Advantages of two- or polyvalent binding of a receptor to the corresponding ligand in comparison to univalent binding

The features of monovalent and bivalent binding of receptors (or antibodies) with a polyvalent ligand (or with an antigen) are considered. It is shown that the rigid connection of the binding sites of the receptor brings to high increase of binding affinity for the corresponding ligand, but only in case if its epitopes are fully complementary to both sites of the receptor binding. If not, then there is no advantage of the binding of bivalent receptor before univalent binding. If the binding sites of the receptor are connected by a flexible linker, then regardless of location of epitopes of the corresponding ligand there is the successful fastening of receptor and ligand. Exactly the connection by a flexible linker is used by Nature in most cases at constructing of polyvalent receptors.     

Interpretation of Scatchard plots for aggregating receptor systems.

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.     

Determination of the binding parameters for a pre-existing antigen-antibody mixture

A new method, which allows to evaluate parameters of interaction between antibodies (or receptors) and an antigen (or ligand) is suggested. The method is based on the use of so-called coordinates of dilution suggested by the author earlier. Representation of the data of the titration curves for the mixtures of antibodies (or receptors) and antigen (or ligand) in these coordinates allows one to determine the affinity of interaction and the concentration of antigen (or ligand), which can reversibly block antibodies (or receptors). Simple formulas, which allow to estimate which part of paratopes or bivalent antibodies is free and which part is blocked by the antigen, depending on dilution of the considered system, are also suggested. Such a method could be useful for characterization of infection and autoimmune processes when the antigen and antibodies circulate together in the bloodstream.     

Cross-linking reconsidered: binding and cross-linking fields and the cellular response.

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.     

ELISA-based method for determining the affinity of bivalent antibodies of two specificities in a mixture

The problem of the evaluation of the affinity for two types of bivalent antibodies in a mixture is considered. It is shown that the binding curve in appropriate coordinates can be used to compose either a system of four equations with four unknowns or a system of two equations with two unknown variables. The numerical solution of these equation systems yields affinity constants for both antibodies and the relationship between concentrations of antibodies studied in the mixture.     

Use of hydrophilic diamines for bridging of two opioid peptide pharmacophores. Synthesis and receptor binding of two new analogues.

The bivalent ligand approach, which assumes that two pharmacophores are connected by a spacer, was used to design receptor type-selective ligands for opioid receptors. The first two opioid peptide bivalent ligands with different spacer lengths containing different numbers of hydroxyl groups, (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-)2 (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-CHOH-)2, were synthesized and their binding to mu, delta, and kappa opioid receptors was characterized. Both analogues were found to possess high opioid in vitro activities. The length of the hydrophilic spacer does not affect the affinity for delta receptors, whereas shorter spacer length increases affinity for mu and even more so for kappa receptors. Thus receptor type-selective peptides for opioid receptors can be designed using the bivalent approach.     

Competition between solution and cell surface receptors for ligand. Dissociation of hapten bound to surface antibody in the presence of solution antibody.

We present a joint theoretical and experimental study on the effects of competition for ligand between receptors in solution and receptors on cell surfaces. We focus on the following experiment. After ligand and cell surface receptors equilibrate, solution receptors are introduced, and the dissociation of surface bound ligand is monitored. We derive theoretical expressions for the dissociation rate and compare with experiment. In a standard dissociation experiment (no solution receptors present) dissociation may be slowed by rebinding, i.e., at high receptor densities a ligand that dissociates from one receptor may rebind to other receptors before separating from the cell. Our theory predicts that rebinding will be prevented when S much greater than N2Kon/(16 pi 2D a4), where S is the free receptor site concentration in solution, N the number of free surface receptor sites per cell, Kon the forward rate constant for ligand-receptor binding in solution, D the diffusion coefficient of the ligand, and a the cell radius. The predicted concentration of solution receptors needed to prevent rebinding is proportional to the square of the cell surface receptor density. The experimental system used in these studies consists of a monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), that reversibly binds to a monoclonal anti-DNP immunoglobulin E (IgE). This IgE is both a solution receptor and, when anchored to its high affinity Fc epsilon receptor on rat basophilic leukemia (RBL) cells, a surface receptor. For RBL cells with 6 x 10(5) binding sites per cell, our theory predicts that to prevent DCT rebinding to cell surface IgE during dissociation requires S much greater than 2,400 nM. We show that for S = 200-1,700 nM, the dissociation rate of DCT from surface IgE is substantially slower than from solution IgE where no rebinding occurs. Other predictions are also tested and shown to be consistent with experiment.     

In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association

Ligand binding to nuclear receptors leads to a conformational change that increases the affinity of the receptors to coactivator proteins. We have developed a ligand sensor assay for farnesoid X receptor (FXR) in which the receptor–coactivator interaction can be directly monitored using surface plasmon resonance biosensor technology. A 25-mer peptide from coactivator SRC1 containing the LXXLL nuclear receptor interaction motif was immobilized on the surface of a BIAcore sensor chip. Injection of the FXR ligand binding domain (FXRLBD) with or without the most potent natural ligand, chenodeoxycholic acid (CDCA), over the surface of the chip resulted in a ligand- and LXXLL motif-dependent interaction. Kinetic analysis revealed that CDCA and its conjugates decreased the equilibrium dissociation constant (K_d) by 8–11-fold, indicating an increased affinity. Using this technique, we found that a synthetic bile acid sulfonate, 3,7-dihydroxy-5β-cholane-24-sulfonate, which was inactive in a FXR response element-driven luciferase assay using CV-1 cells, caused the most potent interaction, comparable to the reaction produced by CDCA. This method provides a rapid and reliable in vitro ligand assay for FXR. This kinetic analysis-featured technique may be applicable to mechanistic studies.     

Theory of equilibrium binding of a bivalent ligand to cell surface antibody: The effect of antibody heterogeneity on cross-linking

We investigate the equilibrium binding of symmetric bivalent ligands to a heterogeneous population of symmetric bivalent cell surface receptors. The receptors are heterogeneous in their binding affinities (equilibrium binding constants) for the ligand. For any distribution of receptor binding affinities we show how to calculate the total concentration of receptors that are cross-linked by the ligand, i.e., the concentration of cell surface aggregates composed of two or more receptors, as well as the concentration of any given aggregate. We show that certain qualitative properties of cross-linking which hold for homogeneous antibody populations fail to hold in the heterogeneous case. We use our results to interpret certain in vitro experiments in which synthetic bivalent haptens are used to trigger histamine release from basophils which have on their surface antibody specific for the hapten.This work was performed under the auspices of the Department of Energy and supported by Grant AI 16465 from the National Institute of Allergy and Infectious Diseases.     

The insulin receptor. Structural basis for high affinity ligand binding

Treatment of the soluble insulin receptor from human placenta with 1.25 mM dithiothreitol and 75 mM Tris at pH 8.5 results in complete reduction of interhalf disulfide bonds (class 1 disulfides) and dissociation of the tetrameric receptor into the dimeric alpha beta form. The alpha beta receptor halves exhibit a reduced affinity for insulin binding (B?ni-Schnetzler, M., Rubin, J. B., and Pilch, P. F. (1986) J. Biol. Chem. 261, 15281-15287). Kinetic experiments reveal that reduction of class 1 disulfides is a faster process than the loss of affinity for ligand, indicating that events subsequent to reduction of interhalf disulfides are responsible for the affinity change. We show that a third class of alpha subunit intrachain disulfides is more susceptible to reduction at pH 7.6 than at pH 8.5 and appears to form part of the ligand binding domain. Reduction of the intrachain disulfide bonds in this part of the alpha subunit leads to a loss of insulin binding. Modification of this putative binding domain by dithiothreitol can be minimized if reduction is carried out at pH 8.5. When the insulin receptor in placental membranes is reduced at pH 8.5, the receptor's affinity for insulin is not changed when binding is measured in the membrane. However, the Kd for insulin binding is reduced 10-fold when alpha beta receptor halves are subsequently solubilized. Scatchard analysis of insulin binding to reduced or intact receptors in the membrane and in soluble form together with sucrose density gradient analysis of soluble receptors suggests that alpha beta receptor halves remain associated in the membrane after reduction, but they are dissociated upon solubilization. We interpret these results to mean that the association of two ligand binding domains, 2 alpha beta receptor halves, is required for the formation of an insulin receptor with high affinity for ligand.     

Essential role of magnesium in oxytocin-receptor affinity and ligand specificity.

We have analysed, with the aid of a novel radioiodinated oxytocin (OT)-receptor antagonist, the role of Mg2+ in uterine OT-receptor function. The antagonist-receptor interaction was characterized by high affinity, reversibility and stereospecificity in Tris/HCl buffer containing 3 mmol of Mg2+/litre as well as buffer free of Mg2+. By contrast, omission of Mg2+ decreased the affinity of the receptor for OT by about 1500-fold; moreover, the stereospecificity of agonist, but not antagonist, binding was lost. Since guanine nucleotides had relatively minor effects in this system (less than or equal to 2-fold decrease in OT affinity), we suggest that the agonist-binding site of OT receptors is directly modulated by Mg2+, unlike other receptors, where the effects of bivalent cations are exerted via guanine-nucleotide-binding (G-) proteins. Thus the ligand recognition mechanism of OT receptors may be novel in this respect.     

Cell-specific targeting by heterobivalent ligands

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 ? is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.     

Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction.

The aggregation of IgE anchored to high-affinity Fc epsilon receptors on rat basophilic leukemia (RBL) cells by multivalent antigens initiates transmembrane signaling and ultimately cellular degranulation. Previous studies have shown that the rate of dissociation of bivalent and multivalent DNP ligands from RBL cells sensitized with anti-DNP IgE decreases with increasing ligand incubation times. One mechanism proposed for this effect is that when IgE molecules are aggregated, a conformational change occurs that results in an increase in the intrinsic affinity of IgE for antigen. This possibility was tested by measuring the equilibrium constant for the binding of monovalent DNP-lysine to anti-DNP IgE under two conditions, where the cell-bound IgE is dispersed and where it has been aggregated into visible patches on the cell surface using anti-IgE and a secondary antibody. No difference in the equilibrium constant in these two cases was observed. We also measured the rate of dissociation of a monovalent ligand from cell surface IgE under these two conditions. Whereas the affinity for monovalent ligand is not altered by IgE aggregation, we observe that the rate of ligand dissociation from IgE in clusters is slower than the rate of ligand dissociation from unaggregated IgE. These results are discussed in terms of recent theoretical developments concerning effects of receptor density on ligand binding to cell surfaces.     

Determination of the stable conformation of GABA(A)-benzodiazepine receptor bivalent ligands by low temperature NMR and X-ray analysis

The stable conformations of GABA(A)-benzodiazepine receptor bivalent ligands 2 and 3 were determined by low temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The linear conformation was important for these dimers to access the binding site and exhibit potent in vitro affinity as illustrated for alpha5 subtype selective ligand 2 (15 nM). Bivalent ligand 3 with the 5 atom linker folded back upon itself both in solution and in the solid state, moreover, it did not bind to Bz receptors.     

Ligand selectivity and affinity of chemokine receptor CXCR1. Role of N-terminal domain

Glu-Leu-Arg ("ELR") CXC chemokines interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA) recruit neutrophils by binding and activating two receptors, CXCR1 and CXCR2. CXCR1 is specific, binding only IL-8 with nanomolar affinity, whereas CXCR2 is promiscuous, binding all ELRCXC chemokines with high affinity. Receptor signaling consists of two events: interactions between the ligand N-terminal loop (N-loop) and receptor N-terminal domain (N-domain) residues (site I), and between the ligand N-terminal ELR and the receptor juxtamembrane domain (J-domain) residues (site II). It is not known how these interactions mediate ligand affinity and selectivity, and whether binding at one site influences binding and function at the other. Sequence analysis and structure-function studies have suggested that the receptor N-domain plays an important role in ligand selectivity. Here, we report ligand-binding properties and structural characteristics of the CXCR1 N-domain in solution and in detergent micelles that mimic the native membrane environment. We find that IL-8 binds the N-domain with significantly higher affinity in micelles than in solution (approximately 1 microM versus approximately 20 microM) and that MGSA does not bind the N-domain in solution but does in micelles with appreciable affinity (approximately 3 microM). We find that the N-domain is structured in micelles and that the entire N-domain interacts with the micelle in an extended fashion. We conclude that the micellar environment constrains the N-domain, and this conformational restraint influences its ligand-binding properties. Most importantly, our data suggest that for both ligands, site I interaction provides similar affinity and that differential coupling between site I and II interactions is responsible for the observed differences in affinity.     

Relative ligand binding to small or large aggregates measured by scanning correlation spectroscopy.

Cell surface receptors transduce signals, required to produce cellular activity, that may be mediated by ligand-induced receptor aggregation. Several receptor systems exhibit both low and high ligand affinities and some models of receptor activation associate receptor clusters with high or low ligand binding affinity. In the present work succinyl concanavalin A, which binds with both high and low affinity to receptors, was studied on 3T3 Swiss mouse fibroblasts, where preaggregation of receptors has been postulated. Scanning fluorescence correlation spectroscopy measurements were used to determine the relationship between the degree of ligand binding and the state of receptor aggregation. Correlation analysis of fluorescence fluctuations across the cell surface reveal that the variance of the fluctuations (quantitated by g[0]) increased when the ligand concentration was varied from 0.33 to 67 mg/L. The g(0) values reached a plateau at concentrations greater than approximately 10 mg/L. These data are incompatible with homogeneous receptor distributions or equal affinity receptor binding but are compatible with a partly aggregated receptor system with high affinity binding to small aggregates, and low affinity binding to large aggregates. Computer simulated scanning fluorescence correlation spectroscopy experiments confirm that background fluorescence from the cell does not account for the experimentally observed effects.     

Multiple receptor states are required to describe both kinetic binding and activation of neutrophils via N-formyl peptide receptor ligands

It is well-established that the binding of N-formyl peptides to the N-formyl peptide receptor on neutrophils can be described by a kinetic scheme that involves two ligand-bound receptor states, both a low affinity ligand-receptor complex and a high affinity ligand-receptor complex, and that the rate constants describing ligand-receptor binding and receptor affinity state interconversion are ligand-specific. Here we examine whether differences due to these rate constants, i.e. differences in the numbers and lifetimes of particular receptor states, are correlated with neutrophil responses, namely actin polymerization and oxidant production. We find that an additional receptor state, one not discerned from kinetic binding assays, is required to account for these responses. This receptor state is interpreted as the number of low affinity bound receptors that are capable of activating G proteins; in other words, the accumulation of these active receptors correlates with the extent of both responses. Furthermore, this analysis allows for the quantification of a parameter that measures the relative strength of a ligand to bias the receptor into the active conformation. A model with this additional receptor state is sufficient to describe response data when two ligands (agonist/agonist or agonist/antagonist pairs) are added simultaneously, suggesting that cells respond to the accumulation of active receptors regardless of the identity of the ligand(s).     

Determination of antibody affinity by ELISA. Theory

New approaches for the measurement of antibody affinity by ELISA are suggested and considered theoretically. It was shown that not only more precise and more convenient in comparison to that suggested earlier, but also more informative graphical representation of the experimental data in the appropriate coordinate could be used for evaluation of antibody affinity. The following cases were considered: (i) determination of antibody affinity for one kind of univalent antibodies, (ii) determination of antibody affinity for one kind of bivalent antibodies, (iii) determination of antibody affinity for two kinds of univalent antibodies, which are in a mixture, and (iv) determination of antibody affinity for two kinds of bivalent antibodies, which are in a mixture. Advantages and disadvantages of the different approaches are discussed.     

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.     

Receptors for insulin and insulin-like growth factor-I can form hybrid dimers. Characterisation of hybrid receptors in transfected cells.

We have demonstrated the formation of hybrid insulin/insulin-like growth factor-I(IGF-I) receptors in transfected rodent fibroblasts, which overexpress human receptors, by examining reactivity with species- and receptor-specific monoclonal antibodies. In NIH 3T3 and Rat 1 fibroblasts, endogenous IGF-I receptors were unreactive with anti-(human insulin receptor)monoclonal antibodies (47-9, 25-49, 83-14, 83-7, 18-44). However, in transfected cells expressing high levels of insulin receptors, 60-80% of high-affinity IGF-I receptors reacted with these antibodies, as assessed either by inhibition of ligand binding in intact cells or by precipitation of solubilized receptors. Conversely, endogenous insulin receptors in NIH 3T3 cells were unreactive with anti-(IGF-I receptor) antibodies alpha IR-3 and 16-13. However, approx. 50% of high-affinity insulin receptors reacted with these antibodies in cells expressing high levels of human IGF-I receptors. The hybrid receptors in transfected cells bound insulin or IGF-I with high affinity. However, responses to these ligands were asymmetrical, in that binding of IGF-I inhibited subsequent binding of insulin, but prior binding of insulin did not affect the affinity for IGF-I. The existence of hybrid receptors in normal tissues could have important implications for metabolic regulation by insulin and IGF-I.