagonists. PMID:23351143

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators

PubMed Central

Redfern, Andrew D.; Colley, Shane M.; Beveridge, Dianne J.; Ikeda, Naoya; Epis, Michael R.; Li, Xia; Foulds, Charles E.; Stuart, Lisa M.; Barker, Andrew; Russell, Victoria J.; Ramsay, Kerry; Kobelke, Simon J.; Li, Xiaotao; Hatchell, Esme C.; Payne, Christine; Giles, Keith M.; Messineo, Adriana; Gatignol, Anne; Lanz, Rainer B.; O’Malley, Bert W.; Leedman, Peter J.

2013-01-01

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing. PMID:23550157

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.

PubMed

Redfern, Andrew D; Colley, Shane M; Beveridge, Dianne J; Ikeda, Naoya; Epis, Michael R; Li, Xia; Foulds, Charles E; Stuart, Lisa M; Barker, Andrew; Russell, Victoria J; Ramsay, Kerry; Kobelke, Simon J; Li, Xiaotao; Hatchell, Esme C; Payne, Christine; Giles, Keith M; Messineo, Adriana; Gatignol, Anne; Lanz, Rainer B; O'Malley, Bert W; Leedman, Peter J

2013-04-16

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

Amino acid substitutions in the hormone-binding domain of the human androgen receptor alter the stability of the hormone receptor complex.

PubMed Central

Marcelli, M; Zoppi, S; Wilson, C M; Griffin, J E; McPhaul, M J

1994-01-01

We have investigated the basis of androgen resistance in seven unrelated individuals with complete testicular feminization or Reifenstein syndrome caused by single amino acid substitutions in the hormone-binding domain of the androgen receptor. Monolayer-binding assays of cultured genital skin fibroblasts demonstrated absent ligand binding, qualitative abnormalities of androgen binding, or a decreased amount of qualitatively normal receptor. The consequences of these mutations were examined by introducing the mutations by site-directed mutagenesis into the androgen receptor cDNA sequence and expressing the mutant cDNAs in mammalian cells. The effects of the amino acid substitutions on the binding of different androgens and on the capacity of the ligand-bound receptors to activate a reporter gene were investigated. Substantial differences were found in the responses of the mutant androgen receptors to incubation with testosterone, 5 alpha-dihydrotestosterone, and mibolerone. In several instances, increased doses of hormone or increased frequency of hormone addition to the incubation medium resulted in normal or near normal activation of a reporter gene by cells expressing the mutant androgen receptors. These studies suggest that the stability of the hormone receptor complex is a major determinant of receptor function in vivo. Images PMID:7929841

Receptor-ligand binding sites and virtual screening.

PubMed

Hattotuwagama, Channa K; Davies, Matthew N; Flower, Darren R

2006-01-01

Within the pharmaceutical industry, the ultimate source of continuing profitability is the unremitting process of drug discovery. To be profitable, drugs must be marketable: legally novel, safe and relatively free of side effects, efficacious, and ideally inexpensive to produce. While drug discovery was once typified by a haphazard and empirical process, it is now increasingly driven by both knowledge of the receptor-mediated basis of disease and how drug molecules interact with receptors and the wider physiome. Medicinal chemistry postulates that to understand a congeneric ligand series, or set thereof, is to understand the nature and requirements of a ligand binding site. Likewise, structural molecular biology posits that to understand a binding site is to understand the nature of ligands bound therein. Reality sits somewhere between these extremes, yet subsumes them both. Complementary to rules of ligand design, arising through decades of medicinal chemistry, structural biology and computational chemistry are able to elucidate the nature of binding site-ligand interactions, facilitating, at both pragmatic and conceptual levels, the drug discovery process.

The role of receptor binding specificity in interspecies transmission of influenza viruses

PubMed Central

Imai, Masaki; Kawaoka, Yoshihiro

2017-01-01

Influenza A virus infection begins with the binding of the hemagglutinin (HA) glycoprotein to sialic acid-containing receptors on the surface of the target cell. Avian influenza viruses, including avian H5N1, H7, and H9N2 viruses, can occasionally cross the species barrier and infect humans; however, these viruses do not spread efficiently from person to person, perhaps, in part, due to differences in the receptor-binding specificities of human and avian influenza viruses. The HAs of avian influenza viruses must adapt to receptors in humans to acquire efficient human-to-human transmissibility. In this review, we discuss the receptor binding specificity of influenza A viruses and its role in interspecies transmission. PMID:22445963

Substance P receptor binding sites are expressed by glia in vivo after neuronal injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mantyh, P.W.; Johnson, D.J.; Boehmer, C.G.

1989-07-01

In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, the authors examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve andmore » tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.« less

Receptor-Binding Profiles of H7 Subtype Influenza Viruses in Different Host Species

PubMed Central

Gambaryan, Alexandra S.; Matrosovich, Tatyana Y.; Philipp, Jennifer; Munster, Vincent J.; Fouchier, Ron A. M.; Cattoli, Giovanni; Capua, Ilaria; Krauss, Scott L.; Webster, Robert G.; Banks, Jill; Bovin, Nicolai V.; Klenk, Hans-Dieter

2012-01-01

Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.e., preferential binding to sulfated oligosaccharides Neu5Acα2-3Galβ1-4(6-O-HSO3)GlcNAc and Neu5Acα2-3Galβ1-4(Fucα1-3)(6-O-HSO3)GlcNAc. This phenotype correlated with the unique amino acid sequence of the amino acid 185 to 189 loop of H7 HA and seemed to be dependent on ionic interactions between the sulfate group of the receptor and Lys193 and on the lack of sterical clashes between the fucose residue and Gln222. Many North American and Eurasian H7 influenza viruses displayed weak but detectable binding to the human-type receptor moiety Neu5Acα2-6Galβ1-4GlcNAc, highlighting the potential of H7 influenza viruses for avian-to-human transmission. Equine H7 influenza viruses differed from other viruses by preferential binding to the N-glycolyl form of sialic acid. Our data suggest that the receptor-binding site of contemporary H7 influenza viruses in aquatic and terrestrial birds was formed after the introduction of their common precursor from ducks to a new host, presumably, gallinaceous poultry. The uniformity of the receptor-binding profile of H7 influenza viruses in various wild and domestic birds indicates that there is no strong receptor-mediated host range restriction in birds on viruses with this HA subtype. This notion agrees with repeated interspecies transmission of H7 influenza viruses from aquatic birds to poultry. PMID:22345462

Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors.

PubMed

Hedlund, P B; Carson, M J; Sutcliffe, J G; Thomas, E A

1999-12-01

Oleamide belongs to a family of amidated lipids with diverse biological activities, including sleep induction and signaling modulation of several 5-hydroxytryptamine (5-HT) receptor subtypes, including 5-HT1A, 5-HT2A/2C, and 5-HT7. The 5-HT7 receptor, predominantly localized in the hypothalamus, hippocampus, and frontal cortex, stimulates cyclic AMP formation and is thought to be involved in the regulation of sleep-wake cycles. Recently, it was proposed that oleamide acts at an allosteric site on the 5-HT7 receptor to regulate cyclic AMP formation. We have further investigated the interaction between oleamide and 5-HT7 receptors by performing radioligand binding assays with HeLa cells transfected with the 5-HT7 receptor. Methiothepin, clozapine, and 5-HT all displaced specific [3H]5-HT (100 nM) binding, with pK(D) values of 7.55, 7.85, and 8.39, respectively. Oleamide also displaced [3H]5-HT binding, but the maximum inhibition was only 40% of the binding. Taking allosteric (see below) cooperativity into account, a K(D) of 2.69 nM was calculated for oleamide. In saturation binding experiments, oleamide caused a 3-fold decrease in the affinity of [3H]5-HT for the 5-HT7 receptor, without affecting the number of binding sites. A Schild analysis showed that the induced shift in affinity of [3H]5-HT reached a plateau, unlike that of a competitive inhibitor, illustrating the allosteric nature of the interaction between oleamide and the 5-HT7 receptor. Oleic acid, the product of oleamide hydrolysis, had a similar effect on [3H]5-HT binding, whereas structural analogs of oleamide, trans-9,10-octadecenamide, cis-8,9-octadecenamide, and erucamide, did not alter [3H]5-HT binding significantly. The findings support the hypothesis that oleamide acts via an allosteric site on the 5-HT7 receptor regulating receptor affinity.

Delineation of the peptide binding site of the human galanin receptor.

PubMed Central

Kask, K; Berthold, M; Kahl, U; Nordvall, G; Bartfai, T

1996-01-01

Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th Images PMID:8617199

Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bray, J.J.; Drachman, D.B.

1982-01-01

Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the rangemore » of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.« less

Neurotensin receptor binding levels in basal ganglia are not altered in Huntington's chorea or schizophrenia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palacios, J.M.; Chinaglia, G.; Rigo, M.

1991-02-01

Autoradiographic techniques were used to examine the distribution and levels of neurotensin receptor binding sites in the basal ganglia and related regions of the human brain. Monoiodo ({sup 125}I-Tyr3)neurotensin was used as a ligand. High amounts of neurotensin receptor binding sites were found in the substantia nigra pars compacta. Lower but significant quantities of neurotensin receptor binding sites characterized the caudate, putamen, and nucleus accumbens, while very low quantities were seen in both medial and lateral segments of the globus pallidus. In Huntington's chorea, the levels of neurotensin receptor binding sites were found to be comparable to those of controlmore » cases. Only slight but not statistically significant decreases in amounts of receptor binding sites were detected in the dorsal part of the head and in the body of caudate nucleus. No alterations in the levels of neurotensin receptor binding sites were observed in the substantia nigra pars compacta and reticulata. These results suggest that a large proportion of neurotensin receptor binding sites in the basal ganglia are located on intrinsic neurons and on extrinsic afferent fibers that do not degenerate in Huntington's disease.« less

(/sup 3/H)Ethylketocyclazocine binding to mouse brain membranes: evidence for a kappa opioid receptor type

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 portionmore » 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.« less

Structural Analysis on the Pathologic Mutant Glucocorticoid Receptor Ligand-Binding Domains.

PubMed

Hurt, Darrell E; Suzuki, Shigeru; Mayama, Takafumi; Charmandari, Evangelia; Kino, Tomoshige

2016-02-01

Glucocorticoid receptor (GR) gene mutations may cause familial or sporadic generalized glucocorticoid resistance syndrome. Most of the missense forms distribute in the ligand-binding domain and impair its ligand-binding activity and formation of the activation function (AF)-2 that binds LXXLL motif-containing coactivators. We performed molecular dynamics simulations to ligand-binding domain of pathologic GR mutants to reveal their structural defects. Several calculated parameters including interaction energy for dexamethasone or the LXXLL peptide indicate that destruction of ligand-binding pocket (LBP) is a primary character. Their LBP defects are driven primarily by loss/reduction of the electrostatic interaction formed by R611 and T739 of the receptor to dexamethasone and a subsequent conformational mismatch, which deacylcortivazol resolves with its large phenylpyrazole moiety and efficiently stimulates transcriptional activity of the mutant receptors with LBP defect. Reduced affinity of the LXXLL peptide to AF-2 is caused mainly by disruption of the electrostatic bonds to the noncore leucine residues of this peptide that determine the peptide's specificity to GR, as well as by reduced noncovalent interaction against core leucines and subsequent exposure of the AF-2 surface to solvent. The results reveal molecular defects of pathologic mutant receptors and provide important insights to the actions of wild-type GR.

Dimensionality of Motion and Binding Valency Govern Receptor-Ligand Kinetics As Revealed by Agent-Based Modeling.

PubMed

Lehnert, Teresa; Figge, Marc Thilo

2017-01-01

Mathematical modeling and computer simulations have become an integral part of modern biological research. The strength of theoretical approaches is in the simplification of complex biological systems. We here consider the general problem of receptor-ligand binding in the context of antibody-antigen binding. On the one hand, we establish a quantitative mapping between macroscopic binding rates of a deterministic differential equation model and their microscopic equivalents as obtained from simulating the spatiotemporal binding kinetics by stochastic agent-based models. On the other hand, we investigate the impact of various properties of B cell-derived receptors-such as their dimensionality of motion, morphology, and binding valency-on the receptor-ligand binding kinetics. To this end, we implemented an algorithm that simulates antigen binding by B cell-derived receptors with a Y-shaped morphology that can move in different dimensionalities, i.e., either as membrane-anchored receptors or as soluble receptors. The mapping of the macroscopic and microscopic binding rates allowed us to quantitatively compare different agent-based model variants for the different types of B cell-derived receptors. Our results indicate that the dimensionality of motion governs the binding kinetics and that this predominant impact is quantitatively compensated by the bivalency of these receptors.

The Roles of Hemagglutinin Phe-95 in Receptor Binding and Pathogenicity of Influenza B Virus

PubMed Central

Ni, Fengyun; Mbawuike, Innocent Nnadi; Kondrashkina, Elena; Wang, Qinghua

2014-01-01

Diverged ~4,000 years ago, influenza B virus has several important differences from influenza A virus, including lower receptor-binding affinity and highly restricted host range. Based on our prior structural studies, we hypothesized that a single-residue difference in the receptor-binding site of hemagglutinin (HA), Phe-95 in influenza B virus versus Tyr-98 in influenza A/H1~H15, is possibly a key determinant for the low receptor-binding affinity. Here we demonstrate that the mutation Phe95→Tyr in influenza B virus HA restores all three hydrogen bonds made by Tyr-98 in influenza A/H3 HA and has the potential to enhance receptor binding. However, the full realization of this potential is influenced by the local environment into which the mutation is introduced. The binding and replication of the recombinant viruses correlate well with the receptor-binding capabilities of HA. These results are discussed in relation to the roles of Phe-95 in receptor binding and pathogenicity of influenza B virus. PMID:24503069

FOLLITROPIN RECEPTORS CONTAIN CRYPTIC LIGAND BINDING SITES1

PubMed Central

Lin, Win; Bernard, Michael P.; Cao, Donghui; Myers, Rebecca V.; Kerrigan, John E.; Moyle, William R.

2007-01-01

Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with an FSHR/LHR chimera having only two unique LHR residues similar to the manners in which they dock with LHR and FSHR, respectively. This shows that although the FSHR does not normally bind hCG, it contains a cryptic lutropin binding site that has the potential to recognize hCG in a manner similar to the LHR. The presence of this cryptic site may explain why equine lutropins bind many mammalian FSHR and why mutations in the transmembrane domain distant from the extracellular domain enable the FSHR to bind hCG. The leucine-rich repeat domain (LRD) of the FSHR also appears to contain a cryptic FSH binding site that is obscured by other parts of the extracellular domain. This will explain why contacts seen in crystals of hFSH complexed with an LRD fragment of the human FSHR are hard to reconcile with the abilities of FSH analogs to interact with membrane G-protein coupled FSHR. We speculate that cryptic lutropin binding sites in the FSHR, which are also likely to be present in thyrotropin receptors (TSHR), permit the physiological regulation of ligand binding specificity. Cryptic FSH binding sites in the LRD may enable alternate spliced forms of the FSHR to interact with FSH. PMID:17059863

Transforming growth factor β suppresses peroxisome proliferator-activated receptor γ expression via both SMAD binding and novel TGF-β inhibitory elements.

PubMed

Lakshmi, Sowmya P; Reddy, Aravind T; Reddy, Raju C

2017-04-24

Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other's expression, and such PPARγ down-regulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here, we show that TGF-β induces the association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive co-repressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated the partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Demonstration of muscarinic and nicotinic receptor binding activities of distigmine to treat detrusor underactivity.

PubMed

Harada, Taketsugu; Fushimi, Kazumi; Kato, Aya; Ito, Yoshihiko; Nishijima, Saori; Sugaya, Kimio; Yamada, Shizuo

2010-01-01

The present study was undertaken to examine whether distigmine, a therapeutic agent used to treat detrusor underactivity, binds directly to muscarinic and nicotinic receptors. We used radioreceptor binding assays and compared the effects of distigmine with those of neostigmine and donepedil. The inhibitory effect of distigmine on the blood acetylcholinesterase (AChE) activity was significantly weaker than that of neostigmine. Distigmine, neostigmine, and donepezil competed for specific binding sites of [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS ) and [(3)H]oxotremorine-M in the bladder, submaxillary gland and cerebral cortex of rats in a concentration-dependent manner, indicating significant binding activity of muscarinic receptors. Distigmine displayed significantly higher affinity for binding sites of [(3)H]oxotremorine-M compared with those of [(3)H]NMS as revealed by large ratios of its K(i) value for [(3)H]NMS to that for [(3)H]oxotremorine-M, suggesting that it has preferential affinity for agonist sites of muscarinic receptors. Distigmine seemed to bind to the agonist sites of muscarinic receptors in a competitive manner. Repeated oral administration of distigmine caused a significant decrease in the maximal number of binding sites (B(max)) for [(3)H]NMS in the bladder and submaxillary gland but not cerebral cortex. Distigmine also bound to nicotinic receptors in the rat cerebral cortex. In conclusion, distigmine shows direct binding to muscarinic receptors in the rat bladder, and repeated oral administration of distigmine causes downregulation of muscarinic receptors in the rat bladder. The observed direct interaction of distigmine with the bladder muscarinic receptors may partly contribute to the therapeutic and/or side effects seen in the treatment of detrusor underactivity.

Hormone Binding to Recombinant Estrogen Receptors from Human, Alligator, Quail, Salamander, and Fathead Minnow

EPA Science Inventory

In this work, a 96-well plate estrogen receptor binding assay was developed to facilitate the direct comparison of chemical binding to full-length recombinant estrogen receptors across vertebrate classes. Receptors were generated in a baculovirus expression system. This approach ...

Sequences Flanking the Gephyrin-Binding Site of GlyRβ Tune Receptor Stabilization at Synapses

PubMed Central

Grünewald, Nora; Salvatico, Charlotte; Kress, Vanessa

2018-01-01

Abstract The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the β-subunit (β-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 310-helix C-terminal to the β-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR β-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells. PMID:29464196

Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors.

PubMed

Prokop, Susanne; Perry, Nicole A; Vishnivetskiy, Sergey A; Toth, Andras D; Inoue, Asuka; Milligan, Graeme; Iverson, Tina M; Hunyady, Laszlo; Gurevich, Vsevolod V

2017-08-01

Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M 2 muscarinic receptor, so that agonist activation of the M 2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M 2 , whereas its interactions with other receptors, including the β 2 -adrenergic receptor and the D 1 and D 2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the β 2 -adrenergic and D 2 dopamine receptors, while reducing its interaction with the D 1 dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes. Copyright © 2017. Published by Elsevier Inc.

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

McVittie, L.D.; Sibley, D.R.

1989-01-01

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either (/sup 3/H)-N-(1-(2-thienyl)cyclohexyl)piperidine ((/sup 3/H)TCP) or (/sup 3/H)MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4/degrees/C. In the presence of detergent, (/sup 3/H)TCP binding exhibitsmore » a K/sub d/ of 250 nM, a B/sub max/ of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor (/sup 3/H)TCP binding: K/sub d/ = 48 nM, M/sub max/ = 1.13 pmol/mg protein.« less

Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez, Elisabeth D.; Pattabiraman, Nagarajan; Department of Oncology, Georgetown University School of Medicine, Washington, DC 20057

2005-08-15

The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increasedmore » ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids.« less

Reassessment of the Unique Mode of Binding between Angiotensin II Type 1 Receptor and Their Blockers

PubMed Central

Matsuo, Yoshino; Saku, Keijiro; Karnik, Sadashiva S.

2013-01-01

While the molecular structures of angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are very similar, they are also slightly different. Although each ARB has been shown to exhibit a unique mode of binding to AT1 receptor, different positions of the AT1 receptor have been analyzed and computational modeling has been performed using different crystal structures for the receptor as a template and different kinds of software. Therefore, we systematically analyzed the critical positions of the AT1 receptor, Tyr113, Tyr184, Lys199, His256 and Gln257 using a mutagenesis study, and subsequently performed computational modeling of the binding of ARBs to AT1 receptor using CXCR4 receptor as a new template and a single version of software. The interactions between Tyr113 in the AT1 receptor and the hydroxyl group of olmesartan, between Lys199 and carboxyl or tetrazole groups, and between His256 or Gln257 and the tetrazole group were studied. The common structure, a tetrazole group, of most ARBs similarly bind to Lys199, His256 and Gln257 of AT1 receptor. Lys199 in the AT1 receptor binds to the carboxyl group of EXP3174, candesartan and azilsartan, whereas oxygen in the amidecarbonyl group of valsartan may bind to Lys199. The benzimidazole portion of telmisartan may bind to a lipophilic pocket that includes Tyr113. On the other hand, the n-butyl group of irbesartan may bind to Tyr113. In conclusion, we confirmed that the slightly different structures of ARBs may be critical for binding to AT1 receptor and for the formation of unique modes of binding. PMID:24260317

Exploration of N-arylpiperazine Binding Sites of D2 Dopaminergic Receptor.

PubMed

Soskic, Vukic; Sukalovic, Vladimir; Kostic-Rajacic, Sladjana

2015-01-01

The crystal structures of the D3 dopamine receptor and several other G-protein coupled receptors (GPCRs) were published in recent times. Those 3D structures are used by us and other scientists as a template for the homology modeling and ligand docking analysis of related GPCRs. Our main scientific interest lies in the field of pharmacologically active N-arylpiperazines that exhibit antipsychotic and/or antidepressant properties, and as such are dopaminergic and serotonergic receptor ligands. In this short review article we are presenting synthesis and biological data on the new N-arylpipereazine as well our results on molecular modeling of the interactions of those N-arylpiperazines with the model of D2 dopamine receptors. To obtain that model the crystal structure of the D3 dopamine receptor was used. Our results show that the N-arylpiperazines binding site consists of two pockets: one is the orthosteric binding site where the N-arylpiperazine part of the ligand is docked and the second is a non-canonical accessory binding site for N-arylpipereazine that is formed by a second extracellular loop (ecl2) of the receptor. Until now, the structure of this receptor region was unresolved in crystal structure analyses of the D3 dopamine receptor. To get a more complete picture of the ligand - receptor interaction, DFT quantum mechanical calculations on N-arylpiperazine were performed and the obtained models were used to examine those interactions.

Conserved Receptor-Binding Domains of Lake Victoria Marburgvirus and Zaire Ebolavirus Bind a Shared Receptor

DTIC Science & Technology

2006-04-14

virion, because of the functional importance of and limited variation in this region (44, 45). In some cases, such as murine and feline leukemia viruses ...murine leukemia virus ; PBS, phos- phate-buffered saline; RBD, receptor-binding domain; SARS, severe acute respiratory syndrome; VSV, vesicular stomatitis...entryofpseudotypedret- roviruses. A Moloney murine leukemia virus vector expressing GFP was pseudotyped with the GP1,2 of MARV-Mus (MARV/MLV), a mucin-like

Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins.

PubMed

Vishnivetskiy, Sergey A; Gimenez, Luis E; Francis, Derek J; Hanson, Susan M; Hubbell, Wayne L; Klug, Candice S; Gurevich, Vsevolod V

2011-07-08

Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements.

Few Residues within an Extensive Binding Interface Drive Receptor Interaction and Determine the Specificity of Arrestin Proteins*

PubMed Central

Vishnivetskiy, Sergey A.; Gimenez, Luis E.; Francis, Derek J.; Hanson, Susan M.; Hubbell, Wayne L.; Klug, Candice S.; Gurevich, Vsevolod V.

2011-01-01

Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements. PMID:21471193

The epidermal growth factor receptor (EGFR) promotes uptake of influenza A viruses (IAV) into host cells.

PubMed

Eierhoff, Thorsten; Hrincius, Eike R; Rescher, Ursula; Ludwig, Stephan; Ehrhardt, Christina

2010-09-09

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake.

The Epidermal Growth Factor Receptor (EGFR) Promotes Uptake of Influenza A Viruses (IAV) into Host Cells

PubMed Central

Eierhoff, Thorsten; Hrincius, Eike R.; Rescher, Ursula; Ludwig, Stephan; Ehrhardt, Christina

2010-01-01

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake. PMID:20844577

Central phencyclidine (PCP) receptor binding is glutamate dependent: evidence for a PCP/excitatory amino acid receptor (EAAR) complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loo, P.; Braunwalder, A.; Lehmann, J.

PCP and other dissociative anesthetica block the increase in neuronal firing rate evoked by the EAAR agonist, N-methyl-Daspartate. NMDA and other EAAs such as glutamate (glu) have not been previously shown to affect PCP ligand binding. In the present study, using once washed rat forebrain membranes, 10 ..mu..M-glu was found to increase the binding of (/sup 3/H)TCP, a PCP analog, to defined PCP recognition sites by 20%. Removal of glu and aspartate (asp) by extensive washing decreased TCP binding by 75-90%. In these membranes, 10 ..mu..M L-glu increased TCP binding 3-fold. This effect was stereospecific and evoked by other EAAsmore » with the order of activity, L-glu > D-asp > L- asp > NMDA > D-glu > quisqualate. Kainate, GABA, NE, DA, 5-HT, 2-chloroadenosine, oxotremorine and histamine had no effect on TCP binding at concentrations up to 100 ..mu..M. The effects of L-glu were attenuated by the NMDA-type receptor antagonist, 2-amino-7--phosphonoheptanoate (AP7; 10 ..mu..M-1 mM). These findings indicate that EAAS facilitate TCP binding, possibly through NMDA-type receptors. The observed interaction between the PCP receptor and EAARs may reflect the existence of a macromolecular receptor complex similar to that demonstrated for the benzodiazepines and GABA.« less

Binding of phycoerythrin and its conjugates to murine low affinity receptors for immunoglobulin G.

PubMed

Takizawa, F; Kinet, J P; Adamczewski, M

1993-06-18

Conjugates of R-phycoerythrin are widely used for immunohistochemistry, especially for two-color flow cytometry. Their use is however limited by their apparent tendency to bind non-specifically. Using cells transfected with cDNAs for the murine low affinity receptors for immunoglobulin G (Fc gamma RII and -III) and cells naturally expressing these receptors, we demonstrate that R-phycoerythrin and its conjugates bind specifically and inhibitably to Fc gamma RII and -III. Immunofluorescence stainings of cells bearing these receptors, such as macrophages, monocytes, neutrophils, mast cells, subsets of T cells, and natural killer cells, may therefore not reflect the binding of antibody to antigen, but rather the binding of R-phycoerythrin to the receptors.

Atrial natriuretic factor receptor heterogeneity in rat tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andresen, J.W.; Kuno, T.; Kamisaki, Y.

1986-03-01

Rat /sup 125/I-atrial natriuretic factor (ANF, 8-33) was used to identify ANF receptors in membrane preparations from rat adrenal gland and lung. When solubilized with Lubrol-PX, the receptors retained a binding profile and properties that correspond to the high affinity and specificity found in crude membranes. Single peaks of binding activity were observed in gel permeation HPLC and density gradient centrifugation analysis of the solubilized preparations. However, when membranes and solubilized preparations were labeled with /sup 125/I-ANF, treated with crosslinking reagent (disuccinimidyl suberate), and analyzed by SDS gel electrophoresis several specifically labeled bands (120,000, 70,000, and 60,000 daltons) were identifiedmore » by autoradiography. The relative distribution of the specifically labeled proteins varied significantly between rat adrenal gland and lung. In adrenal glands the 120K dalton band was the most prominent specifically labeled protein, while the 60K and 70K dalton proteins were labeled to a lesser degree. In lung membranes the lower molecular weight proteins were more prominent. These results suggest the presence of multiple ANF receptor subtypes, the distribution of which varies among tissues. Chromatographic separation and further characterization of these receptors are currently in progress, and preliminary purification studies support this hypothesis.« less

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors.

PubMed

Koshimizu, Taka-Aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-05-03

Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors

PubMed Central

Koshimizu, Taka-aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-01-01

Reducing Na+ in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na+-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na+ sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na+ increased cell surface [3H]AVP binding and decreased receptor internalization. Substitution of Na+ by Cs+ or NH4+ inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na+ over Cs+. Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations. PMID:27138239

Synthesis of heteroaromatic tropeines and heterogeneous binding to glycine receptors.

PubMed

Maksay, Gábor; Vincze, Zoltán; Nemes, Péter

2009-10-01

Heteroaromatic carboxylic esters of (nor)tropine were synthesized. Tropine esters displaced [(3)H]strychnine binding to glycine receptors of rat spinal cord with low Hill slopes. Two-site displacement resulted in nanomolar IC(50,1) and micromolar IC(50,2) values, and IC(50,2)/IC(50,1) ratios up to 615 depending on the heteroaromatic rings and N-methyl substitution. Nortropeines displayed high affinity and low heterogeneity. IC(50,1) and IC(50,2) values of tropeines did not correlate suggesting different binding modes/sites. Glycine potentiated only the nanomolar displacement reflecting positive allosteric interactions and potentiation of ionophore function. Affinities of three (nor)tropeines were different for glycine receptors but identical for 5-HT(3) receptors.

Characterization of glucagon-like peptide-1 receptor-binding determinants.

PubMed

Xiao, Q; Jeng, W; Wheeler, M B

2000-12-01

Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.

Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

PubMed

Miao, Yinglong; McCammon, J Andrew

2018-03-20

Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

Characterization of (/sup 3/H)pirenzepine binding to muscarinic cholinergic receptors solubilized from rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luthin, G.R.; Wolfe, B.B.

Membranes prepared from rat cerebral cortex were solubilized in buffer containing 1% digitonin. Material present in the supernatant after centrifugation at 147,000 X g was shown to contain binding sites for both (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) and (/sup 3/H)pirenzepine ((/sup 3/H)PZ). Recovery of binding sites was approximately 25% of the initial membrane-bound (/sup 3/H)QNB binding sites. The Kd values for (/sup 3/H)QNB and (/sup 3/H)PZ binding to solubilized receptors were 0.3 nM and 0.1 microM, respectively. As has been observed previously in membrane preparations, (/sup 3/H)PZ appeared to label fewer solubilized binding sites than did (/sup 3/H)QNB. Maximum bindingmore » values for (/sup 3/H)PZ and (/sup 3/H)QNB binding to solubilized receptors were approximately 400 and 950 fmol/mg of protein, respectively. Competition curves for PZ inhibiting the binding of (/sup 3/H)QNB, however, had Hill slopes of 1, with a Ki value of 0.24 microM. The k1 and k-1 for (/sup 3/H)PZ binding were 3.5 X 10(6) M-1 min-1 and 0.13 min-1, respectively. The muscarinic receptor antagonists atropine, scopolamine and PZ inhibited the binding of (/sup 3/H)QNB and (/sup 3/H)PZ to solubilized receptors with Hill slopes of 1, as did the muscarinic receptor agonist oxotremorine. The muscarinic receptor agonist carbachol competed for (/sup 3/H)QNB and (/sup 3/H)PZ binding with a Hill slope of less than 1 in cerebral cortex, but not in cerebellum. GTP did not alter the interactions of carbachol or oxotremorine with the solubilized receptor. Together, these data suggest that muscarinic receptor sites solubilized from rat brain retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.« less

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

PubMed

Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

1992-08-05

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

Cloning retinoid and peroxisome proliferator-activated nuclear receptors of the Pacific oyster and in silico binding to environmental chemicals

PubMed Central

Vogeler, Susanne; Galloway, Tamara S.; Isupov, Michail

2017-01-01

Disruption of nuclear receptors, a transcription factor superfamily regulating gene expression in animals, is one proposed mechanism through which pollution causes effects in aquatic invertebrates. Environmental pollutants have the ability to interfere with the receptor’s functions through direct binding and inducing incorrect signals. Limited knowledge of invertebrate endocrinology and molecular regulatory mechanisms, however, impede the understanding of endocrine disruptive effects in many aquatic invertebrate species. Here, we isolated three nuclear receptors of the Pacific oyster, Crassostrea gigas: two isoforms of the retinoid X receptor, CgRXR-1 and CgRXR-2, a retinoic acid receptor ortholog CgRAR, and a peroxisome proliferator-activated receptor ortholog CgPPAR. Computer modelling of the receptors based on 3D crystal structures of human proteins was used to predict each receptor’s ability to bind to different ligands in silico. CgRXR showed high potential to bind and be activated by 9-cis retinoic acid and the organotin tributyltin (TBT). Computer modelling of CgRAR revealed six residues in the ligand binding domain, which prevent the successful interaction with natural and synthetic retinoid ligands. This supports an existing theory of loss of retinoid binding in molluscan RARs. Modelling of CgPPAR was less reliable due to high discrepancies in sequence to its human ortholog. Yet, there are suggestions of binding to TBT, but not to rosiglitazone. The effect of potential receptor ligands on early oyster development was assessed after 24h of chemical exposure. TBT oxide (0.2μg/l), all-trans retinoic acid (ATRA) (0.06 mg/L) and perfluorooctanoic acid (20 mg/L) showed high effects on development (>74% abnormal developed D-shelled larvae), while rosiglitazone (40 mg/L) showed no effect. The results are discussed in relation to a putative direct (TBT) disruption effect on nuclear receptors. The inability of direct binding of ATRA to CgRAR suggests either a

The high mobility group protein 1 enhances binding of the estrogen receptor DNA binding domain to the estrogen response element.

PubMed

Romine, L E; Wood, J R; Lamia, L A; Prendergast, P; Edwards, D P; Nardulli, A M

1998-05-01

We have examined the ability of the high-mobility group protein 1 (HMG1) to alter binding of the estrogen receptor DNA-binding domain (DBD) to the estrogen response element (ERE). HMG1 dramatically enhanced binding of purified, bacterially expressed DBD to the consensus vitellogenin A2 ERE in a dose-dependent manner. The ability of HMG1 to stabilize the DBD-ERE complex resulted in part from a decrease in the dissociation rate of the DBD from the ERE. Antibody supershift experiments demonstrated that HMG1 was also capable of forming a ternary complex with the ERE-bound DBD in the presence of HMG1-specific antibody. HMG1 did not substantially affect DBD-ERE contacts as assessed by methylation interference assays, nor did it alter the ability of the DBD to induce distortion in ERE-containing DNA fragments. Because HMG1 dramatically enhanced estrogen receptor DBD binding to the ERE, and the DBD is the most highly conserved region among the nuclear receptor superfamily members, HMG1 may function to enhance binding of other nuclear receptors to their respective response elements and act in concert with coactivator proteins to regulate expression of hormone-responsive genes.

Aging-induced changes in brain regional serotonin receptor binding: Effect of Carnosine.

PubMed

Banerjee, S; Poddar, M K

2016-04-05

Monoamine neurotransmitter, serotonin (5-HT) has its own specific receptors in both pre- and post-synapse. In the present study the role of carnosine on aging-induced changes of [(3)H]-5-HT receptor binding in different brain regions in a rat model was studied. The results showed that during aging (18 and 24 months) the [(3)H]-5-HT receptor binding was reduced in hippocampus, hypothalamus and pons-medulla with a decrease in their both Bmax and KD but in cerebral cortex the [(3)H]-5-HT binding was increased with the increase of its only Bmax. The aging-induced changes in [(3)H]-5-HT receptor binding with carnosine (2.0 μg/kg/day, intrathecally, for 21 consecutive days) attenuated in (a) 24-month-aged rats irrespective of the brain regions with the attenuation of its Bmax except hypothalamus where both Bmax and KD were significantly attenuated, (b) hippocampus and hypothalamus of 18-month-aged rats with the attenuation of its Bmax, and restored toward the [(3)H]-5-HT receptor binding that observed in 4-month-young rats. The decrease in pons-medullary [(3)H]-5-HT binding including its Bmax of 18-month-aged rats was promoted with carnosine without any significant change in its cerebral cortex. The [(3)H]-5-HT receptor binding with the same dosages of carnosine in 4-month-young rats (a) increased in the cerebral cortex and hippocampus with the increase in their only Bmax whereas (b) decreased in hypothalamus and pons-medulla with a decrease in their both Bmax and KD. These results suggest that carnosine treatment may (a) play a preventive role in aging-induced brain region-specific changes in serotonergic activity (b) not be worthy in 4-month-young rats in relation to the brain regional serotonergic activity. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising "Hot-Spot" Lysine Residues.

PubMed

van den Biggelaar, Maartje; Madsen, Jesper J; Faber, Johan H; Zuurveld, Marleen G; van der Zwaan, Carmen; Olsen, Ole H; Stennicke, Henning R; Mertens, Koen; Meijer, Alexander B

2015-07-03

Lysine residues are implicated in driving the ligand binding to the LDL receptor family. However, it has remained unclear how specificity is regulated. Using coagulation factor VIII as a model ligand, we now study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and LRP1 occurs over an extended surface containing multiple lysine residues. None of the individual lysine residues account completely for LRP1 binding, suggesting an additive binding model. Together with structural docking studies, our data suggest that FVIII interacts with LRP1 via an extended surface of multiple lysine residues that starts at the bottom of the C1 domain and winds around the FVIII molecule. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural basis for the inhibition of insulin-like growth factors by insulin-like growth factor-binding proteins

PubMed Central

Sitar, Tomasz; Popowicz, Grzegorz M.; Siwanowicz, Igor; Huber, Robert; Holak, Tad A.

2006-01-01

Insulin-like growth factor-binding proteins (IGFBPs) control bioavailability, activity, and distribution of insulin-like growth factor (IGF)1 and -2 through high-affinity IGFBP/IGF complexes. IGF-binding sites are found on N- and C-terminal fragments of IGFBPs, the two conserved domains of IGFBPs. The relative contributions of these domains to IGFBP/IGF complexation has been difficult to analyze, in part, because of the lack of appropriate three-dimensional structures. To analyze the effects of N- and C-terminal domain interactions, we determined several x-ray structures: first, of a ternary complex of N- and C-terminal domain fragments of IGFBP4 and IGF1 and second, of a “hybrid” ternary complex using the C-terminal domain fragment of IGFBP1 instead of IGFBP4. We also solved the binary complex of the N-terminal domains of IGFBP4 and IGF1, again to analyze C- and N-terminal domain interactions by comparison with the ternary complexes. The structures reveal the mechanisms of IGF signaling regulation via IGFBP binding. This finding supports research into the design of IGFBP variants as therapeutic IGF inhibitors for diseases of IGF disregulation. In IGFBP4, residues 1–38 form a rigid disulphide bond ladder-like structure, and the first five N-terminal residues bind to IGF and partially mask IGF residues responsible for the type 1 IGF receptor binding. A high-affinity IGF1-binding site is located in a globular structure between residues 39 and 82. Although the C-terminal domains do not form stable binary complexes with either IGF1 or the N-terminal domain of IGFBP4, in the ternary complex, the C-terminal domain contacts both and contributes to blocking of the IGF1 receptor-binding region of IGF1. PMID:16924115

Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

PubMed Central

Spurny, Radovan; Debaveye, Sarah; Farinha, Ana; Veys, Ken; Vos, Ann M.; Gossas, Thomas; Atack, John; Bertrand, Sonia; Bertrand, Daniel; Danielson, U. Helena; Tresadern, Gary; Ulens, Chris

2015-01-01

The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential. PMID:25918415

GABAergic control of neostriatal dopamine D2 receptor binding and behaviors in the rat.

PubMed

Nikolaus, Susanne; Beu, Markus; de Souza Silva, Maria Angelica; Huston, Joseph P; Antke, Christina; Müller, Hans-Wilhelm; Hautzel, Hubertus

2017-02-01

The present study assessed the influence of the GABA A receptor agonist muscimol and the GABA A receptor antagonist bicuculline on neostriatal dopamine D 2 receptor binding in relation to motor and exploratory behaviors in the rat. D 2 receptor binding was measured in baseline and after challenge with either 1mg/kg muscimol or 1mg/kg bicuculline. In additional rats, D 2 receptor binding was measured after injection of saline. After treatment with muscimol, bicuculline and saline, motor and exploratory behaviors were assessed for 30min in an open field prior to administration of [ 123 I]S-3-iodo-N-(1-ethyl-2-pyrrolidinyl)methyl-2-hydroxy-6-methoxybenzamide ([ 123 I]IBZM). For baseline and challenges, striatal equilibrium ratios (V 3 ″) were computed as estimation of the binding potential. Muscimol but not bicuculline reduced D 2 receptor binding relative to baseline and to saline. Travelled distance, duration of rearing and frequency of rearing and of head-shoulder motility were lower after muscimol compared to saline. In contrast, duration of rearing and grooming and frequency of rearing, head-shoulder motility and grooming were elevated after bicuculline relative to saline. Moreover, bicuculline decreased duration of sitting and head-shoulder motility. The muscimol-induced decrease of motor/exploratory behaviors can be related to an elevation of striatal dopamine levels. In contrast, bicuculline is likely to elicit a decline of synaptic dopamine, which, however, is compensated by the time of D 2 receptor imaging studies. The results indicate direct GABAergic control over D 2 receptor binding in the neostriatum in relation to behavioral action, and, thus, complement earlier pharmacological studies. Copyright © 2016. Published by Elsevier Inc.

Characterization of the receptor-binding domain of Ebola glycoprotein in viral entry.

PubMed

Wang, Jizhen; Manicassamy, Balaji; Caffrey, Michael; Rong, Lijun

2011-06-01

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.

Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

NASA Technical Reports Server (NTRS)

Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

1995-01-01

A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morrison, W.J.

1988-01-01

The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF ormore » thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).« less

Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.

PubMed

André, S; Ortega, P J; Perez, M A; Roy, R; Gabius, H J

1999-11-01

Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential

Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

PubMed Central

Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

2012-01-01

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

Receptor binding kinetics equations: Derivation using the Laplace transform method.

PubMed

Hoare, Sam R J

Measuring unlabeled ligand receptor binding kinetics is valuable in optimizing and understanding drug action. Unfortunately, deriving equations for estimating kinetic parameters is challenging because it involves calculus; integration can be a frustrating barrier to the pharmacologist seeking to measure simple rate parameters. Here, a well-known tool for simplifying the derivation, the Laplace transform, is applied to models of receptor-ligand interaction. The method transforms differential equations to a form in which simple algebra can be applied to solve for the variable of interest, for example the concentration of ligand-bound receptor. The goal is to provide instruction using familiar examples, to enable investigators familiar with handling equilibrium binding equations to derive kinetic equations for receptor-ligand interaction. First, the Laplace transform is used to derive the equations for association and dissociation of labeled ligand binding. Next, its use for unlabeled ligand kinetic equations is exemplified by a full derivation of the kinetics of competitive binding equation. Finally, new unlabeled ligand equations are derived using the Laplace transform. These equations incorporate a pre-incubation step with unlabeled or labeled ligand. Four equations for measuring unlabeled ligand kinetics were compared and the two new equations verified by comparison with numerical solution. Importantly, the equations have not been verified with experimental data because no such experiments are evident in the literature. Equations were formatted for use in the curve-fitting program GraphPad Prism 6.0 and fitted to simulated data. This description of the Laplace transform method will enable pharmacologists to derive kinetic equations for their model or experimental paradigm under study. Application of the transform will expand the set of equations available for the pharmacologist to measure unlabeled ligand binding kinetics, and for other time

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain.

PubMed

Speranskiy, Kirill; Kurnikova, Maria

2005-08-30

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.

Renal atrial natriuretic factor receptors in hamster cardiomyopathy.

PubMed

Mukaddam-Daher, S; Jankowski, M; Dam, T V; Quillen, E W; Gutkowska, J

1995-12-01

Hamsters with cardiomyopathy (CMO), an experimental model of congestive heart failure, display stimulated renin-angiotensin-aldosterone and enhanced sympathetic nervous activity, all factors that lead to sodium retention, volume expansion and subsequent elevation of plasma atrial natriuretic factor (ANF) by the cardiac atria. However, sodium and water retention persist in CMO, indicating hyporesponsiveness to endogenous ANF. These studies were undertaken to fully characterize renal ANF receptor subtypes in normal hamsters and to evaluate whether alterations in renal ANF receptors may contribute to renal resistance to ANF in cardiomyopathy. Transcripts of the guanylyl cyclase-A (GC-A) and guanylyl cyclase B (GC-B) receptors were detected by quantitative polymerase chain reaction (PCR) in renal cortex, and outer and inner medullas. Compared to normal controls, the cardiomyopathic hamster's GC-A mRNA was similar in cortex but significantly increased in outer and inner medulla. Levels of GC-B mRNA were not altered by the disease. On the other hand, competitive binding studies, autoradiography, and affinity cross-linking demonstrated the absence of functional GC-B receptors in the kidney glomeruli and inner medulla. Also, C-type natriuretic peptide (CNP), the natural ligand for the GC-B receptors, failed to stimulate glomerular production of its second messenger cGMP. In CMO, sodium and water excretion were significantly reduced despite elevated plasma ANF (50.5 +/- 11.1 vs. 309.4 +/- 32.6 pg/ml, P < 0.001). Competitive binding studies of renal glomerular ANF receptors revealed no change in total receptor density, Bmax (369.6 +/- 27.4 vs. 282.8 +/- 26.2 fmol/mg protein), nor in dissociation constant, Kd (647.4 +/- 79.4 vs. 648.5 +/- 22.9 pM). Also, ANF-C receptor density (254.3 +/- 24.8 vs. 233.8 +/- 23.5 fmol/mg protein), nor affinity were affected by heart failure. Inner medullary receptors were exclusively of the GC-A subtype with Bmax (153.2 +/- 26.4 vs. 134

Antagonism of human CC-chemokine receptor 4 can be achieved through three distinct binding sites on the receptor

PubMed Central

Slack, Robert J; Russell, Linda J; Barton, Nick P; Weston, Cathryn; Nalesso, Giovanna; Thompson, Sally-Anne; Allen, Morven; Chen, Yu Hua; Barnes, Ashley; Hodgson, Simon T; Hall, David A

2013-01-01

Chemokine receptor antagonists appear to access two distinct binding sites on different members of this receptor family. One class of CCR4 antagonists has been suggested to bind to a site accessible from the cytoplasm while a second class did not bind to this site. In this report, we demonstrate that antagonists representing a variety of structural classes bind to two distinct allosteric sites on CCR4. The effects of pairs of low-molecular weight and/or chemokine CCR4 antagonists were evaluated on CCL17- and CCL22-induced responses of human CCR4+ T cells. This provided an initial grouping of the antagonists into sets which appeared to bind to distinct binding sites. Binding studies were then performed with radioligands from each set to confirm these groupings. Some novel receptor theory was developed to allow the interpretation of the effects of the antagonist combinations. The theory indicates that, generally, the concentration-ratio of a pair of competing allosteric modulators is maximally the sum of their individual effects while that of two modulators acting at different sites is likely to be greater than their sum. The low-molecular weight antagonists could be grouped into two sets on the basis of the functional and binding experiments. The antagonistic chemokines formed a third set whose behaviour was consistent with that of simple competitive antagonists. These studies indicate that there are two allosteric regulatory sites on CCR4. PMID:25505571

Negatively Cooperative Binding of High Density Lipoprotein to the HDL Receptor SR-BI†

PubMed Central

Nieland, Thomas J.F.; Xu, Shangzhe; Penman, Marsha; Krieger, Monty

2011-01-01

Scavenger receptor class B, type I (SR-BI) is a high-density lipoprotein (HDL) receptor, which also binds low density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a co-receptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high affinity binding sites (one site model). We have re-investigated the ligand concentration dependence of 125I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [3H]CE from [3H]CE-HDL using an expanded range of ligand concentrations (<1 µg protein/ml, lower than previously reported). Scatchard and non-linear least squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites, or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects (‘ lattice model’). Similar results were observed for LDL. Application of the ‘infinite dilution’ dissociation rate method established that the binding of 125I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport and cell signaling. PMID:21254782

Recent Progress in the Design and Discovery of RXR Modulators Targeting Alternate Binding Sites of the Receptor.

PubMed

Su, Ying; Zeng, Zhiping; Chen, Ziwen; Xu, Dan; Zhang, Weidong; Zhang, Xiao-Kun

2017-01-01

Retinoid X receptors (RXRs) occupy a central position within the nuclear receptor superfamily. They not only function as important transcriptional factors but also exhibit diverse nongenomic biological activities. The pleiotropic actions of RXRs under both physiological and pathophysiological conditions confer RXRs important drug targets for the treatment of cancer, and metabolic and neurodegenerative diseases. RXR modulators have been studied for the purpose of developing both drug molecules and chemical tools for biological investigation of RXR. Development of RXR modulators has focused on small molecules targeting the canonical ligand-binding pocket. However, accumulating results have demonstrated that there are other binding mechanisms by which small molecules interact with RXR to act as RXR modulators. This review discusses the recent development in the design and discovery of RXR modulators with a focus on those targeting novel binding sites on RXR.

Simultaneous Multiple MS Binding Assays Addressing D1 and D2 Dopamine Receptors.

PubMed

Schuller, Marion; Höfner, Georg; Wanner, Klaus T

2017-10-09

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D 1 and D 2 dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D 1 and D 2 receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D 1 and D 2 receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Binding Pathway of Opiates to μ-Opioid Receptors Revealed by Machine Learning

NASA Astrophysics Data System (ADS)

Barati Farimani, Amir; Feinberg, Evan; Pande, Vijay

2018-02-01

Many important analgesics relieve pain by binding to the $\\mu$-Opioid Receptor ($\\mu$OR), which makes the $\\mu$OR among the most clinically relevant proteins of the G Protein Coupled Receptor (GPCR) family. Despite previous studies on the activation pathways of the GPCRs, the mechanism of opiate binding and the selectivity of $\\mu$OR are largely unknown. We performed extensive molecular dynamics (MD) simulation and analysis to find the selective allosteric binding sites of the $\\mu$OR and the path opiates take to bind to the orthosteric site. In this study, we predicted that the allosteric site is responsible for the attraction and selection of opiates. Using Markov state models and machine learning, we traced the pathway of opiates in binding to the orthosteric site, the main binding pocket. Our results have important implications in designing novel analgesics.

Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain.

PubMed Central

Stevenson, S C; Rollence, M; White, B; Weaver, L; McClelland, A

1995-01-01

The adenovirus fiber protein is responsible for attachment of the virion to cell surface receptors. The identity of the cellular receptor which mediates binding is unknown, although there is evidence suggesting that two distinct adenovirus receptors interact with the group C (adenovirus type 5 [Ad5]) and the group B (Ad3) adenoviruses. In order to define the determinants of adenovirus receptor specificity, we have carried out a series of competition binding experiments using recombinant native fiber polypeptides from Ad5 and Ad3 and chimeric fiber proteins in which the head domains of Ad5 and Ad3 were exchanged. Specific binding of fiber to HeLa cell receptors was assessed with radiolabeled protein synthesized in vitro, and by competition analysis with baculovirus-expressed fiber protein. Fiber produced in vitro was found as both monomer and trimer, but only the assembled trimers had receptor binding activity. Competition data support the conclusion that Ad5 and Ad3 interact with different cellular receptors. The Ad5 receptor distribution on several cell lines was assessed with a fiber binding flow cytometric assay. HeLa cells were found to express high levels of receptor, while CHO and human diploid fibroblasts did not. A chimeric fiber containing the Ad5 fiber head domain blocked the binding of Ad5 fiber but not Ad3 fiber. Similarly, a chimeric fiber containing the Ad3 fiber head blocked the binding of labeled Ad3 fiber but not Ad5 fiber. In addition, the isolated Ad3 fiber head domain competed effectively with labeled Ad3 fiber for binding to HeLa cell receptors. These results demonstrate that the determinants of receptor binding are located in the head domain of the fiber and that the isolated head domain is capable of trimerization and binding to cellular receptors. Our results also show that it is possible to change the receptor specificity of the fiber protein by manipulation of sequences contained in the head domain. Modification or replacement of the fiber

Binding Mode Analysis of Zerumbone to Key Signal Proteins in the Tumor Necrosis Factor Pathway

PubMed Central

Fatima, Ayesha; Abdul, Ahmad Bustamam Hj.; Abdullah, Rasedee; Karjiban, Roghayeh Abedi; Lee, Vannajan Sanghiran

2015-01-01

Breast cancer is the second most common cancer among women worldwide. Several signaling pathways have been implicated as causative and progression agents. The tumor necrosis factor (TNF) α protein plays a dual role in promoting and inhibiting cancer depending largely on the pathway initiated by the binding of the protein to its receptor. Zerumbone, an active constituent of Zingiber zerumbet, Smith, is known to act on the tumor necrosis factor pathway upregulating tumour necrosis factor related apoptosis inducing ligand (TRAIL) death receptors and inducing apoptosis in cancer cells. Zerumbone is a sesquiterpene that is able to penetrate into the hydrophobic pockets of proteins to exert its inhibiting activity with several proteins. We found a good binding with the tumor necrosis factor, kinase κB (IKKβ) and the Nuclear factor κB (NF-κB) component proteins along the TNF pathway. Our results suggest that zerumbone can exert its apoptotic activities by inhibiting the cytoplasmic proteins. It inhibits the IKKβ kinase that activates the NF-κB and also binds to the NF-κB complex in the TNF pathway. Blocking both proteins can lead to inhibition of cell proliferating proteins to be downregulated and possibly ultimate induction of apoptosis. PMID:25629232

Structure of the bacteriophage T4 long tail fiber receptor-binding tip

PubMed Central

Bartual, Sergio G.; Otero, José M.; Garcia-Doval, Carmela; Llamas-Saiz, Antonio L.; Kahn, Richard; Fox, Gavin C.; van Raaij, Mark J.

2010-01-01

Bacteriophages are the most numerous organisms in the biosphere. In spite of their biological significance and the spectrum of potential applications, little high-resolution structural detail is available on their receptor-binding fibers. Here we present the crystal structure of the receptor-binding tip of the bacteriophage T4 long tail fiber, which is highly homologous to the tip of the bacteriophage lambda side tail fibers. This structure reveals an unusual elongated six-stranded antiparallel beta-strand needle domain containing seven iron ions coordinated by histidine residues arranged colinearly along the core of the biological unit. At the end of the tip, the three chains intertwine forming a broader head domain, which contains the putative receptor interaction site. The structure reveals a previously unknown beta-structured fibrous fold, provides insights into the remarkable stability of the fiber, and suggests a framework for mutations to expand or modulate receptor-binding specificity. PMID:21041684

Participation of Water in the Binding of Estrogen Receptor with Estrogen Responsive Element in vitro.

PubMed

Zhu, Guo-Zhang; Tang, Guo-Qing; Ruan, Kang-Cheng; Gong, Yue-Ting; Zhang, Yong-Lian

1998-01-01

Many reports have showed that bound water was involved in the interaction between/among the macromolecules. However, it has not been reported whether bound water is also involved in the binding of trans-factors and cis-elements in the regulation of the eukaryotic gene trans-cription or not. Preliminary studies have been made on the effect of bound water on the binding of estrogen receptor with estrogen responsive element in vitro. In the gel retardation assay using the cytosol extract of rat uterus as the supplier of estrogen receptor and 32 bp oligonucleotide containing a concensus vitellogenin A(2) ERE as the probe, various cosolvents, such as glycerol, sucrose, N-dimethylformamide and dimethylsulfoxide, were added respectively to the reaction mixture in varying concentrations to regulate the osmotic pressure. The results indicated that the binding of ER-ERE was enhanced with the increase in the final concentration of these individual cosolvents. On the other hand, when the reaction was carried out under an increasing hydrostatic pressure, the ER-ERE binding was decreased sharply. After decompression the binding of ER-ERE was gradually restored to the normal level with the lapse of time. These results suggested that bound water was directly involved in the binding of ER-ERE and may play an important role in the regulation of the eukaryotic gene transcription.

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding.

PubMed

Jung, Seung-Ryoung; Kushmerick, Christopher; Seo, Jong Bae; Koh, Duk-Su; Hille, Bertil

2017-07-11

Binding of agonists to G-protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M 1 muscarinic acetylcholine receptors (M 1 Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M 1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M 1 R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane.

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding

PubMed Central

Jung, Seung-Ryoung; Kushmerick, Christopher; Seo, Jong Bae; Koh, Duk-Su

2017-01-01

Binding of agonists to G-protein–coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M1 muscarinic acetylcholine receptors (M1Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M1R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane. PMID:28652372

Impact of linker and conjugation chemistry on antigen binding, Fc receptor binding and thermal stability of model antibody-drug conjugates

PubMed Central

Acchione, Mauro; Kwon, Hyewon; Jochheim, Claudia M.; Atkins, William M.

2012-01-01

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHISNLC and aHISTPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate. PMID:22531451

A dopamine D2 receptor mutant capable of G protein-mediated signaling but deficient in arrestin binding.

PubMed

Lan, Hongxiang; Liu, Yong; Bell, Michal I; Gurevich, Vsevolod V; Neve, Kim A

2009-01-01

Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D(2) and D(3) receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D(2) and D(3) receptors other than phosphorylation sites and to create a D(2) receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D(2) and D(3) receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D(2) receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D(2)-IC3 and D(3)-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D(2) and D(3) receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D(2) receptor is a key element of the arrestin binding site.

Muscarinic and alpha 1-adrenergic receptor binding characteristics of saw palmetto extract in rat lower urinary tract.

PubMed

Suzuki, Mayumi; Oki, Tomomi; Sugiyama, Tomomi; Umegaki, Keizo; Uchida, Shinya; Yamada, Shizuo

2007-06-01

To elucidate the in vitro and ex vivo effects of saw palmetto extract (SPE) on autonomic receptors in the rat lower urinary tract. The in vitro binding affinities for alpha 1-adrenergic, muscarinic, and purinergic receptors in the rat prostate and bladder were measured by radioligand binding assays. Rats received vehicle or SPE (0.6 to 60 mg/kg/day) orally for 4 weeks, and alpha 1-adrenergic and muscarinic receptor binding in tissues of these rats were measured. Saw palmetto extract inhibited specific binding of [3H]prazosin and [N-methyl-3H]scopolamine methyl chloride (NMS) but not alpha, beta-methylene adenosine triphosphate [2,8-(3)H]tetrasodium salt in the rat prostate and bladder. The binding activity of SPE for muscarinic receptors was four times greater than that for alpha 1-adrenergic receptors. Scatchard analysis revealed that SPE significantly reduced the maximal number of binding sites (Bmax) for each radioligand in the prostate and bladder under in vitro condition. Repeated oral administration of SPE to rats brought about significant alteration in Bmax for prostatic [3H]prazosin binding and for bladder [3H]NMS binding. Such alteration by SPE was selective to the receptors in the lower urinary tract. Saw palmetto extract exerts significant binding activity on autonomic receptors in the lower urinary tract under in vitro and in vivo conditions.

Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Lianying; College of Life Science, Dezhou University, Dezhou 253023; Ren, Xiao-Min

2014-09-15

Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group.more » For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.« less

Receptor Signaling Directs Global Recruitment of Pre-existing Transcription Factors to Inducible Elements.

PubMed

Cockerill, Peter N

2016-12-01

Gene expression programs are largely regulated by the tissue-specific expression of lineage-defining transcription factors or by the inducible expression of transcription factors in response to specific stimuli. Here I will review our own work over the last 20 years to show how specific activation signals also lead to the wide-spread re-distribution of pre-existing constitutive transcription factors to sites undergoing chromatin reorganization. I will summarize studies showing that activation of kinase signaling pathways creates open chromatin regions that recruit pre-existing factors which were previously unable to bind to closed chromatin. As models I will draw upon genes activated or primed by receptor signaling in memory T cells, and genes activated by cytokine receptor mutations in acute myeloid leukemia. I also summarize a hit-and-run model of stable epigenetic reprograming in memory T cells, mediated by transient Activator Protein 1 (AP-1) binding, which enables the accelerated activation of inducible enhancers.

Development of Gamma-Emitting Receptor Binding Radiopharmace

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reba, Richard

2003-02-20

The long-term objective is to develop blood-brain barrier (BBB) permeable m2-selective (relative to m1, m3, and m4) receptor-binding radiotracers and utilize these radiotracers for quantifying receptor concentrations obtained from PET or SPECT images of human brain. In initial studies, we concluded that the lipophilicity and high affinity prevented (R,S)-I-QNB from reaching a flow-independent and receptor-dependent state in a reasonable time. Thus, it was clear that (R,S)-I-QNB should be modified. Therefore, during the last portion of this funded research, we proposed that more polar heterocycles should help accomplish that. Since reports of others concluded that radiobromination and radiofluorination of the unactivatedmore » phenyl ring is not feasible (Newkome et al,,1982), we, therefore, explored during this grant period a series of analogues of (R)-QNB in which one or both of the six-membered phenyl rings is replaced by a five-membered thienyl (Boulay et al., 1995), or furyl ring. The chemistry specific aims were to synthesize novel compounds designed to be m2-selective mAChR ligands capable of penetrating into the CNS, and develop methods for efficient radiolabeling of promising m2-selective muscarinic ligands. The pharmacology specific aims were to determine the affinity and subtype-selectivity of the novel compounds using competition binding studies with membranes from cells that express each of the five muscarinic receptor subtypes, to determine the ability of the promising non-radioactive compounds and radiolabeled novel compounds to cross the BBB, to determine the biodistribution, in-vivo pharmacokinetics, and in-vitm kinetics of promising m2-selective radioligands and to determine the distribution of receptors for the novel m2-selective radioligands using quantitative autoradiography of rat brain, and compare this distribution to the distribution of known m2-selective compounds.« less

Chemokine receptor binding and signal transduction in native cells of the central nervous system.

PubMed

Davis, Christopher N; Chen, Shuzhen; Boehme, Stefen A; Bacon, Kevin B; Harrison, Jeffrey K

2003-04-01

Chemokine receptors belong to the superfamily of seven-transmembrane-spanning, G-protein-coupled receptors, and their expression by central nervous system cells is clearly documented. As this gene family has become the target of novel therapeutic development, the analysis of these receptors requires radioligand binding techniques as well as methods that entail assessing receptor stimulation of signal transduction pathways. Herein, we describe specific protocols for measuring radiolabeled chemokine binding to their cognate receptors on cultured glial cells as well as to receptors expressed in heterologous cell systems. Multiple downstream signaling pathways, including intracellular calcium influx and receptor-dependent kinase activation, are associated with chemokine receptor stimulation. Protocols for measuring these signaling events in chemokine-receptor-expressing cells are also presented.

High Affinity Binding of Epibatidine to Serotonin Type 3 Receptors*

PubMed Central

Drisdel, Renaldo C.; Sharp, Douglas; Henderson, Tricia; Hales, Tim G.; Green, William N.

2008-01-01

Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of 125I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT3Rs) block the remaining 25%. 125I-Epibatidine binds with a high affinity to native 5-HT3Rs of N1E-115 cells and to receptors composed of only 5-HT3A subunits expressed in HEK cells. In these cells, serotonin, the 5-HT3R-specific antagonist MDL72222, and the 5-HT3R agonist chlorophenylbiguanide readily competed with 125I-epibatidine binding to 5-HT3Rs. Nicotine was a poor competitor for 125I-epibatidine binding to 5-HT3Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of 125I-epibatidine binding to 5-HT3Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT3Rs in neuroblastoma cell lines and 5-HT3ARs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT3Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT3R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT3Rs. PMID:17702741

G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na/sup +/ channel interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

1988-01-12

The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinicallymore » induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.« less

Selectivity in progesterone and androgen receptor binding of progestagens used in oral contraceptives

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kloosterboer, H.J.; Vonk-Noordegraaf, C.A.; Turpijn, E.W.

1988-09-01

The relative binding affinities (RBAs) of four progestational compounds (norethisterone, levonorgestrel, 3-keto-desogestrel and gestodene) for the human progesterone and androgen receptors were measured in MCF-7 cytosol and intact MCF-7 cells. For the binding to the progesterone receptor, both Org 2058 and Org 3236 (or 3-keto-desogestrel) were used as labelled ligands. The following ranking (low to high) for the RBA of the nuclear (intact cells) progesterone receptor irrespective of the ligand used is found: norethisterone much less than levonorgestrel less than 3-keto-destogestrel less than gestodene. The difference between the various progestagens is significant with the exception of that between 3-keto-desogestrel andmore » gestodene, when Org 2058 is used as ligand. For the cytosolic progesterone receptor, the same order is found with the exception that similar RBAs are found for gestodene and 3-keto-desogestrel. The four progestagens clearly differ with respect to binding to the androgen receptor using dihydrotestosterone as labelled ligand in intact cells; the ranking (low to high) is: norethisterone less than 3 keto-desogestrel less than levonorgestrel and gestodene. The difference between 3-keto-desogestrel and levonorgestrel or gestodene is significant. The selectivity indices (ratio of the mean RBA for the progesterone receptor to that of androgen receptor) in intact cells are significantly higher for 3-keto-desogestrel and gestodene than for levonorgestrel and norethisterone. From these results we conclude that the introduction of the 18-methyl in norethisterone (levonorgestel) increases both the binding to the progesterone and androgen receptors.« less

The Receptor-Binding Site of the Measles Virus Hemagglutinin Protein Itself Constitutes a Conserved Neutralizing Epitope

PubMed Central

Ohno, Shinji; Sakai, Kouji; Ito, Yuri; Fukuhara, Hideo; Komase, Katsuhiro; Brindley, Melinda A.; Rota, Paul A.; Plemper, Richard K.; Maenaka, Katsumi; Takeda, Makoto

2013-01-01

Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature. PMID:23283964

BiFC Assay to Detect Calmodulin Binding to Plant Receptor Kinases.

PubMed

Fischer, Cornelia; Sauter, Margret; Dietrich, Petra

2017-01-01

Plant receptor-like kinases (RLKs) are regulated at various levels including posttranscriptional modification and interaction with regulatory proteins. Calmodulin (CaM) is a calcium-sensing protein that was shown to bind to some RLKs such as the PHYTOSULFOKINE RECEPTOR1 (PSKR1). The CaM-binding site is embedded in subdomain VIa of the kinase domain. It is possible that many more of RLKs interact with CaM than previously described. To unequivocally confirm CaM binding, several methods exist. Bimolecular fluorescence complementation (BiFC) and pull-down assays have been successfully used to study CaM binding to PSKR1 and are described in this chapter (BiFC) and in Chapter 15 (pull down). The two methods are complementary. BiFC is useful to show localization and interaction of soluble as well as of membrane-bound proteins in planta.

Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

PubMed

Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

2018-05-15

All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret

Potent haloperidol derivatives covalently binding to the dopamine D2 receptor.

PubMed

Schwalbe, Tobias; Kaindl, Jonas; Hübner, Harald; Gmeiner, Peter

2017-10-01

The dopamine D 2 receptor (D 2 R) is a common drug target for the treatment of a variety of neurological disorders including schizophrenia. Structure based design of subtype selective D 2 R antagonists requires high resolution crystal structures of the receptor and pharmacological tools promoting a better understanding of the protein-ligand interactions. Recently, we reported the development of a chemically activated dopamine derivative (FAUC150) designed to covalently bind the L94C mutant of the dopamine D 2 receptor. Using FAUC150 as a template, we elaborated the design and synthesis of irreversible analogs of the potent antipsychotic drug haloperidol forming covalent D 2 R-ligand complexes. The disulfide- and Michael acceptor-functionalized compounds showed significant receptor affinity and an irreversible binding profile in radioligand depletion experiments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Receptor-binding region in human choriogonadotropin/lutropin. beta. subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keutmann, H.T.; Charlesworth, M.C.; Mason, K.A.

1987-04-01

Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. The authors prepared a series of peptides representing the intercysteine loop sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) ..beta.. subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCG..beta..-(38-57) and hLH..beta..-(38-57) peptides inhibited binding of /sup 125/I-labeled hCG half-maximally at 1.51 x 10/sup -4/ and 2.03 x 10/sup -5/ M, respectively,more » while other peptide hormones and fragments from elsewhere in the ..beta.. subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 x 10/sup -5/ M (hCG) and 2.18 x 10/sup -5/ M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCG..beta..-(38-57) peptide, native hCG and ..beta.. subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. These results indicate that the 38-57 region of ..beta.. subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones.« less

Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

PubMed Central

Coopman, K.; Wallis, R.; Robb, G.; Brown, A. J. H.; Wilkinson, G. F.; Timms, D.

2011-01-01

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues. PMID:21868452

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

PubMed

Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

2011-12-01

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

Arrestin binds to different phosphorylated regions of the thyrotropin-releasing hormone receptor with distinct functional consequences.

PubMed

Jones, Brian W; Hinkle, Patricia M

2008-07-01

Arrestin binding to agonist-occupied phosphorylated G protein-coupled receptors typically increases the affinity of agonist binding, increases resistance of receptor-bound agonist to removal with high acid/salt buffer, and leads to receptor desensitization and internalization. We tested whether thyrotropin-releasing hormone (TRH) receptors lacking phosphosites in the C-terminal tail could form stable and functional complexes with arrestin. Fibroblasts from mice lacking arrestins 2 and 3 were used to distinguish between arrestin-dependent and -independent effects. Arrestin did not promote internalization or desensitization of a receptor that had key Ser/Thr phosphosites mutated to Ala (4Ala receptor). Nevertheless, arrestin greatly increased acid/salt resistance and the affinity of 4Ala receptor for TRH. Truncation of 4Ala receptor just distal to the key phosphosites (4AlaStop receptor) abolished arrestin-dependent acid/salt resistance but not the effect of arrestin on agonist affinity. Arrestin formed stable complexes with activated wild-type and 4Ala receptors but not with 4AlaStop receptor, as measured by translocation of arrestin-green fluorescent protein to the plasma membrane or chemical cross-linking. An arrestin mutant that does not interact with clathrin and AP2 did not internalize receptor but still promoted high affinity TRH binding, acid/salt resistance, and desensitization. A sterically restricted arrestin mutant did not cause receptor internalization or desensitization but did promote acid/salt resistance and high agonist affinity. The results demonstrate that arrestin binds to proximal or distal phosphosites in the receptor tail. Arrestin binding at either site causes increased agonist affinity and acid/salt resistance, but only the proximal phosphosites evoke the necessary conformational changes in arrestin for receptor desensitization and internalization.

Glycine Hinges with Opposing Actions at the Acetylcholine Receptor-Channel Transmitter Binding SiteS⃞

PubMed Central

Purohit, Prasad

2011-01-01

The extent to which agonists activate synaptic receptor-channels depends on both the intrinsic tendency of the unliganded receptor to open and the amount of agonist binding energy realized in the channel-opening process. We examined mutations of the nicotinic acetylcholine receptor transmitter binding site (α subunit loop B) with regard to both of these parameters. αGly147 is an “activation” hinge where backbone flexibility maintains high values for intrinsic gating, the affinity of the resting conformation for agonists and net ligand binding energy. αGly153 is a “deactivation” hinge that maintains low values for these parameters. αTrp149 (between these two glycines) serves mainly to provide ligand binding energy for gating. We propose that a concerted motion of the two glycine hinges (plus other structural elements at the binding site) positions αTrp149 so that it provides physiologically optimal binding and gating function at the nerve-muscle synapse. PMID:21115636

The DNA replication licensing factor miniature chromosome maintenance 7 is essential for RNA splicing of epidermal growth factor receptor, c-Met, and platelet-derived growth factor receptor.

PubMed

Chen, Zhang-Hui; Yu, Yan P; Michalopoulos, George; Nelson, Joel; Luo, Jian-Hua

2015-01-16

Miniature chromosome maintenance 7 (MCM7) is an essential component of DNA replication licensing complex. Recent studies indicate that MCM7 is amplified and overexpressed in a variety of human malignancies. In this report, we show that MCM7 binds SF3B3. The binding motif is located in the N terminus (amino acids 221-248) of MCM7. Knockdown of MCM7 or SF3B3 significantly increased unspliced RNA of epidermal growth factor receptor, platelet-derived growth factor receptor, and c-Met. A dramatic drop of reporter gene expression of the oxytocin exon 1-intron-exon 2-EGFP construct was also identified in SF3B3 and MCM7 knockdown PC3 and DU145 cells. The MCM7 or SF3B3 depleted cell extract failed to splice reporter RNA in in vitro RNA splicing analyses. Knockdown of SF3B3 and MCM7 leads to an increase of cell death of both PC3 and DU145 cells. Such cell death induction is partially rescued by expressing spliced c-Met. To our knowledge, this is the first report suggesting that MCM7 is a critical RNA splicing factor, thus giving significant new insight into the oncogenic activity of this protein. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

PubMed

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

2012-12-04

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins

PubMed Central

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M.; Zakarian, Armen; Molgó, Jordi

2014-01-01

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility. PMID:23131021

Ondansetron and granisetron binding orientation in the 5-HT(3) receptor determined by unnatural amino acid mutagenesis.

PubMed

Duffy, Noah H; Lester, Henry A; Dougherty, Dennis A

2012-10-19

The serotonin type 3 receptor (5-HT(3)R) is a ligand-gated ion channel found in the central and peripheral nervous systems. The 5-HT(3)R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT(3)A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket.

Expression of serum insulin-like growth factors, insulin-like growth factor-binding proteins, and the growth hormone-binding protein in heterozygote relatives of Ecuadorian growth hormone receptor deficient patients.

PubMed

Fielder, P J; Guevara-Aguirre, J; Rosenbloom, A L; Carlsson, L; Hintz, R L; Rosenfeld, R G

1992-04-01

Recently, an isolated population of apparent GH-receptor deficient (GHRD) patients has been identified in the Loja province of southern Ecuador. These individuals presented many of the physical and biochemical phenotypes characteristic of Laron-Syndrome and are believed to have a defect in the GH-receptor gene. In this study, we have compared the biochemical phenotypes between the affected individuals and their parents, considered to be obligate heterozygotes for the disorder. Serum GH, insulin-like growth factor I and II (IGF-I and IGF-II) levels were measured by RIA Insulin-like growth factor binding proteins. (IGFBPs) were measured by Western ligand blotting (WLB) of serum samples, following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and relative quantitation of serum IGFBPs was performed with a scanning laser densitometer. Serum GH-binding protein (GHBP) levels were measured with a ligand-mediated immunofunctional assay using a monoclonal antibody raised against the GHBP. These values were then compared to values obtained from normal, sex-matched adult Ecuadorian controls, to determine if the above parameters were abnormal in the heterozygotes. The serum IGF-I levels of the GHRD patients were less than 13% of control values for adults and 2% for children. However, the IGF-I levels of both the mothers and fathers were not significantly different from that of the control population. The serum IGF-II levels of the GHRD patients were approximately 20% of control values for adults and 12% for the children. The IGF-II levels of the mothers were reduced, but were not significantly different from that of the control population. However, IGF-II levels of the fathers were significantly lower than those of controls (64% of control male levels). WLB analysis of serum IGFBP levels of the affected subjects demonstrated increased IGFBP-2 and decreased IGFBP-3, suggesting an inverse relationship between these IGFBPs. The GHRD patients who had the

Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.

PubMed

Schrevel, Marlies; Osse, E Michelle; Prins, Frans A; Trimbos, J Baptist M Z; Fleuren, Gert Jan; Gorter, Arko; Jordanova, Ekaterina S

2017-06-01

In cervical cancer, the epidermal growth factor receptor (EGFR) is overexpressed in 70-90% of the cases and has been associated with poor prognosis. EGFR-based therapy is currently being explored in cervical cancer. We investigated which EGFR ligand is primarily expressed in cervical cancer and which cell type functions as the major source of this ligand. We hypothesized that macrophages are the main source of EGFR ligands and that a paracrine loop between tumor cells and macrophages is responsible for ligand expression. mRNA expression analysis was performed on 32 cervical cancer cases to determine the expression of the EGFR ligands amphiregulin, β-cellulin, epidermal growth factor (EGF), epiregulin, heparin-binding EGF-like growth factor (HB‑EGF) and transforming growth factor α (TGFα). Subsequently, protein expression was determined immunohistochemically on 36 additional cases. To assess whether macrophages are the major source of EGFR ligands, immunohistochemical double staining was performed on four representative tissue slides. Expression of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif ligand 2 (CCL2) was determined by mRNA in situ hybridization. Of the known EGFR ligands, HB‑EGF had the highest mRNA expression and HB‑EGF and EGFR protein expression were highly correlated. Tumor specimens with high EGFR expression showed higher numbers of macrophages, and higher expression of GM-CSF and CCL2, but only a small subset (9%) of macrophages was found to be HB‑EGF-positive. Strikingly, 78% of cervical cancer specimens were found to express HB‑EGF. Standardized assessment of staining intensity, using spectral imaging analysis, showed that HB‑EGF expression was higher in the tumor compartment than in the stromal compartment. These results suggest that HB‑EGF is an important EGFR ligand in cervical cancer and that cervical cancer cells are the predominant source of HB‑EGF. Therefore, we propose an autocrine

Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

NASA Technical Reports Server (NTRS)

Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

1999-01-01

Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

Reversibly bound chloride in the atrial natriuretic peptide receptor hormone-binding domain: possible allosteric regulation and a conserved structural motif for the chloride-binding site.

PubMed

Ogawa, Haruo; Qiu, Yue; Philo, John S; Arakawa, Tsutomu; Ogata, Craig M; Misono, Kunio S

2010-03-01

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(-)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(-) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(-) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis.

Reversibly Bound Chloride in the Atrial Natriuretic Peptide Receptor Hormone Binding Domain: Possible Allosteric Regulation and a Conserved Structural Motif for the Chloride-binding Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogawa, H.; Qiu, Y; Philo, J

2010-01-01

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(-)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. Amore » new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(-) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(-) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis.« less

Reversibly bound chloride in the atrial natriuretic peptide receptor hormone-binding domain: Possible allosteric regulation and a conserved structural motif for the chloride-binding site

PubMed Central

Ogawa, Haruo; Qiu, Yue; Philo, John S; Arakawa, Tsutomu; Ogata, Craig M; Misono, Kunio S

2010-01-01

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(−)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(−) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(−) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis. PMID:20066666

Role of G protein-coupled receptors (GPCR), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), heparin-binding epidermal growth factor-like growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) in trenbolone acetate-stimulated bovine satellite cell proliferation.

PubMed

Thornton, K J; Kamange-Sollo, E; White, M E; Dayton, W R

2015-09-01

Implanting cattle with steroids significantly enhances feed efficiency, rate of gain, and muscle growth. However, the mechanisms responsible for these improvements in muscle growth have not been fully elucidated. Trenbolone acetate (TBA), a testosterone analog, has been shown to increase proliferation rate in bovine satellite cell (BSC) cultures. The classical genomic actions of testosterone have been well characterized; however, our results indicate that TBA may also initiate a quicker, nongenomic response that involves activation of G protein-coupled receptors (GPCR) resulting in activation of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) that release membrane-bound heparin-binding epidermal growth factor-like growth factor (hbEGF), which then binds to and activates the epidermal growth factor receptor (EGFR) and/or erbB2. Furthermore, the EGFR has been shown to regulate expression of the IGF-1 receptor (IGF-1R), which is well known for its role in modulating muscle growth. To determine whether this nongenomic pathway is potentially involved in TBA-stimulated BSC proliferation, we analyzed the effects of treating BSC with guanosine 5'-O-2-thiodiphosphate (GDPβS), an inhibitor of all GPCR; a MMP2 and MMP9 inhibitor (MMPI); CRM19, a specific inhibitor of hbEGF; AG1478, a specific EGFR tyrosine kinase inhibitor; AG879, a specific erbB2 kinase inhibitor; and AG1024, an IGF-1R tyrosine kinase inhibitor on TBA-stimulated proliferation rate (H-thymidine incorporation). Assays were replicated at least 9 times for each inhibitor experiment using BSC cultures obtained from at least 3 different animals. Bovine satellite cell cultures were obtained from yearling steers that had no previous exposure to androgenic or estrogenic compounds. As expected, BSC cultures treated with 10 n TBA showed ( < 0.05) increased proliferation rate when compared with control cultures. Additionally, treatment with 5 ng hbEGF/mL stimulated proliferation in BSC cultures ( < 0.05). Treatment

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.

PubMed

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-05-01

The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor. The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X(7) receptors. Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. These data demonstrate that human P2X(7) receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.

Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

PubMed

Kim, Ji-Hun; Sim, Dae-Won; Park, Dongsun; Jung, Tai-Geun; Lee, Seonghwan; Oh, Taeheun; Ha, Jong-Ryul; Seok, Seung-Hyeon; Seo, Min-Duk; Kang, Ho Chul; Kim, Young Pil; Won, Hyung-Sik

2016-12-01

Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Hua; Chen, Li-Mei; Carney, Paul J.

2012-02-21

Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avianmore » receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type ({alpha}2-3) receptor binding profile, with only moderate binding to human-type ({alpha}2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.« less

Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation

PubMed Central

Koland, John G.

2014-01-01

Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR

The effects of abnormalities of glucose homeostasis on the expression and binding of muscarinic receptors in cerebral cortex of rats.

PubMed

Sherin, Antony; Peeyush, Kumar T; Naijil, George; Nandhu, Mohan Sobhana; Jayanarayanan, Sadanandan; Jes, Paul; Paulose, Cheramadathikudiyil Skaria

2011-01-25

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Both hypo and hyperglycemia contributes to neuronal functional deficit. In the present study, effect of insulin induced hypoglycemia and streptozotocin induced diabetes on muscarinic receptor binding, cholinergic enzymes; AChE, ChAT expression and GLUT3 in the cerebral cortex of experimental rats were analysed. Total muscarinic, muscarinic M(1) receptor showed a significant decrease and muscarinic M(3) receptor subtype showed a significant increased binding in the cerebral cortex of hypoglycemic rats compared to diabetic and control. Real-Time PCR analysis of muscarinic M(1), M(3) receptor subtypes confirmed the receptor binding studies. Immunohistochemistry of muscarinic M(1), M(3) receptors using specific antibodies were also carried out. AChE and GLUT3 expression up regulated and ChAT expression down regulated in hypoglycemic rats compared to diabetic and control rats. Our results showed that hypo/hyperglycemia caused impaired glucose transport in neuronal cells as shown by altered expression of GLUT3. Increased AChE and decreased ChAT expression is suggested to alter cortical acetylcholine metabolism in experimental rats along with altered muscarinic receptor binding in hypo/hyperglycemic rats, impair cholinergic transmission, which subsequently lead to cholinergic dysfunction thereby causing learning and memory deficits. We observed a prominent cholinergic functional disturbance in hypoglycemic condition than in hyperglycemia. Hypoglycemia exacerbated the neurochemical changes in cerebral cortex induced by hyperglycemia. These findings have implications for both therapy and identification of causes contributing to neuronal dysfunction in diabetes. Copyright © 2010 Elsevier B.V. All rights reserved.

NMR spectroscopy of the ligand binding core of ionotropic glutamate receptor 2 bound to 5-substituted willardiine partial agonists

PubMed Central

Fenwick, Michael K.; Oswald, Robert E.

2008-01-01

Glutamate receptors mediate neuronal intercommunication in the central nervous system by coupling extracellular neurotransmitter-receptor interactions to ion channel conductivity. To gain insight into structural and dynamical factors that underlie this coupling, solution NMR experiments were performed on the bi-lobed ligand-binding core of glutamate receptor 2 in complexes with a set of willardiine partial agonists. These agonists are valuable for studying structure-function relationships because their 5-position substituent size is correlated with ligand efficacy and extent of receptor desensitization whereas the substituent electronegativity is correlated with ligand potency. NMR results show that the protein backbone amide chemical shift deviations correlate mainly with efficacy and extent of desensitization. Pronounced deviations occur at specific residues in the ligand-binding site and in the two helical segments that join the lobes by a disulfide bond. Experiments detecting conformational exchange show that micro- to millisecond timescale motions also occur near the disulfide bond and vary largely with efficacy and extent of desensitization. These results thus identify regions displaying structural and dynamical dissimilarity arising from differences in ligand-protein interactions and lobe closure which may play a critical role in receptor response. Furthermore, measures of line broadening and conformational exchange for a portion of the ligand-binding site correlate with ligand EC50 data. These results do not have any correlate in the currently available crystal structures and thus provide a novel view of ligand-binding events that may be associated with agonist potency differences. PMID:18387631

Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism).

PubMed

Daughaday, W H; Trivedi, B

1987-07-01

It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, we have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of 125I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Nonspecific binding was determined when 125I-hGH was incubated with serum in the presence of an excess of GH. Results are expressed as percent of specifically bound 125I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. We suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor.

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

PubMed Central

Reedijk, M; Liu, X; van der Geer, P; Letwin, K; Waterfield, M D; Hunter, T; Pawson, T

1992-01-01

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha. Images PMID:1314163

Binding constant of cell adhesion receptors and substrate-immobilized ligands depends on the distribution of ligands

NASA Astrophysics Data System (ADS)

Li, Long; Hu, Jinglei; Xu, Guangkui; Song, Fan

2018-01-01

Cell-cell adhesion and the adhesion of cells to tissues and extracellular matrix, which are pivotal for immune response, tissue development, and cell locomotion, depend sensitively on the binding constant of receptor and ligand molecules anchored on the apposing surfaces. An important question remains of whether the immobilization of ligands affects the affinity of binding with cell adhesion receptors. We have investigated the adhesion of multicomponent membranes to a flat substrate coated with immobile ligands using Monte Carlo simulations of a statistical mesoscopic model with biologically relevant parameters. We find that the binding of the adhesion receptors to ligands immobilized on the substrate is strongly affected by the ligand distribution. In the case of ligand clusters, the receptor-ligand binding constant can be significantly enhanced due to the less translational entropy loss of lipid-raft domains in the model cell membranes upon the formation of additional complexes. For ligands randomly or uniformly immobilized on the substrate, the binding constant is rather decreased since the receptors localized in lipid-raft domains have to pay an energetic penalty in order to bind ligands. Our findings help to understand why cell-substrate adhesion experiments for measuring the impact of lipid rafts on the receptor-ligand interactions led to contradictory results.

1-Methyl-beta-carboline (harmane), a potent endogenous inhibitor of benzodiazepine receptor binding.

PubMed

Rommelspacher, H; Nanz, C; Borbe, H O; Fehske, K J; Müller, W E; Wollert, U

1980-10-01

The interaction of several beta-carbolines with specific [3H]-flunitrazepam binding to benzodiazepine receptors in rat brain membranes was investigated. Out of the investigated compounds, harmane and norharmane were the most potent inhibitors of specific [3H]-flunitrazepam binding, with IC50-values in the micromolar range. All other derivatives, including harmine, harmaline, and several tetrahydroderivatives were at least ten times less potent. Harmane has been previously found in rat brain and human urine, so it is the most potent endogenous inhibitor of specific [3H]-flunitrazepam binding known so far, with a several fold higher affinity for the benzodiazepine receptor than inosine and hypoxanthine. Thus, we suggest that harmane or other related beta-carbolines could be potential candidates as endogenous ligands of the benzodiazepine receptor.

Ondansetron and Granisetron Binding Orientation in the 5-HT3 Receptor Determined by Unnatural Amino Acid Mutagenesis

PubMed Central

Duffy, Noah H.; Lester, Henry A.; Dougherty, Dennis A.

2012-01-01

The serotonin type 3 receptor (5-HT3R) is a ligand-gated ion channel that mediates fast synaptic transmission in the central and peripheral nervous systems. The 5-HT3R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT3A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket. PMID:22873819

The effects of pargyline and 2-phenylethylamine on D1-like dopamine receptor binding.

PubMed

Berry, Mark D

2011-07-01

2-Phenylethylamine (PE) potentiates neuronal responses to dopamine by an unknown post-synaptic mechanism. Here, whether PE modifies D1-like receptor binding was examined. An unexpected effect of the monoamine oxidase inhibitor pargyline was observed, which did not involve competition for ligand binding. PE did not affect ligand binding in the presence or absence of pargyline. It is concluded that the effect of pargyline does not involve elevation of endogenous PE, and PE effects on dopaminergic neurotransmission are not due to altered D1-like receptor binding.

Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

PubMed Central

Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

1992-01-01

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

Activation of erythropoietin receptor in the absence of hormone by a peptide that binds to a domain different from the hormone binding site

PubMed Central

Naranda, Tatjana; Wong, Kenneth; Kaufman, R. Ilene; Goldstein, Avram; Olsson, Lennart

1999-01-01

Applying a homology search method previously described, we identified a sequence in the extracellular dimerization site of the erythropoietin receptor, distant from the hormone binding site. A peptide identical to that sequence was synthesized. Remarkably, it activated receptor signaling in the absence of erythropoietin. Neither the peptide nor the hormone altered the affinity of the other for the receptor; thus, the peptide does not bind to the hormone binding site. The combined activation of signal transduction by hormone and peptide was strongly synergistic. In mice, the peptide acted like the hormone, protecting against the decrease in hematocrit caused by carboplatin. PMID:10377456

Gab-family adapter molecules in signal transduction of cytokine and growth factor receptors, and T and B cell antigen receptors.

PubMed

Hibi, M; Hirano, T

2000-04-01

Gab1 and Gab2 (Grb2 associated binder 1 and 2) are scaffolding adapter molecules that display sequence similarity with Drosophila DOS (daughter of sevenless), which is a potential substrate for the protein tyrosine phosphatase, Corkscrew, Both Gab1 and Gab2, like DOS, have a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab1 and Gab2 are phosphorylated on tyrosine upon the stimulation of various cytokines, growth factors, and antigen receptors, and interact with signaling molecules, such as Grb2, SHP-2, and PI-3 kinase. Overexpression of Gab1 or Gab2 mimics or enhances growth factor or cytokine-mediated biological processes and activates ERK MAP kinase. These data imply that Gab1 and Gab2 act downstream of a broad range of cytokine and growth factor receptors, as well as T and B antigen receptors, and link these receptors to ERK MAP kinase and biological actions.

Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding.

PubMed

Churn, Severn B; Rana, Aniruddha; Lee, Kangmin; Parsons, J Travis; De Blas, Angel; Delorenzo, Robert J

2002-09-01

gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.

Structure and Receptor Binding of the Hemagglutinin from a Human H6N1 Influenza Virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tzarum, Netanel; de Vries, Robert P.; Zhu, Xueyong

Avian influenza viruses that cause infection and are transmissible in humans involve changes in the receptor binding site (RBS) of the viral hemagglutinin (HA) that alter receptor preference from α2-3-linked (avian-like) to α2-6-linked (human-like) sialosides. A human case of avian-origin H6N1 influenza virus was recently reported, but the molecular mechanisms contributing to it crossing the species barrier are unknown. We find that, although the H6 HA RBS contains D190V and G228S substitutions that potentially promote human receptor binding, recombinant H6 HA preferentially binds α2-3-linked sialosides, indicating no adaptation to human receptors. Crystal structures of H6 HA with avian and humanmore » receptor analogs reveal that H6 HA preferentially interacts with avian receptor analogs. Lastly, this binding mechanism differs from other HA subtypes due to a unique combination of RBS residues, highlighting additional variation in HA-receptor interactions and the challenges in predicting which influenza strains and subtypes can infect humans and cause pandemics.« less

Structure and Receptor Binding of the Hemagglutinin from a Human H6N1 Influenza Virus

DOE PAGES

Tzarum, Netanel; de Vries, Robert P.; Zhu, Xueyong; ...

2015-03-11

Avian influenza viruses that cause infection and are transmissible in humans involve changes in the receptor binding site (RBS) of the viral hemagglutinin (HA) that alter receptor preference from α2-3-linked (avian-like) to α2-6-linked (human-like) sialosides. A human case of avian-origin H6N1 influenza virus was recently reported, but the molecular mechanisms contributing to it crossing the species barrier are unknown. We find that, although the H6 HA RBS contains D190V and G228S substitutions that potentially promote human receptor binding, recombinant H6 HA preferentially binds α2-3-linked sialosides, indicating no adaptation to human receptors. Crystal structures of H6 HA with avian and humanmore » receptor analogs reveal that H6 HA preferentially interacts with avian receptor analogs. Lastly, this binding mechanism differs from other HA subtypes due to a unique combination of RBS residues, highlighting additional variation in HA-receptor interactions and the challenges in predicting which influenza strains and subtypes can infect humans and cause pandemics.« less

Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling

PubMed Central

Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

2017-01-01

Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr52, which then promoted the dephosphorylation of CAR at Thr38 by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR. PMID:23652203

Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

PubMed

Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

2013-05-07

Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

One amino acid in mouse activated factor VII defines its endothelial protein C receptor (EPCR) binding and modulates its EPCR-dependent hemostatic activity in vivo.

PubMed

Pavani, G; Zintner, S M; Ivanciu, L; Small, J C; Stafford, K A; Szeto, J H; Margaritis, P

2017-03-01

Essentials The lack of factor (F) VIIa-endothelial protein C receptor (EPCR) binding in mice is unresolved. A single substitution of Leu4 to Phe in mouse FVIIa (mFVIIa) enables its interaction with EPCR. mFVIIa with a Phe4 shows EPCR binding-dependent enhanced hemostatic function in vivo vs. mFVIIa. Defining the FVIIa-EPCR interaction in mice allows for further investigating its biology in vivo. Background Human activated factor VII (hFVIIa), which is used in hemophilia treatment, binds to the endothelial protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the activated FVII (FVIIa)-EPCR interaction. Therefore, to investigate the hemostatic consequences of this interaction in hemophilia, we previously engineered a mouse FVIIa (mFVIIa) molecule that bound mouse EPCR (mEPCR) by using three substitutions from mouse PC (mPC), i.e. Leu4→Phe, Leu8→Met, and Trp9→Arg. The resulting molecule, mFVIIa-FMR, modeled the EPCR-binding properties of hFVIIa and showed enhanced hemostatic capacity in hemophilic mice versus mFVIIa. These data implied a role of EPCR in the action of hFVIIa in hemophilia treatment. However, the substitutions in mFVIIa-FMR only broadly defined the sequence determinants for its mEPCR interaction and enhanced function in vivo. Objectives To determine the individual contributions of mPC Phe4, Met8 and Arg9 to the in vitro/in vivo properties of mFVIIa-FMR. Methods The mEPCR-binding properties of single amino acid variants of mFVIIa or mPC at position 4, 8 or 9 were investigated. Results and conclusions Phe4 in mFVIIa or mPC was solely critical for interaction with mEPCR. In hemophilic mice, administration of mFVIIa harboring a Phe4 resulted in a 1.9-2.5-fold increased hemostatic capacity versus mFVIIa that was EPCR binding-dependent. This recapitulated previous observations made with triple-mutant mFVIIa-FMR. As Leu8 is crucial for hFVIIa-EPCR binding, we describe the sequence divergence of this interaction in

Molecular mechanism of ATP binding and ion channel activation in P2X receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hattori, Motoyuki; Gouaux, Eric

P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure ofmore » the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.« less

Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies.

PubMed

Wu, Qing-Ping; Zhang, Lei; Shao, Xiao-Xia; Wang, Jia-Hui; Gao, Yu; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

2016-04-01

Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-receptor binding assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1.

In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles.

PubMed Central

Beltzer, J P; Spiess, M

1991-01-01

The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitrocellulose. The receptor was found to interact with proteins of approximately 100 kDa. The cytoplasmic domain of the ASGP receptor subunit H1 fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the beta adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the beta adaptin contains the binding site for receptors. Images PMID:1935897

Binding, Thermodynamics, and Selectivity of a Non-peptide Antagonist to the Melanocortin-4 Receptor

PubMed Central

Saleh, Noureldin; Kleinau, Gunnar; Heyder, Nicolas; Clark, Timothy; Hildebrand, Peter W.; Scheerer, Patrick

2018-01-01

The melanocortin-4 receptor (MC4R) is a potential drug target for treatment of obesity, anxiety, depression, and sexual dysfunction. Crystal structures for MC4R are not yet available, which has hindered successful structure-based drug design. Using microsecond-scale molecular-dynamics simulations, we have investigated selective binding of the non-peptide antagonist MCL0129 to a homology model of human MC4R (hMC4R). This approach revealed that, at the end of a multi-step binding process, MCL0129 spontaneously adopts a binding mode in which it blocks the agonistic-binding site. This binding mode was confirmed in subsequent metadynamics simulations, which gave an affinity for human hMC4R that matches the experimentally determined value. Extending our simulations of MCL0129 binding to hMC1R and hMC3R, we find that receptor subtype selectivity for hMC4R depends on few amino acids located in various structural elements of the receptor. These insights may support rational drug design targeting the melanocortin systems.

Characterization of the Binding Site of Aspartame in the Human Sweet Taste Receptor

PubMed Central

Maillet, Emeline L.; Cui, Meng; Jiang, Peihua; Mezei, Mihaly; Hecht, Elizabeth; Quijada, Jeniffer; Osman, Roman; Max, Marianna

2015-01-01

The sweet taste receptor, a heterodimeric G protein-coupled receptor comprised of T1R2 and T1R3, binds sugars, small molecule sweeteners, and sweet proteins to multiple binding sites. The dipeptide sweetener, aspartame binds in the Venus Flytrap Module (VFTM) of T1R2. We developed homology models of the open and closed forms of human T1R2 and human T1R3 VFTMs and their dimers and then docked aspartame into the closed form of T1R2’s VFTM. To test and refine the predictions of our model, we mutated various T1R2 VFTM residues, assayed activity of the mutants and identified 11 critical residues (S40, Y103, D142, S144, S165, S168, Y215, D278, E302, D307, and R383) in and proximal to the binding pocket of the sweet taste receptor that are important for ligand recognition and activity of aspartame. Furthermore, we propose that binding is dependent on 2 water molecules situated in the ligand pocket that bridge 2 carbonyl groups of aspartame to residues D142 and L279. These results shed light on the activation mechanism and how signal transmission arising from the extracellular domain of the T1R2 monomer of the sweet receptor leads to the perception of sweet taste. PMID:26377607

Modulation of GABAergic receptor binding by activation of calcium and calmodulin-dependent kinase II membrane phosphorylation.

PubMed

Churn, S B; DeLorenzo, R J

1998-10-26

gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Because of the important role that GABA plays in the CNS, alteration of GABAA receptor function would significantly affect neuronal excitability. Protein phosphorylation is a major mechanism for regulating receptor function in the brain and has been implicated in modulating GABAA receptor function. Therefore, this study was initiated to determine the role of calmodulin-dependent kinase II (CaM kinase II) membrane phosphorylation on GABAA receptor binding. Synaptosomal membrane fractions were tested for CaM kinase II activity towards endogenous substrates. In addition, muscimol binding was evaluated under equilibrium conditions in synaptosomal membrane fractions subjected to either basal (Mg2+ alone) or maximal CaM kinase II-dependent phosphorylation. Activation of endogenous CaM kinase II-dependent phosphorylation resulted in a significant enhancement of the apparent Bmax for muscimol binding without significantly altering the apparent binding affinity. The enhanced muscimol binding could be increased further by the addition of exogenous CaM kinase II to synaptosomal membrane fractions. Co-incubation with inhibitors of kinase activity during the phosphorylation reactions blocked the CaM kinase II-dependent increase in muscimol binding. The data support the hypothesis that activation of CaM kinase II-dependent phosphorylation caused an increased GABAA receptor binding and may play an important role in modulating the function of this inhibitory receptor/chloride ion channel complex. Copyright 1998 Elsevier Science B.V.

THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS

EPA Science Inventory

THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS. JG Teeguarden1 and HA Barton2. 1ENVIRON International, Ruston LA; 2US EPA, ORD, NHEERL, ETD, Pharmacokinetics Branch, RTP, NC.

One measure of th...

Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism).

PubMed Central

Daughaday, W H; Trivedi, B

1987-01-01

It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, we have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of 125I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Nonspecific binding was determined when 125I-hGH was incubated with serum in the presence of an excess of GH. Results are expressed as percent of specifically bound 125I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. We suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor. PMID:3474620

Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors

PubMed Central

1984-01-01

Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites. PMID:6088662

Substitution of synthetic chimpanzee androgen receptor for human androgen receptor in competitive binding and transcriptional activation assays for EDC screening

EPA Science Inventory

The potential effect of receptor-mediated endocrine modulators across species is of increasing concern. In attempts to address these concerns we are developing androgen and estrogen receptor binding assays using recombinant hormone receptors from a number of species across differ...

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding

PubMed Central

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-01-01

Background and Purpose: The P2X7 receptor exhibits complex pharmacological properties. In this study, binding of a [3H]-labelled P2X7 receptor antagonist to human P2X7 receptors has been examined to further understand ligand interactions with this receptor. Experimental Approach: The P2X7 receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X7 receptors. Key Results: Binding of [3H]-compound-17 was higher in membranes prepared from cells expressing P2X7 receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X7 receptors. Binding was reversible, saturable and modulated by P2X7 receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. Conclusions: These data demonstrate that human P2X7 receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X7 receptor complex enhances subsequent binding to other P2X7 subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X7 receptor. PMID:17339830

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

PubMed Central

Lou, Meizhen; Garrett, Thomas P. J.; McKern, Neil M.; Hoyne, Peter A.; Epa, V. Chandana; Bentley, John D.; Lovrecz, George O.; Cosgrove, Leah J.; Frenkel, Maurice J.; Ward, Colin W.

2006-01-01

The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1–CR–L2) of human IR at 2.3 Å resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) β-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more α-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R. PMID:16894147

Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

PubMed Central

Jenkins, Jeremy L; Dean, Donald H

2001-01-01

Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm) aminopeptidase N (APN) and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM), and unusually tight binding to the cadherin-like receptor (2.6 nM), which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac) was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research. PMID:11722800

Novel Yeast-based Strategy Unveils Antagonist Binding Regions on the Nuclear Xenobiotic Receptor PXR*

PubMed Central

Li, Hao; Redinbo, Matthew R.; Venkatesh, Madhukumar; Ekins, Sean; Chaudhry, Anik; Bloch, Nicolin; Negassa, Abdissa; Mukherjee, Paromita; Kalpana, Ganjam; Mani, Sridhar

2013-01-01

The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple “gain-of-function” mutants that were “resistant” to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications. PMID:23525103

SH3 domain-mediated binding of the Drk protein to Dos is an important step in signaling of Drosophila receptor tyrosine kinases.

PubMed

Feller, Stephan M; Wecklein, Heike; Lewitzky, Marc; Kibler, Eike; Raabe, Thomas

2002-08-01

Activation of the Sevenless (Sev) receptor tyrosine kinase (RTK) in the developing Drosophila eye is required for the specification of the R7 photoreceptor cell fate. Daughter of Sevenless (Dos), a putative multi-site adaptor protein, is a substrate of the Sev kinase and is known to associate with the tyrosine phosphatase Corkscrew (Csw). Binding of Csw to Dos depends on the Csw Src homology 2 (SH2) domains and is an essential step for signaling by the Sev RTK. Dos, however, lacks a recognizable phosphotyrosine interaction domain and it was previously unclear how it is recruited to the Sev receptor. Here it is shown that the SH2/SH3 domain adaptor protein Drk can provide this link. Drk binds with its SH2 domain to the autophosphorylated Sev receptor while the C-terminal SH3 domain is able to associate with Dos. The Drk SH3 domain binding motifs on Dos were mapped to two sites which do not conform the known Drk SH3 domain binding motif (PxxPxR) but instead have the consensus PxxxRxxKP. Mutational analysis in vitro and in vivo provided evidence that both Drk binding sites fulfil an important function in the context of Sev and Drosophila epidermal growth factor receptor mediated signaling processes.

Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS.

PubMed

Lee, Sang-Min; Hay, Debbie L; Pioszak, Augen A

2016-04-15

Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists.

PubMed

Mochizuki, Michiyo; Kori, Masakuni; Kobayashi, Katsumi; Yano, Takahiko; Sako, Yuu; Tanaka, Maiko; Kanzaki, Naoyuki; Gyorkos, Albert C; Corrette, Christopher P; Cho, Suk Young; Pratt, Scott A; Aso, Kazuyoshi

2016-03-24

Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 μM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 μmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.

Estrogen Receptor Binding Affinity of Food Contact Material Components Estimated by QSAR.

PubMed

Sosnovcová, Jitka; Rucki, Marián; Bendová, Hana

2016-09-01

The presented work characterized components of food contact materials (FCM) with potential to bind to estrogen receptor (ER) and cause adverse effects in the human organism. The QSAR Toolbox, software application designed to identify and fill toxicological data gaps for chemical hazard assessment, was used. Estrogen receptors are much less of a lock-and-key interaction than highly specific ones. The ER is nonspecific enough to permit binding with a diverse array of chemical structures. There are three primary ER binding subpockets, each with different requirements for hydrogen bonding. More than 900 compounds approved as of FCM components were evaluated for their potential to bind on ER. All evaluated chemicals were subcategorized to five groups with respect to the binding potential to ER: very strong, strong, moderate, weak binder, and no binder to ER. In total 46 compounds were characterized as potential disturbers of estrogen receptor. Among the group of selected chemicals, compounds with high and even very high affinity to the ER binding subpockets were found. These compounds may act as gene activators and cause adverse effects in the organism, particularly during pregnancy and breast-feeding. It should be considered to carry out further in vitro or in vivo tests to confirm their potential to disturb the regulation of physiological processes in humans by abnormal ER signaling and subsequently remove these chemicals from the list of approved food contact materials. Copyright© by the National Institute of Public Health, Prague 2016

RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

EPA Science Inventory

Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay
Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. Wilson
U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

Time course of the estradiol-dependent induction of oxytocin receptor binding in the ventromedial hypothalamic nucleus of the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, A.E.; Ball, G.F.; Coirini, H.

1989-09-01

Oxytocin (OT) transmission is involved in the steroid-dependent display of sexual receptivity in rats. One of the biochemical processes stimulated by the ovarian steroid 17 beta-estradiol (E2) that is relevant to reproduction is the induction of OT receptor binding in the ventromedial hypothalamic nucleus (VMN). The purpose of these experiments was to determine if E2-induced changes in OT receptor binding in the VMN occur within a time frame relevant to cyclic changes in ovarian steroid secretion. OT receptor binding was measured in the VMN of ovariectomized rats implanted for 0-96 h with E2-containing Silastic capsules. The rate of decay ofmore » OT receptor binding was measured in another group of animals 6-48 h after capsule removal. Receptors were labeled with the specific OT receptor antagonist ({sup 125}I)d(CH2)5(Tyr(Me)2,Thr4,Tyr-NH2(9))OVT, and binding was measured with quantitative autoradiographic methods. In addition, plasma E2 levels and uterine weights were assessed in animals from each treatment condition. Significant increases in E2-dependent OT receptor binding and uterine weight occurred within 24 h of steroid treatment. After E2 withdrawal, OT receptor binding and uterine weight decreased significantly within 24 h. These results are consistent with the hypothesis that steroid modulation of OT receptor binding is necessary for the induction of sexual receptivity.« less

Differential effects of chronic lorazepam and alprazolam on benzodiazepine binding and GABAA-receptor function.

PubMed Central

Galpern, W. R.; Miller, L. G.; Greenblatt, D. J.; Shader, R. I.

1990-01-01

1. Chronic benzodiazepine administration has been associated with tolerance and with downregulation of gamma-aminobutyric acidA (GABAA)-receptor binding and function. However, effects of individual benzodiazepines on brain regions have varied. 2. To compare the effects of chronic lorazepam and alprazolam, we have administered these drugs to mice for 1 and 7 days (2 mg kg-1 day-1) and determined benzodiazepine receptor binding in vivo with and without administration of CL 218,872, 25 mg kg-1 i.p., and GABA-dependent chloride uptake in 3 brain regions at these time points. 3. Benzodiazepine binding was decreased in the cortex and hippocampus at day 7 compared to day 1 of lorazepam, with an increase in CL 218,872-resistant (Type 2) sites in both regions. Maximal GABA-dependent chloride uptake was also decreased in the cortex and hippocampus at day 7. 4. Binding was decreased only in the cortex after 7 days of alprazolam, with no significant change in Type 2 binding. Maximal GABA-dependent chloride uptake was also decreased only in the cortex. 5. These data suggest that the effects of chronic benzodiazepine administration on the GABAA-receptor may be both region-specific and receptor subtype-specific. PMID:1964820

Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ritvos, O.; Ranta, T.; Jalkanen, J.

1988-05-01

The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purifiedmore » 34 K IGF-BP specifically bound (125I)iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of (125I) iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of (125I) iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with (125I)iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-(3H)aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells.« less

GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

PubMed Central

Yasumoto, Hiroaki; Meng, Lingjun; Lin, Tao; Zhu, Qubo; Tsai, Robert Y.L.

2010-01-01

Summary Guanine-nucleotide binding protein 3-like (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1p-null yeasts. To determine the unique function of GNL3L, we identified estrogen receptor-related protein-γ (ERRγ) as a GNL3L-specific binding protein. GNL3L and ERRγ are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The interaction between GNL3L and ERRγ requires the intermediate domain of GNL3L and the AF2-domain of ERRγ. Gain- and loss-of-function experiments show that GNL3L can inhibit the transcriptional activities of ERR genes in a cell-based reporter system, which does not require the nucleolar localization of GNL3L. We further demonstrate that GNL3L is able to reduce the steroid receptor coactivator (SRC) binding and the SRC-mediated transcriptional coactivation of ERRγ. This work reveals a novel mechanism that negatively regulates the transcriptional function of ERRγ by GNL3L through coactivator competition. PMID:17623774

Regulation of atrial natriuretic peptide clearance receptors in mesangial cells by growth factors.

PubMed

Paul, R V; Wackym, P S; Budisavljevic, M; Everett, E; Norris, J S

1993-08-25

Rat mesangial cells can express both 130-kDa guanylyl cyclase-coupled and 66-kDa non-coupled atrial natriuretic peptide (ANP) receptors (ANPR-A and ANPR-C, respectively). Exposure of mesangial cells, grown in 20% fetal calf serum, to 0.1% serum for 24 h increased total ANP receptor density more than 2-fold (Bmax = 87 versus 37 fmol/mg of cell protein) without changing binding affinity (Kd = 94 versus 88 pM). Radioligand binding and cross-linking studies demonstrated that up-regulation of ANP binding after serum deprivation was entirely due to an increase in ANPR-C, with little or no change in ANPR-A. Inhibition of protein synthesis with cycloheximide blocked up-regulation after serum deprivation. Steady-state ANPR-C mRNA level was increased 15-fold by serum deprivation, as judged by Northern blotting. There was no change in ANPR-A mRNA. Platelet-derived growth factor and phorbol myristate acetate, when added to low serum medium, blocked or reversed the effect of serum deprivation on ANPR-C. We conclude that synthesis and expression of ANPR-C but not ANPR-A is suppressed by serum, platelet-derived growth factor, and phorbol myristate acetate. Suppression of ANPR-C in vivo could contribute to mesangial cell proliferative responses to growth factors.

An automated system for the analysis of G protein-coupled receptor transmembrane binding pockets: alignment, receptor-based pharmacophores, and their application.

PubMed

Kratochwil, Nicole A; Malherbe, Pari; Lindemann, Lothar; Ebeling, Martin; Hoener, Marius C; Mühlemann, Andreas; Porter, Richard H P; Stahl, Martin; Gerber, Paul R

2005-01-01

G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Here, a comprehensive and automated method allowing fast analysis and comparison of these putative binding pockets across the entire GPCR family is presented. The method relies on a robust alignment algorithm based on conservation indices, focusing on pharmacophore-like relationships between amino acids. Analysis of conservation patterns across the GPCR family and alignment to the rhodopsin X-ray structure allows the extraction of the amino acids lining the TM binding pocket in a so-called ligand binding pocket vector (LPV). In a second step, LPVs are translated to simple 3D receptor pharmacophore models, where each amino acid is represented by a single spherical pharmacophore feature and all atomic detail is omitted. Applications of the method include the assessment of selectivity issues, support of mutagenesis studies, and the derivation of rules for focused screening to identify chemical starting points in early drug discovery projects. Because of the coarseness of this 3D receptor pharmacophore model, however, meaningful scoring and ranking procedures of large sets of molecules are not justified. The LPV analysis of the trace amine-associated receptor family and its experimental validation is discussed as an example. The value of the 3D receptor model is demonstrated for a class C GPCR family, the metabotropic glutamate receptors.

Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280).

PubMed

Li, Ming; Li, Chuanyu; Weingarten, Paul; Bunzow, James R; Grandy, David K; Zhou, Qun Yong

2002-03-01

Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.

Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

PubMed

Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

1993-06-25

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

Serotonin 2A receptor agonist binding in the human brain with [11C]Cimbi-36

PubMed Central

Ettrup, Anders; da Cunha-Bang, Sophie; McMahon, Brenda; Lehel, Szabolcs; Dyssegaard, Agnete; Skibsted, Anine W; Jørgensen, Louise M; Hansen, Martin; Baandrup, Anders O; Bache, Søren; Svarer, Claus; Kristensen, Jesper L; Gillings, Nic; Madsen, Jacob; Knudsen, Gitte M

2014-01-01

[11C]Cimbi-36 was recently developed as a selective serotonin 2A (5-HT2A) receptor agonist radioligand for positron emission tomography (PET) brain imaging. Such an agonist PET radioligand may provide a novel, and more functional, measure of the serotonergic system and agonist binding is more likely than antagonist binding to reflect 5-HT levels in vivo. Here, we show data from a first-in-human clinical trial with [11C]Cimbi-36. In 29 healthy volunteers, we found high brain uptake and distribution according to 5-HT2A receptors with [11C]Cimbi-36 PET. The two-tissue compartment model using arterial input measurements provided the most optimal quantification of cerebral [11C]Cimbi-36 binding. Reference tissue modeling was feasible as it induced a negative but predictable bias in [11C]Cimbi-36 PET outcome measures. In five subjects, pretreatment with the 5-HT2A receptor antagonist ketanserin before a second PET scan significantly decreased [11C]Cimbi-36 binding in all cortical regions with no effects in cerebellum. These results confirm that [11C]Cimbi-36 binding is selective for 5-HT2A receptors in the cerebral cortex and that cerebellum is an appropriate reference tissue for quantification of 5-HT2A receptors in the human brain. Thus, we here describe [11C]Cimbi-36 as the first agonist PET radioligand to successfully image and quantify 5-HT2A receptors in the human brain. PMID:24780897

Muscarinic cholinergic receptor binding: in vivo depiction using single photon emission computed tomography and radioiodinated quinuclidinyl benzilate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Drayer, B.; Jaszczak, R.; Coleman, E.

1982-06-01

An attempt was made to characterize, in vivo, specific binding to the muscarinic cholinergic receptor in the calf using the radioiodinated ligand quinuclidinyl benzilate (/sup 123/I-OH-QNB) and single photon detection emission computed tomography (SPECT). The supratentorial brain activity was significantly increased after the intravenous infusion of /sup 123/I-OH-QNB as compared to free /sup 123/I. Scopolamine, a muscarinic cholinergic receptor antagonist, decreased the measured brain activity when infused prior to /sup 123/I-OH-QNB consistent with pharmacologic blockade of specific receptor binding. Quantitative in vitro tissue distribution studies obtained following SPECT imaging were consistent with regionally distinct specific receptor binding in the striatummore » and cortical gray matter, nonspecific binding in the cerebellum, and pharmacologic blockade of specific binding sites with scopolamine. Although /sup 123/I-OH-QNB is not the ideal radioligand, our limited success will hopefully encourage the development of improved binding probes for SPECT imaging and quantitation.« less

The Ligand Binding Region of the Sigma-1 Receptor: Studies Utilizing Photoaffinity Probes, Sphingosine and N-Alkylamines

PubMed Central

Ruoho, Arnold E.; Chu, Uyen B.; Ramachandran, Subramaniam; Fontanilla, Dominique; Mavlyutov, Timur; Hajipour, Abdol R.

2015-01-01

The sigma-1 receptor is a 26 kDa endoplasmic reticulum resident membrane protein that has been shown to have chaperone activity in addition to its promiscuous binding to pharmacological agents. Ligand binding domain(s) of the sigma-1 receptor have been identified using the E. coli expressed and purified receptor protein and novel radioiodinated azido photoaffinity probes followed by pro-teolytic and chemical cleavage strategies. The outcome of these experiments indicates that the sigma-1 receptor ligand binding regions are formed primarily by juxtaposition of its second and third hydrophobic domains, regions where the protein shares considerable homology with the fungal enzyme, sterol isomerase that is essential for the biosynthesis of ergosterol. Data indicate that these hydrophobic steroid binding domain like (SBDL) regions on the sigma-1 receptor are likely to interact selectively with N-alkyl amines such as the endogenous sphingolipids and with synthetic N-alkylamines and N-aralkylamines derivatives. A proposed model for the sigma-1 receptor is presented. PMID:22288412

Structural analysis of binding functionality of folic acid-PEG dendrimers against folate receptor.

PubMed

Sampogna-Mireles, Diana; Araya-Durán, Ingrid D; Márquez-Miranda, Valeria; Valencia-Gallegos, Jesús A; González-Nilo, Fernando D

2017-03-01

Dendrimers functionalized with folic acid (FA) are drug delivery systems that can selectively target cancer cells with folate receptors (FR-α) overexpression. Incorporation of polyethylene glycol (PEG) can enhance dendrimers solubility and pharmacokinetics, but ligand-receptor binding must not be affected. In this work we characterized, at atomic level, the binding functionality of conventional site-specific dendrimers conjugated with FA with PEG 750 or PEG 3350 as a linker. After Molecular Dynamics simulation, we observed that both PEG's did not interfere over ligand-receptor binding functionality. Although binding kinetics could be notably affected, the folate fragment from both dendrimers remained exposed to the solvent before approaching selectively to FR-α. PEG 3350 provided better solubility and protection from enzymatic degradation to the dendrimer than PEG 750. Also, FA-PEG3350 dendrimer showed a slightly better interaction with FR-α than FA-PEG750 dendrimer. Therefore, theoretical evidence supports that both dendrimers are suitable as drug delivery systems for cancer therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

PubMed

Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

2013-09-17

Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

Is the isolated ligand binding domain a good model of the domain in the native receptor?