共查询到20条相似文献,搜索用时 15 毫秒
1.
D R Hurwitz S L Emanuel M H Nathan N Sarver A Ullrich S Felder I Lax J Schlessinger 《The Journal of biological chemistry》1991,266(32):22035-22043
The binding of epidermal growth factor (EGF) to its cell surface receptor (EGF-R) results in a number of intracellular responses including the activation of the receptor intracellular tyrosine kinase. Receptor oligomerization induced by ligand binding has been suggested to play an important role in signal transduction. However, the mechanisms involved in oligomerization and signal transduction are poorly understood. We have produced and purified several milligrams of recombinant extracellular domain of the EGF receptor (EGF-Rx) using the baculovirus/insect cell expression system. The baculovirus-generated EGF-Rx is glycosylated, has had its signal peptide correctly cleaved, and exhibits a dissociation constant for EGF similar to that for solubilized full-length receptor, of about 100 nM. The binding of EGF to EGF-Rx leads to the formation of receptor dimers and higher oligomerization states which are irreversibly captured using the covalent cross-linking agent disuccinimidyl suberate. Interestingly, purified receptor monomers and dimers, stabilized by the cross-linker in the presence of EGF, exhibit increased binding affinity toward EGF as compared with receptor monomers which have not been exposed to EGF. It appears that the high affinity state of receptor can be maintained by the covalent cross-linking agent. These results indicate that in addition to ligand binding, the extracellular domain of EGF receptor possesses the inherent ability to undergo ligand-induced dimerization and that the low affinity state is converted to a high affinity state by EGF. 相似文献
2.
Mutational analysis of the ligand binding domain of the low density lipoprotein receptor 总被引:29,自引:0,他引:29
V Esser L E Limbird M S Brown J L Goldstein D W Russell 《The Journal of biological chemistry》1988,263(26):13282-13290
The ligand binding domain of the low density lipoprotein (LDL) receptor contains seven imperfect repeats of a 40-amino acid cysteine-rich sequence. Each repeat contains clustered negative charges that have been postulated as ligand-binding sites. The adjacent region of the protein, the growth factor homology region, contains three cysteine-rich repeats (A-C) whose sequence differs from those in the ligand binding domain. To dissect the contribution of these different cysteine-rich repeats to ligand binding, we used oligonucleotide-directed mutagenesis to alter expressible cDNAs for the human LDL receptor which were then introduced into monkey COS cells by transfection. We measured the ability of the mutant receptors to bind LDL, which contains a single protein ligand for the receptor (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains apoB-100 plus multiple copies of another ligand (apoE). The results show that repeat 1 is not required for binding of either ligand. Repeats 2 plus 3 and repeats 6 plus 7 are required for maximal binding of LDL, but not beta-VLDL. Repeat 5 is required for binding of both ligands. Repeat A in the growth factor homology region is required for binding of LDL, but not beta-VLDL. Repeat B is not required for ligand binding. These results support a model for the LDL receptor in which various repeats play additive roles in ligand binding, each repeat making a separate contribution to the binding event. 相似文献
3.
The mechanism of signal transduction by steroid receptor proteins is complex and not yet understood. We describe here a facile genetic strategy for dissection of the rat glucocorticoid receptor "signaling domain," a region of the protein that binds and transduces the hormonal signal. We found that the characteristics of signal transduction by the receptor expressed in yeast were similar to those of endogenous receptors in mammalian cells. Interestingly, the rank order of particular ligands differed between species with respect to receptor binding and biological efficacy. This suggests that factors in addition to the receptor alone must determine or influence ligand efficacy in vivo. To obtain a collection of receptors with distinct defects in signal transduction, we screened in yeast an extensive series of random point mutations introduced in that region in vitro. Three phenotypic classes were obtained: one group failed to bind hormone, a second displayed altered ligand specificity, and a third bound hormone but lacked regulatory activity. Our results demonstrate that analysis of glucocorticoid receptor action in yeast provides a general approach for analyzing the mechanism of signaling by the nuclear receptor family and may facilitate identification of non-receptor factors that participate in this process. 相似文献
4.
Stem-cell factor (SCF) is a noncovalent homodimeric cytokine that exhibits profound biological function in the early stages of hematopoiesis by binding to a cell surface tyrosine kinase receptor that is encoded by the c-Kit proto-oncogene. The results obtained from a combined implementation of homology-based molecular modeling and computational simulations in the study of species-specific SCF/c-Kit interactions are reported. The structural models of the human and rat SCF ligands are based on the close structural similarity to the cytokine M-CSF, whose Cα structure has recently become available. The constant domains of the human Fc fragment are used as a template for the ligand binding domains of the c-Kit receptor. The factors responsible for the stabilization of the SCF quaternary structure and the molecular determinants for ligand recognition and ligand specificity have been identified by assessing the conformational, topographical, and dynamic features of the isolated ligands and of the ligand-receptor complexes. © 1996 Wiley-Liss, Inc. 相似文献
5.
We study effects of convective transport on a chemical front wave representing a signal propagation at a simple (single layer) epithelium by means of mathematical modeling. Plug flow and laminar flow regimes were considered. We observed a nonmonotonous dependence of the propagation velocity on the ligand receptor binding constant under influence of the convective transport. If the signal propagates downstream, the region of high velocities becomes much broader and spreads over several orders of magnitude of the binding constant. When the convective transport is oriented against the propagating signal, either velocity of the traveling front wave is slowed down or the traveling front wave can stop or reverse the direction of propagation. More importantly, chemical signal in epithelial systems influenced by the convective transport can propagate almost independently of the ligand-receptor binding constant in a broad range of this parameter. Furthermore, we found that the effects of the convective transport becomes more significant in systems where either the characteristic dimension of the extracellular space is larger/comparable with the spatial extent of the ligand diffusion trafficking or the ligand-receptor binding/ligand diffusion rate ratio is high. 相似文献
6.
Owen MR Sherratt JA Myers SR 《Proceedings. Biological sciences / The Royal Society》1999,266(1419):579-585
Juxtacrine signalling is the process of cell communication in which ligand and receptors are both anchored in the cell membrane. We develop three mathematical models for this process, involving different mathematical representations of the dynamics of membrane-bound ligand and free and bound receptors, within an epithelial sheet. We consider the dynamics of this system following a localized disturbance, such as would be provided by a source of ligand or by the generation of a free edge via wounding. We study the ability of the juxtacrine mechanism to transmit a signal away from this disturbance, and show analytically that the spatial half-life of the signal can in fact be arbitrarily large. This result is quite general, since we use a generic reaction kinetic scheme; the key assumption is that ligand and receptor production are both upregulated by binding. Moreover, the result applies to all three of our model formulations. We conclude by discussing applications of the result to the particular case of the transforming growth factor alpha binding to epidermal growth factor receptor in epidermal wound healing. 相似文献
7.
Nosjean O Souchaud S Deniau C Geneste O Cauquil N Boutin JA 《Journal of biomolecular screening》2006,11(8):949-958
Fluorescence polarization is a screening technology that is radioactivity free, homogeneous, and ratiometric. The signal measured with this technology is a weighted value of free and bound ligand. As a consequence, saturation curves are accessible only after calculation of the corresponding concentrations of free and bound ligand. To make this technology more accessible to assay development, the authors propose a simple mathematical model that predicts fluorescence polarization values from ligand and receptor total concentrations, depending on the corresponding dissociation constant. This model was validated using data of Bodipy-NDP-alphaMSH binding to MC(5), obtained after either ligand saturation of a receptor preparation or, conversely, receptor saturation of a ligand solution. These experimental data were also used to calculate the actual concentration of free and bound ligand and receptor and to obtain pharmacological constants by Scatchard analysis. A general method is proposed, which facilitates the design of fluorescence polarization binding assays by relying on the representation of theoretical polarization values. This approach is illustrated by the application to 2 systems of very different affinities. 相似文献
8.
Cytochrome P-450-dependent 14 alpha-demethylation of lanosterol in Candida albicans. 总被引:2,自引:0,他引:2
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Analysis of receptor-ligand binding characteristics can be greatly hampered by the presence of non-specific binding, defined as low-affinity binding to non-receptor domains which is not saturable within the range of ligand concentrations used. Conventional binding analyses, e.g. according to the methods described by Scatchard or Klotz, relate the amount of specific receptor-ligand binding to the concentration of free ligand, and therefore require assumptions on the amount of non-specific binding. In this paper a method is described for determining the parameters of specific receptor-ligand interaction which does not require any assumption or separate determination of the amount of non-specific binding. If the concentration of labelled free ligand is constant, a plot of Fu/(B0*-B*) versus Fu yields a linear relationship, in the case of a single receptor class, in which Fu is the concentration of unlabelled free ligand, B0* is the total amount of labelled bound ligand in the absence of unlabelled ligand and B* is the total amount of labelled bound ligand in the presence of an unlabelled ligand concentration Fu; all of these data are readily obtained from binding studies. This linear relationship holds irrespective of the amount of non-specific binding, and the values for receptor density, ligand dissociation constant and a constant for non-specific binding can be readily obtained from it. If the concentration of labelled free ligand is not a constant for all data points, data are first converted according to a straightforward normalization procedure to permit the use of this relationship. The presence of multiple receptor classes with dissociation constants in the range of the ligand concentrations used results in a negative deviation from this linearity, and therefore the presence of multiple receptor classes can be discriminated unequivocally from non-specific binding. Both theoretical and practical advantages of the present method are described. The method, which will be referred to as the linear subtraction method, is illustrated using the binding of tumour promoters and polypeptide growth factors to their specific cellular receptors. 相似文献
9.
Steric effects on multivalent ligand-receptor binding: exclusion of ligand sites by bound cell surface receptors.
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Steric effects can influence the binding of a cell surface receptor to a multivalent ligand. To account for steric effects arising from the size of a receptor and from the spacing of binding sites on a ligand, we extend a standard mathematical model for ligand-receptor interactions by introducing a steric hindrance factor. This factor gives the fraction of unbound ligand sites that are accessible to receptors, and thus available for binding, as a function of ligand site occupancy. We derive expressions for the steric hindrance factor for various cases in which the receptor covers a compact region on the ligand surface and the ligand expresses sites that are distributed regularly or randomly in one or two dimensions. These expressions are relevant for ligands such as linear polymers, proteins, and viruses. We also present numerical algorithms that can be used to calculate steric hindrance factors for other cases. These theoretical results allow us to quantify the effects of steric hindrance on ligand-receptor kinetics and equilibria. 相似文献
10.
Structure and dynamics of DRD4 bound to an agonist and an antagonist using in silico approaches
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Human dopamine receptor D4 (DRD4), a member of G‐protein coupled receptor (GPCR) family, plays a central role in cell signaling and trafficking. Dysfunctional activity of DRD4 can lead to several psychiatric conditions and, therefore, represents target for many neurological disorders. However, lack of atomic structure impairs our understanding of the mechanism regulating its activity. Here, we report the modeled structure of DRD4 alone and in complex with dopamine and spiperone, its natural agonist and antagonist, respectively. To assess the conformational dynamics induced upon ligand binding, all‐atom explicit solvent molecular dynamics simulations in membrane environment were performed. Comprehensive analyses of simulations reveal that agonist binding triggers a series of conformational changes in the transmembrane region, including rearrangement of residues, characteristic of transmission and tyrosine toggle molecular switches. Further, the trajectories indicate that a loop region in the intracellular region––ICL3, is significantly dynamic in nature, mainly due to the side‐chain movements of conserved proline residues involved in SH3 binding domains. Interestingly, in dopamine‐bound receptor simulation, ICL3 represents an open conformation ideal for G protein binding. The structural and dynamical information presented here suggest a mode of activation of DRD4, upon ligand binding. Our study will help in further understanding of receptor activation, as acquiring structural information is crucial for the design of highly selective DRD4 ligands. Proteins 2014; 83:867–880. © 2014 Wiley Periodicals, Inc. 相似文献
11.
We have discovered the signal initiation structure of the relaxin-like factor and shown its function to be independent of the amino acid side chains in the contact region. Evidence presented in this article suggests that signal induction is a function of the peptide bond and that completion of the signaling contact is initiated by ligand binding to the leucine-rich repeat G-protein coupled receptor 8 (LGR8). The specific mode of binding forces certain peptide bonds into a signaling position. This observation implies that the receiving structures are equally nonspecific so that signaling should occur at any peptide bond of the receptor or the trans-membrane loop that is within reach of the signaling wires of the receptor-bound ligand. Our observations offer an explanation for ligand cross-talk as well as for the ability of some antibodies to elicit the biological response normally associated with a specific ligand. 相似文献
12.
Interdomain dynamics and ligand binding: molecular dynamics simulations of glutamine binding protein
Periplasmic binding proteins from Gram-negative bacteria possess a common architecture, comprised of two domains linked by a hinge region, a fold which they share with the neurotransmitter-binding domains of ionotropic glutamate receptors (GluRs). Glutamine-binding protein (GlnBP) is one such protein, whose crystal structure has been solved in both open and closed forms. Multi-nanosecond molecular dynamics simulations have been used to explore motions about the hinge region and how they are altered by ligand binding. Glutamine binding is seen to significantly reduce inter-domain motions about the hinge region. Essential dynamics analysis of inter-domain motion revealed the presence of both hinge-bending and twisting motions, as has been reported for a related sugar-binding protein. Significantly, the influence of the ligand on GlnBP dynamics is similar to that previously observed in simulations of rat glutamate receptor (GluR2) ligand-binding domain. The essential dynamics analysis of GlnBP also revealed a third class of motion which suggests a mechanism for signal transmission in GluRs. 相似文献
13.
Andrews SS 《Physical biology》2005,2(2):111-122
Serial ligation is the repeated reversible binding of a ligand to one receptor after another. It is a widespread phenomenon throughout biochemical systems, occurring anytime receptors are clustered together and ligand binding is reversible. Computer simulations are used in this work to investigate a representative example, which is the serial ligation of an extracellular aspartate molecule to the membrane-bound chemotaxis receptors of an Escherichia coli bacterium. It is found that the initial binding site of a ligand to a cluster of receptors is more likely to be near the edge of the cluster than near the middle, although there is no overall bias when all rebindings are considered. Serial ligation does not lead directly to signal amplification or attenuation but instead causes binding events to be correlated in both space and time: a ligand is likely to bind many times in rapid succession in a small region of the receptor cluster, but there can also be long intervals between bindings. This leads to an increased level of noise in the received signal but may allow a single ligand to be sensed above a uniform level of background noise. The focus of this paper is on the interpretation of simulation results so they can be generalized to a wide variety of other systems and to allow the identification of systems in which serial ligation is likely to be important. In the process, several characteristic times are identified, as are scaling laws for the spatial and temporal dynamics. 相似文献
14.
M B Jackson 《Biophysical journal》1986,49(3):663-672
Open- and closed-state lifetimes of unliganded acetylcholine receptor channel activity were analyzed by the method of likelihood maximazation. For both open times and closed times, the best-fitting density is most often a sum of two exponentials. These multiple open states cannot depend on the number of receptor binding sites occupied since they are observed in the absence of ligand. The rate of spontaneous opening and the faster decay constant of closing increased as the membrane was hyperpolarized. The voltage dependence of the rate of spontaneous opening is stronger than that for curare-liganded channels. Evidence that the acetylcholine receptor channel can open spontaneously in the absence of ligand has been presented previously (Sanchez et al, 1983; Brehm et al, 1984; Jackson, 1984). To add to this evidence, alpha-bungarotoxin was added to the patch electrode, causing the frequency of openings to decay with time. The rate constant determined from this decay is similar to rate constants reported for the binding of iodinated alpha-bungarotoxin to the acetylcholine receptor. The frequency of unliganded channel opening has been estimated as 2 X 10(-3) s-1 per receptor. A comparison of carbamylcholine-liganded and spontaneous gating transition rates suggests that ligand binding increases the rate of opening by a factor of 1.4 X 10(7). Carbamylcholine binding increases the mean open time by a factor of 5. Thus, a cholinergic agonist activates the acetylcholine receptor by destabilizing the closed state. The liganded and unliganded channel gating rates were used to analyze the energetics of ligand activation of the acetylcholine receptor channel, and to relate the open channel dissociation constant to the closed channel dissociation constant. 相似文献
15.
Rate constants for binding, dissociation, and internalization of EGF: effect of receptor occupancy and ligand concentration 总被引:4,自引:0,他引:4
We measured the kinetic parameters for interaction of epidermal growth factor (EGF) with fetal rat lung (FRL) cells under two sets of experimental conditions and applied sensitivity analysis to see which parameters were well-defined. In the first set of experiments (method 1), the kinetics of internalization and dissociation of radiolabeled EGF were measured with a temperature-shift protocol in medium initially devoid of free ligand. The initial concentration of radiolabeled EGF bound to the cell surface corresponded to levels of receptor occupancy ranging from approximately 200 receptors per cell to approximately 18,000 receptors per cell, a level at which EGF binding approaches saturation. In the second set of experiments (method 2), carried out at a constant temperature, we began with no surface-bound or internalized ligand. The initial free ligand concentration was varied from 0.2 to 50 ng/mL. In both sets of experiments, we measured surface-bound, internalized, and free 125I-EGF as functions of time and evaluated the parameters of a mathematical model of endocytosis. Sensitivity analysis showed that three rate constants were well-defined in this combination of two experimental approaches: ke, the endocytic rate constant; ka, the association rate constant; and kd, the dissociation rate constant. The endocytic parameter ke was found to be independent of initial surface receptor occupancy (method 1); there was some indication that it increased with initial free ligand concentration in method 2. Neither kd nor ka was found to change with extent of initial surface receptor occupancy or initial free ligand concentration, respectively, a finding of significance, since diffusion theory predicts these parameters will vary with surface receptor occupancy.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
Characterization of the receptor binding determinants of granulocyte colony stimulating factor. 总被引:4,自引:3,他引:1
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D. C. Young H. Zhan Q. L. Cheng J. Hou D. J. Matthews 《Protein science : a publication of the Protein Society》1997,6(6):1228-1236
We performed a series of experiments using alanine-scanning mutagenesis to locate side chains within human granulocyte colony-stimulating factor (G-CSF) that are involved in human G-CSF receptor binding. We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G-CSF. The G-CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method. We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay. These studies show that there is a region of five charged residues on helices A and C employed by G-CSF in binding its receptor, with the most important residue in this binding patch being Glu 19. Both wild-type G-CSF and the E19A mutant were expressed in E. coli. The re-folded proteins were found to have proliferative activities similar to the analogous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G-CSF. Although G-CSF is likely to signal through receptor homo-dimerization, we found no compelling evidence for a second receptor binding region. We also found no evidence of self-antagonism at high G-CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G-CSF probably does not signal via a pure 2:1 receptor ligand complex. Thus, G-CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high-affinity interaction with its receptor. 相似文献
17.
A method for determining antigen-antibody binding constants by using enzyme-labeled antigens has been developed. In the measurement, enzyme-labeled and unlabeled antigens (Ag* and Ag) were allowed to compete in binding to the antibody (Ab) under conditions where Ag* much less than Ab much less than Ag. The data were analyzed according to a new theory developed for the analysis of competitive binding of two ligands to a heterogeneous receptor. The theory indicates that the binding degree of a labeled ligand measured at various concentrations of the receptor can be used to prepare a standard curve relating the binding degree of the labeled ligand and the average of the concentrations of the free receptor components which are in binding equilibrium with another unlabeled ligand. For homogeneous receptors, the method gives usual binding constants for the unlabeled ligand, but for heterogeneous receptors, it gives a new type of average binding constant for the unlabeled ligand in which the contribution of each receptor component is amplified in proportion to its affinity against the labeled ligand. This average binding constant was named the "affinity-average binding constant." A rabbit anti-blasticidin S (BLS) antiserum analyzed by the present method using beta-galactosidase-labeled BLS as the labeled ligand was found to be fairly homogeneous with respect to the affinity and to have a binding constant of 1.48 +/- 0.24 (S.D.) X 10(8) M-1 for unlabeled BLS. 相似文献
18.
How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain-EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain. 相似文献
19.
C D Son H Sargsyan G B Hurst F Naider J M Becker 《The journal of peptide research》2005,65(3):418-426
G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable alpha-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to cross-linked fragments of Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an alpha-factor analog with the photoaffinity group p-benzoyl-l-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of alpha-factor and a region of Ste2p covering residues 251-294. 相似文献
20.
We have simulated an odor ligand's dynamic behavior in the binding region of an olfactory receptor (OR). Our short timescale computational studies (up to 200 ps) have helped identify unprecedented postdocking ligand behavior of ligands. From in vacuo molecular dynamics simulations of interactions between models of rat OR I7 and 10 aldehyde ligands, we have identified a dissociative pathway along which the ligand exits and enters the OR-binding pocket--a transit event. The ligand's transit through the receptor's binding region may mark the beginning of a signal transduction cascade leading to odor recognition. We have graphically traced the rotameric changes in key OR amino acid side chains during the transit. Our results have helped substantiate or refute previously held notions of amino acid contribution to ligand stability in the binding pocket. Our observations of ligand activity when compared to those of experimental (electroolfactogram response) OR-activation studies provide a view to predicting the stability of ligands in the binding pocket as a precursor to OR activation by the ligand. 相似文献