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1.
To link quantitatively the cell surface binding of ligand to receptor with the production of cellular responses, it may be necessary to explore early events in signal transduction such as G-protein activation. Two different model frameworks relating receptor/ligand binding to G-protein activation are examined. In the first framework, a simple ordinary differential equation model is used to describe receptor/ligand binding and G-protein activation. In the second framework, the events leading to G-protein activation are simulated using a dynamic Monte Carlo model. In both models, reactions between ligand-bound receptors and G-proteins are assumed to be diffusion-limited. The Monte Carlo model predicts two regimes of G-protein activation, depending upon whether the lifetime of a receptor/ligand complex is long or short compared with the time needed for diffusional encounters of complexes and G-proteins. When the lifetime of a complex is relatively short compared with the diffusion time, the movement of ligand among free receptors by binding and unbinding ("switching") significantly enhances G-protein activation. Receptor antagonists dramatically reduce G-protein activation and, thus, signal transduction in this case, and significant clustering of active G-proteins near receptor/ligand complexes results. The simple ordinary differential equation model poorly predicts G-protein activation for this situation. In the alternative case, when diffusion is relatively fast, ligand movement among receptors is less important and the simple ordinary differential equation model and Monte Carlo model results are similar. In this case, there is little clustering of active G-proteins near receptor/ligand complexes. Results also indicate that as the GTPase activity of the alpha-subunit decreases, the steady-state level of alpha-GTP increases, although temporal sensitivity is compromised. 相似文献
2.
John J. M. Bergeron G. M. Di Guglielmo Patricia C. Baass François Authier Barry I. Posner 《Bioscience reports》1995,15(6):411-418
Upon the binding of insulin or epidermal growth factor to their cognate receptors on the liver parenchymal plasmalemma, signal transduction and receptor internalization are near co-incident. Indeed, the rapidity and extent; of ligand mediated receptor internalization into endosomes in liver as well as other organs predicts that signal transduction is regulated at this intracellular locus. Although internalization has been thought as a mechanism to attenuate ligand mediated signal transduction responses, detailed studies of internalized receptors in isolated liver endosomes suggest an alternative scenario whereby selective signal transduction pathways can be accessed at this locus. 相似文献
3.
We previously described a unique lymphokine that activates macrophage C3 receptors for phagocytosis. The lymphokine is generated when T lymphocytes receive a signal from macrophages that have ingested IgG-coated material. In the present work, we examined the mechanisms by which macrophage Fc receptors must be engaged for macrophages to signal lymphocytes to elaborate the lymphokine. We found that ingestion mediated by any of the three classes of murine macrophage Fc receptors was sufficient to trigger macrophages, and that engagement of macrophage Fc receptors by immobilized immune complexes was effective as well. We also found that ligation of Fc receptors by an anti-Fc receptor IgG antibody or by its F(ab')2 or Fab fragments also triggered macrophages. The ability of monovalent ligation of the receptor to elicit biologic activity suggests that this system may be of value in elucidating general mechanisms by which ligand binding of receptors is transduced into biologic effects. 相似文献
4.
Plasmon-waveguide resonance (PWR) spectroscopy provides a highly sensitive method for characterizing the kinetics, affinities and conformational changes involved in ligand binding to G-protein coupled receptors, without the need for radioactive or other labeling strategies. In the case of the cloned delta-opioid receptor from human brain incorporated into a lipid bilayer, we have shown that affinities determined in this way are consistent with those measured by standard binding procedures using membranes or whole cells containing the receptors, and that the spectral and kinetic properties of the binding processes allow facile distinction between agonist, inverse agonist, and antagonist ligands. We have also shown by direct measurements that G-protein binding affinities and the ability to undergo GTP/GDP exchange are dependent upon the type of ligand pre-bound to the receptor. PWR spectroscopy thus provides a powerful new approach to investigating signal transduction in biological membrane systems. 相似文献
5.
Interrupting autocrine ligand-receptor binding: comparison between receptor blockers and ligand decoys.
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Stimulation of cell behavioral functions by ligand/receptor binding can be accomplished in autocrine fashion, where cells secrete ligand capable of binding to receptors on their own surfaces. This proximal secretion of autocrine ligands near the surface receptors on the secreting cell suggests that control of these systems by inhibitors of receptor/ligand binding may be more difficult than for systems involving exogenous ligands. Hence, it is of interest to predict the conditions under which successful inhibition of cell receptor binding by the autocrine ligand can be expected. Previous theoretical work using a compartmentalized model for autocrine cells has elucidated the conditions under which addition of solution decoys for the autocrine ligand can interrupt cell receptor/ligand binding via competitive binding of the secreted molecules (Forsten, K. E., and D. A. Lauffenburger. 1992. Biophys. J. 61:1-12.) We now apply a similar modeling approach to examine the addition of solution blockers targeted against the cell receptor. Comparison of the two alternative inhibition strategies reveals that a significantly lower concentration of receptor blockers, compared to ligand decoys, will obtain a high degree of inhibition. The more direct interruption scheme characteristic of the receptor blockers may make them a preferred strategy when feasible. 相似文献
6.
Aggregation of cell surface receptors, with each other or with other membrane proteins, occurs in a variety of experimental systems. The list of systems where receptor aggregation appears to be important in understanding ligand binding and cellular responses is growing rapidly. In this paper we explore the interpretation of equilibrium binding data for aggregating receptor systems. The Scatchard plot is a widely used tool for analyzing equilibrium binding data. The shape of the Scatchard plot is often interpreted in terms of multiple noninteracting receptor populations. Such an analysis does not provide a framework for investigating the role of receptor aggregation and will be misleading if there is a relation between receptor aggregation and ligand binding. We present a general model for the equilibrium binding of a ligand with any number of aggregating receptor populations and derive theoretical expressions for observable Scatchard plot features. These can be used to test particular models and estimate model parameters. We develop particular models and apply the general results in the cases of six aggregating receptor systems where ligand binding and receptor aggregation are related: cross-linking of monovalent cell surface proteins by monoclonal antibodies, cross-linking of cell surface antibodies by bivalent ligand, antibody-induced co-cross-linking of cell surface antibodies and Fc gamma receptors, ligand-enhanced aggregation of identical epidermal growth factor receptors, aggregation of heterologous receptors for interleukin 2 to form a high-affinity receptor, and association of receptors, including those for interleukins 5 and 6, with nonbinding accessory proteins that influence receptor affinity or effector function. 相似文献
7.
In this paper, evidence is provided indicating that the blockade of presynchronized CHO 15B cells in prometaphase by nocodazole is fully reversible and efficient enough to allow us to analyze the function of the integrin receptors. Flow cytometry analysis using a specific antibody raised against the fibronectin receptor, and binding studies of the radiolabeled fibronectin on the cell membrane, indicated a stable number of receptors at the cell surface during mitosis. Furthermore, in the mean time, only a slight increase in the Kd value of the fibronectin-receptor interaction was detected. A binding assay designed to test the affinity of the receptor for its extracellular ligand in an insoluble form was used. No difference was observed between mitotic and interphasic cells. Taken together, these results indicate that the rounding up of the cells observed during mitosis is not due to a loss of the receptor affinity for its extracellular ligand. 相似文献
8.
Cross-linking reconsidered: binding and cross-linking fields and the cellular response. 总被引:1,自引:0,他引:1
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We analyze a model for the reversible cross-linking of cell surface receptors by a collection of bivalent ligands with different affinities for the receptor as would be found in a polyclonal anti-receptor serum. We assume that the amount of cross-linking determines, via a monotonic function, the rate at which cells become activated and divide. In addition to the density of receptors on the cell surface, two quantities, the binding field and the cross-linking field, are needed to characterize the cross-linking curve, i.e., the equilibrium concentration of cross-linked receptors plotted as a function of the total ligand site concentration. The binding field is the sum of all ligand site concentrations weighted by their respective binding affinities, and the cross-linking field is the sum of all ligand site concentrations weighted by the product of their respective binding and cross-linking affinity and the total receptor density. Assuming that the cross-linking affinity decreases if the binding affinity decreases, we find that the height of the cross-linking curve decreases, its width narrows, and its center shifts to higher ligand site concentrations as the affinities decrease. Moreover, when we consider cross-linking-induced proliferation, we find that there is a minimum cross-linking affinity that must be surpassed before a clone can expand. We also show that under many circumstances a polyclonal antiserum would be more likely than a monoclonal antibody to lead to cross-linking-induced proliferation. 相似文献
9.
The biological response of interferon gamma is mediated by binding to a specific cell-surface receptor. We investigated the stoichiometry of this binding using soluble receptors produced in prokaryotic and eukaryotic expression systems comprising the extracellular ligand-binding domain of the native protein. The ligand-receptor complexes were analyzed by cross-linking, chromatography, analytical ultracentrifugation and laser-light scattering. Cross-linking and chromatography showed that the stoichiometry of the interaction between ligand and receptor depends on the molar ratios of the two components mixed. All approaches confirmed that mixtures of ligand-receptor complexes are formed with one interferon-gamma dimer bound by one or two receptors. The soluble receptor produced in Escherichia coli mainly showed a ligand/receptor stoichiometry of 1:1, while the receptors produced in eukaryotic cells showed a stoichiometry of binding of 1:2. This apparent discrepancy is most likely due to the conformational heterogeneity of the Escherichia-coli-derived protein. 相似文献
10.
The first event in signal transduction at a synapse is the binding of transmitters to receptors. Because of rapidly changing transmitter levels this binding is unlikely to occur at equilibrium. We describe a mathematical approach that models complex receptor interactions in which the timing and amplitude of transmitter release are noisy. We show that exact solutions for simple bimolecular interactions and receptor transitions can be used to model complex reaction schemes by expressing them in sets of difference equations. Results from the difference equation method to describe binding and channel opening at extended time points compare well with standard solutions using ordinary differential equations. Because it is applicable to noisy systems we used the difference method to investigate the information processing capabilities of GABA receptors and predict how pharmacological agents may modify these properties. As previously demonstrated, the response to a single pulse of GABA is prolonged through entry into a desensitized state. During trains of stimuli the signal to noise ratio can change, and even increase progressively, but the overall transmitted fidelity of the signal decreases with increased driving frequency. The GABA modulator chlorpromazine (primarily affects agonist on and off rates) is predicated to increase receptor signal to noise ratio at all frequencies whereas pregnenolone sulfate (affects receptor desensitization) completely inhibits information transfer. 相似文献
11.
Cytokine receptors and signal transduction. 总被引:16,自引:0,他引:16
Most of the recently cloned cytokine receptors that operate in the immune and hematopoietic systems contain no tyrosine kinase domains in their cytoplasmic regions, unlike the family of growth factor receptors defined earlier. However, they can be assigned to several new types of receptor families based on structural similarities among them. It is characteristic of these receptors that many of them require a receptor-associated molecule in order to achieve high-affinity ligand binding and/or transmission of cytoplasmic signals. Receptor-associated molecules have been found that transduce cytoplasmic signals and are shared by different cytokine receptors. Phosphorylation of the receptors and of various cytoplasmic proteins after ligand stimulation seems to be a common event in cytokine systems. Insight into the pleiotropic and redundant nature of cytokine action is provided by the discovery of several new cytokine receptor families and of shared signal transduction molecules and by the idea that several cytoplasmic kinases may be able to functionally substitute for one another in transmitting cytokine signals. 相似文献
12.
Receptors constitute the interface of cells to their external environment. These molecules bind specific ligands involved in multiple processes, such as signal transduction and nutrient transport. Although a variety of cell surface receptors undergo endocytosis, the systems-level design principles that govern the evolution of receptor trafficking dynamics are far from fully understood. We have constructed a generalized mathematical model of receptor–ligand binding and internalization to understand how receptor internalization dynamics encodes receptor function and regulation. A given signaling or transport receptor system represents a particular implementation of this module with a specific set of kinetic parameters. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptor systems can be characterized as being: i) avidity-controlled where the response control depends primarily on the extracellular ligand capture efficiency, ii) consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii) dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled, and the epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to enhance the accuracy of signaling receptors rather than merely serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulation. 相似文献
13.
N L Incardona 《Journal of theoretical biology》1983,105(4):631-645
A general kinetic mechanism is presented for reversible binding of viruses to cells followed by an irreversible step that initiates the delivery of the viral genome. A novel feature is additional pathways for the release of both virus-occupied and unoccupied receptors from cells. Due to one simplifying assumption, it does not apply at low receptor densities. However, it is sufficiently general to be applicable to ligand binding and internalization for those systems in which ligand diffusion is rate limiting. Three different versions of the model fit the usual kinetic data for the binding of an eclipse mutant of bacteriophage phi X174 to Escherichia coli. However, in each case binding to cell-bound receptors is irreversible. Therefore, this explains the apparent failure of this system to obey the Law of Mass Action. One version of the model also predicts that the release rate of lipopolysaccharide receptors from the outer membrane may be significantly lowered when virus is bound to these receptors. 相似文献
14.
Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling 总被引:16,自引:0,他引:16
B F O'Dowd M Hnatowich J W Regan W M Leader M G Caron R J Lefkowitz 《The Journal of biological chemistry》1988,263(31):15985-15992
Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human beta 2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human beta 2-adrenergic receptor. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail. 相似文献
15.
Pilar García-Peñarrubia Juan J. Gálvez Jesús Gálvez 《Journal of mathematical biology》2014,69(3):553-582
Cell signalling processes involve receptor trafficking through highly connected networks of interacting components. The binding of surface receptors to their specific ligands is a key factor for the control and triggering of signalling pathways. But the binding process still presents many enigmas and, by analogy with surface catalytic reactions, two different mechanisms can be conceived: the first mechanism is related to the Eley–Rideal (ER) mechanism, i.e. the bulk-dissolved ligand interacts directly by pure three-dimensional (3D) diffusion with the specific surface receptor; the second mechanism is similar to the Langmuir–Hinshelwood (LH) process, i.e. 3D diffusion of the ligand to the cell surface followed by reversible ligand adsorption and subsequent two-dimensional (2D) surface diffusion to the receptor. A situation where both mechanisms simultaneously contribute to the signalling process could also occur. The aim of this paper is to perform a computational study of the behavior of the signalling response when these different mechanisms for ligand-receptor interactions are integrated into a model for signal transduction and ligand transport. To this end, partial differential equations have been used to develop spatio-temporal models that show trafficking dynamics of ligands, cell surface components, and intracellular signalling molecules through the different domains of the system. The mathematical modeling developed for these mechanisms has been applied to the study of two situations frequently found in cell systems: (a) dependence of the signal response on cell density; and (b) enhancement of the signalling response in a synaptic environment. 相似文献
16.
Surface plasmon resonance (SPR) biosensors offer a unique opportunity to study the binding activity of G protein-coupled receptors (GPCRs) in real time with minimal sample preparation. Using two chemokine receptors (CXCR4 and CCR5) as model systems, we captured the proteins from crude cell preparations onto the biosensor surface and reconstituted a lipid environment to maintain receptor activity. The conformational states of the receptors were probed using conformationally dependent antibodies, and by characterizing the binding properties of a native chemokine ligand (stromal cell-derived factor 1alpha). The results suggest that the detergent-solubilized receptors are active for ligand binding in the presence and absence of a reconstituted bilayer. There are three advantages to using this receptor-capturing approach: (1) there is no need to purify the receptor prior to immobilization on the biosensor surface, (2) the receptors are homogeneously immobilized through the capturing step, and (3) the receptors can be captured at high enough densities to allow the study of relatively low-molecular-mass ligands (2000-4000Da). We also demonstrated that the receptors are sensitive to the solubilizing conditions, which illustrates the potential for using SPR biosensors to rapidly screen solublization conditions for GPCRs. 相似文献
17.
Cannabinoids produce most of their biochemical and pharmacological effects by interacting with CB1 and CB2 cannabinoid receptors, both of which are G-protein coupled membrane-bound functional proteins. CB1 is found in the central nervous system and in a variety of other organs including heart, vascular endothelium, uterus, vas deferens, testis and small intestine. Conversely, the CB2 receptor appears to be associated exclusively with the immune system and is found in the periphery of the spleen and other cells associated with immunochemical functions. Although both CB1 and CB2 have been cloned and the primary sequences are known, their three dimensional structures and the amino acid residues at the active site, critical for ligand recognition, binding and activation have not been characterized. In the absence of any X-ray crystallographic and NMR data, information on the structural requirements for ligand-receptor interactions is obtained with the help of suitably designed molecular probes. These ligands either interact with the receptor in a reversible fashion (reversible probes) or, alternatively, attach at or near the receptor active site with the formation of a covalent bond (irreversible probes). Subsequently, information related to ligand binding and receptor activation is further amplified with the help of receptor mutants and computer modeling. This review focuses on molecular probes related to the classical and non-classical cannabinoids that have been reported since the discovery of the first cannabinoid receptor over a decade ago. 相似文献
18.
Differences in the binding mechanism of RU486 and progesterone to the progesterone receptor 总被引:4,自引:0,他引:4
D F Skafar 《Biochemistry》1991,30(45):10829-10832
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 [3H]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 (p less than 0.001). This indicated that a difference in the binding mechanism of RU486 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 (p = 0.915). 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. 相似文献
19.
Changing the insulin receptor to possess insulin-like growth factor I ligand specificity 总被引:5,自引:0,他引:5
A S Andersen T Kjeldsen F C Wiberg P M Christensen J S Rasmussen K Norris K B M?ller N P M?ller 《Biochemistry》1990,29(32):7363-7366
To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, we prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the alpha-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor. 相似文献
20.
Autocrine ligand binding to cell receptors. Mathematical analysis of competition by solution "decoys".
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Autocrine ligands have been demonstrated to regulate cell proliferation, cell adhesion, and cell migration in a number of different systems and are believed to be one of the underlying causes of malignant cell transformation. Binding of these ligands to their cellular receptors can be compromised by diffusive transport of ligand away from the secreting cell. Exogenous addition of antibodies or solution receptors capable of competing with cellular receptors for these autocrine ligands has been proposed as a means of inhibiting autocrine-stimulated cell behavioral responses. Such "decoys" complicate cellular binding by offering alternative binding targets, which may also be capable of aiding or abating transport of the ligand away from the cell surface. We present a mathematical model incorporating autocrine ligand production and the presence of competing cellular and solution receptors. We elucidate effects of key system parameters including ligand diffusion rate, binding rate constants, cell density, and secretion rate on the ability of solution receptors to inhibit cellular receptor binding. Both plated and suspension cell systems are considered. An approximate analytical expression relating the key parameters to the critical concentration of solution "decoys" required for inhibition is derived and compared to the numerical calculations. We find that in order to achieve essentially complete inhibition of surface receptor binding, the concentration of decoys may need to be as much as four to eight orders of magnitude greater than the equilibrium disociation constant for ligand binding to surface receptors. 相似文献