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The high affinity interleukin-2 receptor is composed of three cell surface subunits, IL-2Ralpha, IL-2Rbeta, and IL-2Rgamma. Functional forms of the IL-2 receptor exist, however, that enlist only two of the three subunits. On activated T-cells, the alpha- and beta-subunits combine as a preformed heterodimer (the pseudo-high affinity receptor) that serves to capture IL-2. On a subpopulation of natural killer cells, the beta- and gamma-subunits interact in a ligand-dependent manner to form the intermediate affinity receptor site. Previously, we have demonstrated the feasibility of employing coiled-coil molecular recognition for the solution assembly of a heteromeric IL-2 receptor complex. In that study, although the receptor was functional, the coiled-coil complex was a trimer rather than the desired heterodimer. We have now redesigned the hydrophobic heptad sequences of the coiled-coils to generate soluble forms of both the pseudo-high affinity and the intermediate affinity heterodimeric IL-2 receptors. The properties of these complexes were examined and their relevance to the physiological IL-2 receptor mechanism is discussed.

Alefacept is a chimeric protein combining CD58 immunoglobulin-like domain 1 with human IgG1 Fc. Alefacept mediates adhesion by bridging CD2 on T cells to activating Fc receptors on effector cells, but the equilibrium binding parameters have not been determined. Alefacept mediated T cell killing by NK cells and adhesion between CD2- and CD16-expressing cells at an optimum concentration of 100 nM. We introduce novel measurements with supported planer bilayers, from which key two-dimensional and three-dimensional parameters can be determined by data fitting. Alefacept competitively inhibited cell bilayer adhesion mediated by the CD2-CD58 interaction. Alefacept mediated maximal adhesion of CD2(+) T cells to CD16B, an Fc receptor, in planar bilayers at 500 nM. A mechanistic model for alefacept-mediated cell-bilayer adhesion allowed fitting of the data and determination of two-dimensional binding parameters. These included the density of bonds in the adhesion area, which grew to maintain a consistent average bond density of 200 molecules/microm(2) and two-dimensional association constants of 3.1 and 630 microm(2) for bivalently and monovalently bound forms of alefacept, respectively. The maximum number of CD16 bound and the fit value of 4,350 CD2 per cell are much lower than the 40,000 CD2 per cell measured with anti-CD2 Fab. These results suggest that additional information is needed to correctly predict Alefacept-mediated bridge formation.

Cells may discriminate among ligands with different dwell times for receptor binding through a mechanism called kinetic proofreading in which the formation of an activated receptor complex requires a progression of events that is aborted if the ligand dissociates before completion. This mechanism explains how, at equivalent levels of receptor occupancy, a rapidly dissociating ligand can be less effective than a more slowly dissociating analog at generating distal cellular responses. Simple mathematical models predict that kinetic proofreading is limited to the initial complex; once the signal passes to second messengers, the dwell time no longer regulates the signal. This suggests that an assay for kinetic proofreading might be used to determine which activation events occur within the initial signaling complex. In signaling through the high affinity IgE receptor FcεRI, the transmembrane adaptor called linker for activation of T cells (LAT) is thought to nucleate a distinct secondary complex. Experiments in which the concentrations of two ligands with different dwell times are adjusted to equalize the level of LAT phosphorylation in rat basophilic leukemia 2H3 cells show that Erk2 phosphorylation, intracellular Ca2+, and degranulation exhibit kinetic proofreading downstream of LAT phosphorylation. These results suggest that ligand-bound FcεRI and LAT form a complex that is required for effective signal transmission.

We consider a model of early events in signaling by the epidermal growth factor (EGF) receptor (EGFR). The model includes EGF, EGFR, the adapter proteins Grb2 and Shc, and the guanine nucleotide exchange factor Sos, which is activated through EGFinduced formation of EGFR‐Grb2‐Sos and EGFR‐Shc‐Grb2‐Sos assemblies at the plasma membrane. The protein interactions involved in signaling can potentially generate a diversity of protein complexes and phosphoforms; however, this diversity has been largely ignored in models of EGFR signaling. Here, we develop a model that accounts more fully for potential molecular diversity by specifying rules for protein interactions and then using these rules to generate a reaction network that includes all chemical species and reactions implied by the protein interactions. We obtain a model that predicts the dynamics of 356 molecular species, which are connected through 3749 unidirectional reactions. This network model is compared with a previously developed model that includes only 18 chemical species but incorporates the same scope of protein interactions. The predictions of this model are reproduced by the network model, which also yields new predictions. For example, the network model predicts distinct temporal patterns of autophosphorylation for different tyrosine residues of EGFR. A comparison of the two models suggests experiments that could lead to mechanistic insights about competition among adapter proteins for EGFR binding sites and the role of EGFR monomers in signal transduction. © 2005 Elsevier Ireland Ltd. All rights reserved.

Interleukin-2 is the primary T cell growth factor secreted by activated T cells. IL-2 is an alpha-helical cytokine that binds to a multisubunit receptor expressed on the surface of a variety of cell types. IL-2Ralpha, IL-2Rbeta, and IL-2Rgammac receptor subunits expressed on the surface of cells may aggregate to form distinct binding sites of differing affinities. IL-2Rgammac was the last receptor subunit to be identified. It has since been shown to be shared by at least five other cytokine receptors. In this study, we have probed the role of IL-2Rgammac in the assembly of IL-2R complexes and in ligand binding. We demonstrate that in the absence of ligand IL-2Rgammac does not possess detectable affinity for IL-2Ralpha, IL-2Rbeta, or the pseudo-high-affinity binding site composed of preformed IL-2Ralpha/beta. We also demonstrate that IL-2Rgammac possesses an IL-2-dependent affinity for IL-2Rbeta and IL-2Ralpha/beta. We performed a detailed biosensor analysis to examine the interaction of soluble IL-2Rgammac with IL-2-bound IL-2Rbeta and IL-2-bound IL-2Ralpha/beta. The kinetic and equilibrium constants for sIL-2Rgammac binding to these two different liganded complexes were similar, indicating that IL-2Ralpha does not play a role in recruitment of IL-2Rgammac. We also determined that the binding of IL-2 to the isolated IL-2Rgammac was very weak (approximate K(D) = 0.7 mM). The experimental methodologies and principles derived from these studies can be extended to at least five other cytokines that share IL-2Rgammac as a receptor subunit.

Cholera toxin entry into mammalian cells is mediated by binding of the pentameric B subunit (CTB) to ganglioside GM(1) in the cell membrane. We used flow cytometry to quantitatively measure in real time the interactions of fluorescently labeled pentameric cholera toxin B-subunit (FITC-CTB) with its ganglioside receptor on microsphere-supported phospholipid membranes. A model that describes the multiple steps of this mode of recognition was developed to guide our flow cytometric experiments and extract relevant equilibrium and kinetic rate constants. In contrast to previous studies, our approach takes into account receptor cross-linking, an important feature for multivalent interactions. From equilibrium measurements, we determined an equilibrium binding constant for a single subunit of FITC-CTB binding monovalently to GM(1) presented in bilayers of approximately 8 x 10(7) M(-1) while that for binding to soluble GM(1)-pentasaccharide was found to be approximately 4 x 10(6) M(-1). From kinetic measurements, we determined the rate constant for dissociation of a single site of FITC-CTB from microsphere-supported bilayers to be (3.21 +/- 0.03) x 10(-3) s(-1), and the rate of association of a site on FITC-CTB in solution to a GM(1) in the bilayer to be (2.8 +/- 0.4) x 10(4) M(-1) s(-1). These values yield a lower estimate for the equilibrium binding constant of approximately 1 x 10(7) M(-1). We determined the equilibrium surface cross-linking constant [(1.1 +/- 0.1) x 10(-12) cm(2)] and from this value and the value for the rate constant for dissociation derived a value of approximately 3.5 x 10(-15) cm(2) s(-1) for the forward rate constant for cross-linking. We also compared the interaction of the receptor binding B-subunit with that of the whole toxin (A- and B-subunits). Our results show that the whole toxin binds with approximately 100-fold higher avidity than the pentameric B-subunit alone which is most likely due to the additional interaction of the A(2)-subunit with the membrane surface. Interaction of cholera toxin B-subunit and whole cholera toxin with gangliosides other than GM(1) revealed specific binding only to GD1(b) and asialo-GM(1). These interactions, however, are marked by low avidity and require high receptor concentrations to be observed.

Using defined oligomers of IgE, our group previously studied the quantitative relationship between the aggregation of the high affinity receptors for IgE (FcɛRI) and the earliest signals initiated by such aggregation: the phosphorylation of tyrosines on the receptor. Notably, at certain doses of the oligomers such phosphorylation reached a plateau level well before the aggregation of the receptors had reached a maximum. These findings and others led us to propose that aggregates of the receptor were competing for a limited amount of the critical kinase—thought to be Lyn in this system. This paper describes a test of this proposal. We incubated cells with two distinguishable IgEs and examined the effect of aggregating one or the other or both types on the phosphorylation. When receptors binding antigen-specific IgE were aggregated with polyvalent antigen, they became rapidly phosphorylated as expected. Remarkably, however, FcɛRI that had already been phosphorylated by the binding of dimers of IgE, became dephosphorylated simultaneously. Furthermore, when the antigen-driven aggregates were dissociated with hapten, the phosphorylation pattern reverted to that seen prior to the addition of antigen: as the antigen-driven aggregates became dephosphorylated, the receptors stably aggregated by the bound oligomers became rapidly rephosphorylated. Dephosphorylation of oligomer-driven aggregates was also partially reversed during the “spontaneous” dephosphorylation of the antigen-driven receptors seen at longer times after addition of antigen. Thus signal transduction in this system is in part regulated by the shuttling of limited amounts of the kinase that initiates the cascade of phosphorylations.

Previous studies in our laboratory established that the symmetrical bivalent ligand, N,N'-bis-[[epsilon-(2,4-dinitrophenyl)amino]caproyl]-L-tyrosyl]-L-cystin e ((DCT)2-cys), stably cross-links anti-2,4-dinitrophenyl-immunoglobulin E (IgE) bound to high affinity receptors Fc epsilonRI on the surface of RBL-2H3 cells, forming mostly cyclic dimers containing two IgE-Fc epsilonRI and two (DCT)2-cys (Posner et al. (1995) J. Immunol. 155, 3601-3609). These cyclic dimers do not trigger Ca2+ or degranulation responses under a variety of conditions. However, we find that the linearly cross-linked IgE-Fc epsilonRI formed at higher concentrations of (DCT)2-cys do trigger degranulation in the presence of cytochalasin D, an inhibitor of actin polymerization. We further investigated stimulation by (DCT)2-cys of the earliest known events in the functional response, i.e., tyrosine phosphorylation of the beta and gamma subunits of Fc epsilonRI. At the higher (DCT)2-cys concentrations corresponding to linear dimers and maximal degranulation, tyrosine phosphorylation of both beta and gamma are observed. At lower (DCT)2-cys concentrations where cross-linking is maximal and cyclic dimers are overwhelmingly dominant, only gamma tyrosine phosphorylation is observed. Cytochalasin D does not affect these phosphorylation patterns, but instead appears to enhance coupling to downstream signaling events. Phosphorylation of Syk occurs at the higher (DCT)2-cys concentrations in parallel with beta phosphorylation but does not occur in its absence at the lower (DCT)2-cys concentrations. These results suggest that cyclic dimers of IgE-Fc epsilonRI are sterically restricted such that they stimulate tyrosine phosphorylation of gamma but not beta, and this is not sufficient for Syk binding and/or activation.

Previously, we demonstrated that aggregates of the high affinity receptor for IgE (FcϵRI), formed by the binding of chemically cross-linked oligomers of IgE, continue to signal early and late cellular responses long after the formation of new aggregates is blocked. In the present work, we explore quantitatively the relationship between aggregation of the receptors and one of the earliest biochemical changes this initiates. We compare the time course of aggregate formation, inferred from studies of the binding of dimers of IgE, and the time course of phosphorylation of tyrosines on receptor subunits when the receptors are aggregated. A simple model does not fit the data. It appears that aggregates formed late in the response are less effective signaling units than those formed initially. We propose new explanations for the persistence of the response and the unusual kinetics.

In this study, we report the use of coiled-coil (leucine zipper) molecular recognition for the solution assembly of stable, high affinity, heteromeric interleukin-2 receptor complexes. Co-expression of interleukin-2 receptor alpha and beta extracellular domains (ectodomains), each fused to seven coiled-coil heptad repeats, resulted in the formation of heteromeric complexes that bound interleukin-2 in a cooperative fashion and with much higher affinity than similar homomeric complexes. The dissociation constants for these solution complexes are within the range of values reported for the comparable cell surface "pseudo high affinity" interleukin-2 receptor. Ligand-induced cross-linking of homomeric or heteromeric receptor subunits is the common signal transmission mechanism employed by hematopoietin receptors. Individual receptor ectodomains, however, often do not bind ligand with measurable affinity. This is the first study to demonstrate the feasibility of coiled-coil mediated preassembly of cytokine receptor complexes.

We present a model of the early events in mast cell signaling mediated by FcεRI where the plasma membrane is composed of many small ordered lipid domains (rafts), surrounded by a non-order region of lipids consisting of the remaining plasma membrane. The model treats the rafts as transient structures that constantly form and breakup, but that maintain a fixed average number per cell. The rafts have a high propensity for harboring Lyn kinase, aggregated, but not unaggregated receptors, and the linker for the activation of T cells (LAT). Phosphatase activity in the rafts is substantially reduced compared to the nonraft region. We use the model to analyze published experiments on the rat basophilic leukemia (RBL)-2H3 cell line that seem to contradict the notion that rafts offer protection. In these experiments IgE was cross-linked with a multivalent antigen and then excess monovalent hapten was added to break-up cross-links. The dephosphorylation of the unaggregated receptor (nonraft associated) and of LAT (raft associated) were then monitored in time and found to decay at similar rates, leading to the conclusion that rafts offer no protection from dephosphorylation. In the model, because the rafts are transient, a protein that is protected while in a raft will be subject to dephosphorylation when the raft breaks up and the protein finds itself in the nonraft region of the membrane. We show that the model is consistent with the receptor and LAT dephosphorylation experiments while still allowing rafts to enhance signaling by providing substantial protection from phosphatases.

Numerous signaling proteins use multivalent binding to increase the specificity and affinity of their interactions within the cell. Enhancement arises because the effective binding constant for multivalent binding is larger than the binding constants for each individual interaction. We seek to gain both qualitative and quantitative understanding of the multivalent interactions of an adaptor protein, growth factor receptor bound protein-2 (Grb2), containing two SH3 domains interacting with the nucleotide exchange factor son-of-sevenless 1 (Sos1) containing multiple polyproline motifs separated by flexible unstructured regions. Grb2 mediates the recruitment of Sos1 from the cytosol to the plasma membrane where it activates Ras by inducing the exchange of GDP for GTP. First, using a combination of evolutionary information and binding energy calculations, we predict an additional polyproline motif in Sos1 that binds to the SH3 domains of Grb2. This gives rise to a total of five polyproline motifs in Sos1 that are capable of binding to the two SH3 domains of Grb2. Then, using a hybrid method combining molecular dynamics simulations and polymer models, we estimate the enhancement in local concentration of a polyproline motif on Sos1 near an unbound SH3 domain of Grb2 when its other SH3 domain is bound to a different polyproline motif on Sos1. We show that the local concentration of the Sos1 motifs that a Grb2 SH3 domain experiences is approximately 1000 times greater than the cellular concentration of Sos1. Finally, we calculate the intramolecular equilibrium constants for the crosslinking of Grb2 on Sos1 and use thermodynamic modeling to calculate the stoichiometry. With these equilibrium constants, we are able to predict the distribution of complexes that form at physiological concentrations. We believe this is the first systematic analysis that combines sequence, structure, and thermodynamic analyses to determine the stoichiometry of the complexes that are dominant in the cellular environment., Author Summary Many biochemical interactions are mediated by multivalent binding where signaling proteins use relatively weak promiscuous interactions to increase the strength and specificity of complex formation. For a bivalent adaptor protein binding to a multivalent ligand, the tethering of one of the adaptors binding sites to a motif on a multivalent ligand constrains the adaptors second binding site to a region with a high local concentration of ligand binding motifs. Intramolecular equilibrium constants associated with multivalency are difficult to measure. Typically, polymer models are utilized to estimate the enhancement in local concentration and, when the biomolecular equilibrium constants for the individual sites are known, to obtain intramolecular equilibrium constants. However, flexibility of structured regions in proteins that contain the binding motifs restricts the application of simple polymer models for many systems. Here, we develop a hybrid method combining molecular dynamics simulations and polymer models to estimate the intramolecular equilibrium constants. We apply this method to study the multivalent interactions between the widely expressed adaptor protein growth factor receptor bound protein-2 (Grb2) and the nucleotide exchange factor son of sevenless 1 (Sos1).

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 Fcγ 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.

The bivalent ligand N,N'-bis[[epsilon-[(2,4- dinitrophenyl)amino]caproyl]-L-tyrosyl]cystine [(DCT)2-Cys] binds and cross-links anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-receptor complexes on the cell surface of rat basophilic leukemia cells. The rate of dissociation of this bound ligand was monitored by using a fluorescence method under two different conditions. In one case the monovalent ligand DCT was added in large excess to prevent the dissociating ligand from rebinding to unoccupied antibody combining sites. Under these conditions, dissociation of the bivalent ligand from IgE-sensitized cells proceeds to completion with kinetics that are well described by two rate constants that are independent of the time of preincubation of the bivalent ligand with the cells. In the second case, dissociation of (DCT)2-Cys from cell-bound anti-DNP IgE was monitored in the presence of a large excess of anti-DNP IgE in solution that acts as a sink to absorb the dissociated ligand. Under these conditions, the bivalent ligand becomes more resistant to dissociation as the preincubation time of the bivalent ligand with the cells is increased. An increasing fraction of the bound ligand does not dissociate on a measurable time scale in the presence of this sink. The results indicate that cell-associated IgE-receptor complexes undergo a time-dependent change that facilitates the reformation of the cross-linked state when one end of the ligand dissociates to break up the existing cross-link. The possible physical basis and functional implications of these results are discussed.

A series of synthetic ligands consisting of poly(ethylene glycol) (PEG), capped on one or both ends with the hapten 2,4-dinitrophenyl (DNP), were previously shown to be potent inhibitors of cellular activation in RBL mast cells stimulated by a multivalent antigen [Baird, E. J., Holowka, D., Coates, G. W., and Baird, B. (2003) Biochemistry 42, 12739-12748]. In this study, we systematically investigated the effect of increasing length of the PEG scaffold on the binding of these monovalent and bivalent ligands to anti-DNP IgE in solution. Our analysis reveals evidence for an energetically favorable interaction between two monovalent ligands bound to the same receptor, when the PEG molecular mass exceeds approximately 5 kDa. Additionally, for ligands with much higher molecular masses (>10 kDa PEG), the binding of a single ligand apparently leads to a steric exclusion of the second binding site by the bulky PEG scaffold. These results are further corroborated by data from an alternate fluorescence-based assay that we developed to quantify the capacity of these ligands to displace a small hapten bound to IgE. This new assay monitors the displacement of a small, receptor-bound hapten by a competitive monovalent ligand and thus quantifies the competitive inhibition offered by a monovalent ligand. We also show that, for bivalent ligands, inhibitory capacity is correlated with the capacity to form effective intramolecular cross-links with IgE.

Summary: BioNetGen allows a user to create a computational model that characterizes the dynamics of a signal transduction system, and that accounts comprehensively and precisely for specified enzymatic activities, potential post-translational modifications and interactions of the domains of signaling molecules. The output defines and parameterizes the network of molecular species that can arise during signaling and provides functions that relate model variables to experimental readouts of interest. Models that can be generated are relevant for rational drug discovery, analysis of proteomic data and mechanistic studies of signal transduction. Availability: http://cellsignaling.lanl.gov/bionetgen

Many activities of cells are controlled by cell-surface receptors, which in response to ligands, trigger intracellular signaling reactions that elicit cellular responses. A hallmark of these signaling reactions is the reversible nucleation of multicomponent complexes, which typically begin to assemble when ligand-receptor binding allows an enzyme, often a kinase, to create docking sites for signaling molecules through chemical modifications, such as tyrosine phosphorylation. One function of such docking sites is the co-localization of enzymes with their substrates, which can enhance both enzyme activity and specificity. The directed assembly of complexes can also influence the sensitivity of cellular responses to ligand-receptor binding kinetics and determine whether a cellular response is up- or downregulated in response to a ligand stimulus. The full functional implications of ligand-stimulated complex formation are difficult to discern intuitively. Complex formation is governed by conditional interactions among multivalent signaling molecules and influenced by quantitative properties of both the components in a system and the system itself. Even a simple list of the complexes that can potentially form in response to a ligand stimulus is problematic because of the number of ways signaling molecules can be modified and combined. Here, we review the role of multicomponent complexes in signal transduction and advocate the use of mathematical models that incorporate detail at the level of molecular domains to study this important aspect of cellular signaling.

Signaling by the T cell receptor (TCR), and the related immunoreceptor Fc epsilon RI, is sensitive to ligand-receptor binding kinetics. Differences in the rate at which a ligand dissociates from a receptor cause disproportionate differences in signaling events and cellular responses to ligand-receptor engagement. Analysis of a simple mathematical model, developed by McKeithan (1995, Proc. Natl. Acad. Sci. USA, 92, 5042-5046), has indicated that such sensitivity to binding kinetics is expected if a bound receptor must complete a cascade of modifications before generating a productive signal. However, recent experiments show that some cellular responses mediated by immunoreceptors escape from the control of kinetic proofreading, in the sense that these responses do not exhibit the expected sensitivity to the lifetime of a ligand-receptor bond. Here, we use an extended form of the McKeithan model to investigate possible explanations for such exceptions to the kinetic proofreading rule. We examine cellular responses triggered by cytosolic messengers, which are activated by modified receptors, and responses triggered by receptors in intermediate states of modification, i.e., receptors that have not progressed through the full series of potential modifications. Receptor aggregation is also considered. We find that the expected relationship between ligand-receptor binding kinetics and cellular responses can change significantly when signal transduction depends on a messenger or a partially modified receptor. In particular, cellular responses triggered by a messenger, such as a transcription factor that translocates from the membrane to the nucleus after receptor-mediated activation, can be sensitive or insensitive to a change in the lifetime of a ligand-receptor bond, depending on the parameters that govern the activation and decay of a messenger.

In response to antigenic stimuli, the multisubunit immune recognition receptors become aggregated and then phosphorylated on their cytoplasmic tyrosines. For the clonotypic receptors of B and T cells and for Fc receptors such as the high-affinity receptor for IgE (FcepsilonRI), a Src family kinase initiates this phosphorylation. We ask whether aggregation of the initiating kinase itself is required for signal transduction or whether, alternatively, a single associated kinase molecule can phosphorylate the receptors in an aggregate. We formulate the alternative molecular mechanisms mathematically and compare predictions with experimental findings on FcepsilonRI-bearing cells expressing varying amounts of the transfected Src family kinase Lyn. The data are consistent with the requirement of only a single Lyn molecule per FcepsilonRI aggregate to initiate signaling and are inconsistent with a mechanism requiring more than one Lyn molecule.

Important immune responses are linked to Src-mediated phosphorylation of cytoplasmic tyrosine-based motifs (ITAMs). However, the mechanism of how receptor ligation translates into ITAM phosphorylation remains elusive. Here, we use molecular dynamic simulations to explore the potential regulatory involvement of lipid membranes and their influence on the structure and behavior of the cytoplasmic portion of the CD3ɛ chain of the T-cell receptor. It has been hypothesized that the accessibility of ITAM motifs, such as those in the CD3ɛ cytoplasmic tail, can be blocked by ionic interactions between positively charged amino acids in receptor tails and negatively charged lipid head groups. This interesting hypothesis represents a previously unrecognized mechanism for control of receptor activation. Our simulations support the notion that the net charge of the lipids present in the membrane can affect peptide-membrane interactions. Results are consistent with experimental findings that show increase interfacial absorption in negatively charged lipid bilayer. Our simulations revealed the conformational variability of the disordered tail, which led to an additional focus on quantifying the interaction by free energy calculations, combined with long time-scale simulations using coarse-grained (CG) approaches. These studies will be extended to address how changes in ionic conditions can modulate phosphorylation of ITAM motifs and lead to regulation of activation of the TCR and other ITAM-bearing immunoreceptors.

It is often difficult to induce dissociation of multivalent antigens that are bound to cells by surface-associated immunoglobulin (sIg), even when high concentrations of monovalent hapten are used to fill all free binding sites. Here, Byron Goldstein and Carla Wofsy discuss the evidence that this appears to be due to a cellular response triggered by sIg crosslinking and propose that a receptor-desensitization process is responsible for the transition of multivalent antigens to a dissociation-resistant state.

Publisher Summary Interleukin-2 (IL-2) is a multifunctional cytokine that represents ligand recognition among the hematopoietin receptor family. To facilitate structure–function analysis of IL-2 and its receptor, the chapter describes a strategy of directed and stable solution assembly of receptor ectodomains. This strategy helps to prepare stable complexes of cytokine receptor ectodomains of defined composition and stoichiometry and that mimic the ligand binding characteristics of the equivalent cell surface receptor sites. The chapter demonstrates that it is possible to direct the assembly of cytokine receptor subunits in solution by employing coiled-coil molecular recognition. Heteromeric complexes can be prepared where the subunits function cooperatively to bind ligand. The use of idealized coiled-coil designs rather that naturally occurring sequences result in sub-nanomolar complex stabilities. The techniques of analytical ultracentrifugation and surface plasmon resonance help in quantitatively characterizing the complexes. Using these approaches and the present knowledge of coiled-coil molecular recognition, it should be possible to prepare soluble receptor complexes of defined stoichiometries and subunit composition.

The linker for activation of T cells (LAT), the linker for activation of B cells (LAB), and the linker for activation of X cells (LAX) form a family of transmembrane adaptor proteins widely expressed in lymphocytes. These scaffolding proteins have multiple binding motifs that, when phosphorylated, bind the SH2 domain of the cytosolic adaptor Grb2. Thus, the valence of LAT, LAB and LAX for Grb2 is variable, depending on the strength of receptor activation that initiates phosphorylation. During signaling, the LAT population will exhibit a time-varying distribution of Grb2 valences from zero to three. In the cytosol, Grb2 forms 1:1 and 2:1 complexes with the guanine nucleotide exchange factor SOS1. The 2:1 complex can bridge two LAT molecules when each Grb2, through their SH2 domains, binds to a phosphorylated site on a separate LAT. In T cells and mast cells, after receptor engagement, receptor phosphoyrlation is rapidly followed by LAT phosphorylation and aggregation. In mast cells, aggregates containing more than one hundred LAT molecules have been detected. Previously we considered a homogeneous population of trivalent LAT molecules and showed that for a range of Grb2, SOS1 and LAT concentrations, an equilibrium theory for LAT aggregation predicts the formation of a gel-like phase comprising a very large aggregate (superaggregate). We now extend this theory to investigate the effects of a distribution of Grb2 valence in the LAT population on the formation of LAT aggregates and superaggregate and use stochastic simulations to calculate the fraction of the total LAT population in the superaggregate.

Many ligands stimulate cellular responses by aggregating the cell-surface receptors to which they are bound. We investigated several mechanistic questions related to aggregation of receptors by using the high-affinity receptor for IgE (Fc epsilon RI) on mast cells as a model system. We briefly exposed cells to covalently cross-linked oligomers of IgE and then added excess monomeric IgE to prevent further aggregation. Early events were examined by monitoring the phosphorylation of protein tyrosines; later events were examined by monitoring secretion. We found that aggregated receptors continue to signal both late and early events in the absence of formation of new aggregates. Additional experiments suggested that the clustered receptors undergo a dynamic process of phosphorylation and dephosphorylation. Our findings suggest that for these and related receptors that function by aggregation, the persistence of signal transduction is directly related to the intrinsic affinity of the ligand for the individual receptor.

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.

The high affinity receptor for IL-2 is composed of at least two chains, a p55 chain that binds IL-2 with low affinity and a p75 chain that binds with intermediate affinity. Two molecular mechanisms have been proposed for the formation of the high affinity receptor-ligand complex: The affinity conversion model proposes that the high affinity receptor is formed via stepwise binding in which IL-2 first binds to the p55 chain and the resulting complex then associates with the p75 chain to form a high affinity ternary complex. In the performed heterodimer model the p55 and p75 chains form a non-covalently linked high affinity heterodimer in the absence of IL-2. We show that these two models can be distinguished on the basis of equilibrium binding experiments using cell lines expressing different numbers of p55 chains. To make this distinction we develop a general model for the interaction of IL-2 with its various receptors. We than analyze the case in which heterodimers exist in the absence of IL-2 and the case in which no preformed heterodimers exist. For both cases we predict the shape of equilibrium Scatchard plots. We then show that published IL-2 binding studies are consistent with a model in which a large concentration of preformed heterodimers is present on the cell surface and inconsistent with a model in which preformed heterodimers are absent from the cell surface. The models that we develop should have general applicability to the entire class of receptor systems in which low and intermediate affinity chains interact to constitute a high affinity receptor.

We study the dissociation of preformed bivalent ligand-bivalent receptor aggregates in solution, where the ligand is a symmetric bivalent hapten with two identical 2,4-dinitrophenyl (DNP) groups and the receptor is a fluorescein-labeled monoclonal anti-DNP IgE. We promote dissociation in two ways: by the addition of high concentrations of a monovalent hapten that competes for IgE binding sites with the bivalent hapten and by the addition of high concentrations of unlabeled IgE that binds almost all ligand binding sites that dissociate from labeled IgE. We investigate both theoretically and experimentally the two types of dissociation and find them to be quite different. Theory predicts that their kinetics will depend differently on the fundamental rate constants that characterize binding and aggregation. Using monovalent ligand to promote dissociation, we find that the fraction of labeled IgE sites bound to bivalent ligand decays with a slow and fast component. The fast decay corresponds to the dissociation of a singly bound DNP hapten. The interpretation of the slow decay depends on the detailed way in which ligand-receptor aggregates break up. We show that one possible explanation of these data is that small stable rings form before the addition of monovalent ligand. Other possible explanations are also presented.

In the autoimmune disease, Systemic Lupus Erythematosus, an individual produces antibodies that bind to his or her own DNA. In this paper we consider a single, long DNA-like molecule in a solution containing bivalent antibodies that can bind to the DNA molecule at regularly spaced sites. The antibody can be attached to DNA by either one or two binding sites. We assume that, when an antibody molecule binds through both its sites, it spans a fixed number of free sites that remain accessible to antibody binding. In this model, antibody molecules can interdigitate along the DNA molecule. We allow steric hindrance within such interdigitating clusters of bound antibodies. We derive analytical expressions for the average number of free, monovalently bound and bivalently bound antibodies, and see how this distribution is influenced by steric hindrance and by the relative binding strengths of the singly and doubly bound antibody.

Bretscher (1983) has shown that on uniformly spread giant HeLa cells, the receptors for low density lipoprotein (LDL) and transferrin are concentrated toward the periphery of the cells. To explain these nonuniform distributions, he proposed that on giant HeLa cells, recycling receptors return to the cell surface at the cell's leading edge. Since the distribution of coated pits on these cells is uniform, Bretscher and Thomson (1983) proposed that there is a bulk membrane flow toward the cell centers. Here we present a mathematical model that allows us to predict the distribution of cell surface proteins on a thin circular cell, when exocytosis occurs at the cell periphery and endocytosis occurs uniformly over the cell surface. We show that on such a cell, a bulk membrane flow will be generated, whose average velocity is zero at the cell center and increases linearly with the distance from the cell center. Our model predicts that proteins that aggregate in coated pits will have concentrations that are maximal at the cell periphery. We fit our theory to the data of Bretscher and Thomson (1983) on the distribution of ferritin receptors for the following cases: the receptors move by diffusion alone; they move by bulk membrane flow alone; they move by a combination of diffusion and bulk membrane flow. From our fits we show that tau m greater than 3.5 tau p, where tau m and tau p are the lifetimes of the membrane and the ferritin receptor on the cell surface, and that tau pD less than 6.9 X 10(-7) cm2, where D is the ferritin receptor diffusion coefficient. Surprisingly, we obtain the best fits to the data when we neglect membrane flow. Our model predicts that for proteins that are excluded from coated pits, the protein concentration will be Gaussian, being maximal at the cell center and decreasing with the distance from the cell center. If on giant HeLa cells a protein with such a distribution could be found, it would strongly support Bretcher's proposal that there is an inward membrane flow.

We extend the mathematical theory of hemolytic plaque growth to include plaques produced by cells secreting antibodies in layers of finite thickness. Previous theories have assumed that the layer was either two-dimensional or of infinite thickness. By using the method of images we derive an equation for the plaque radius as a function of time for layers of any thickness. We show that at short times and at long times the equation reduces to the appropriate infinite three-dimensional and two-dimensional limiting forms, and obtain expressions for estimating the range of times for which these limiting results are valid. For the liquid monolayer technique we obtain a new limiting result. The equation for the plaque radius is a transcendental equation which we solve numerically for a number of cases of interest. These results illustrate a variety of different features of plaque growth associated with the finite thickness of the layer. Experimental studies are usually carried out in layers whose thicknesses are not standardized. In the assays commonly used the thickness h can vary more than six hundred fold, i.e. 1 × 10 −3 cm ⪅ h ⪅ 6.5 × 10 −1 cm. Such variation in h will cause widely different kinetics of plaque growth. For typical plaque experiments of one hour duration the two-dimensional limit is valid when h ⪅ 3 × 10 −3 cm while the infinite thickness limit is valid when h ⪆ 10 −1 cm. For thicknesses in between these values the finite layer results must be used.

We are interested in the effect of receptor clustering on k+, the diffusion-limited forward rate constant for the binding of a ligand to a cell surface receptor. Here we estimate the reduction in k+ when receptors are clustered in various configurations. We obtain two alternative expressions for the flux of ligands into receptors distributed on a surface. Next we show through a variational principle that these provide both upper and lower bounds on the flux when evaluated for trial concentration functions which satisfy only the boundary conditions of the Laplace equation. We use an analogy with electrostatics to calculate rigorous bounds within approx. 10% of the exact result for a variety of planar clusters of hemispherical receptor sites. We also obtain an exact result for the flux into a spheroidal receptor and use this result to obtain bounds on the flux into certain receptor clusters.

The Zimm-Bragg theory is extended to treat the melting of the triple helix poly (A + 2U) for a solution with a 1 : 2 mole ratio of poly A to poly U. Only the case for long chains is considered. For a given set of parameters the theory predicts the fraction of segments in the triple helix, double helix, and random coil states as a function of temperature. Four nucleation parameters are introduced to describe the two order–disorder transitions (poly (A + 2U) ⇄ poly A + 2 poly U and poly (A + U) ⇄ poly A + poly U) and the single order–order transition (poly (A + 2U) ⇄ poly (A + U) + poly U). A relation between the nucleation parameters is obtained which reduces the number of independent parameters to three. A method for determining these parameters from experiment is presented. From the previously published data of Blake, Massoulie and Fresco8 for [Na+] = 0.04, we find σT = 6.0 × 10−4, σD = 1.0 × 10−3, and σσ* = 1.5 × 10−3. σT and σD are the nucleation parameters for nucleating a triple helix and double helix, respectively, from a random coil region. σσ* is the nucleation parameter for nucleating a triple helix from a double helix and a single strand. Melting curves are generated from the theory and compared with the experimental melting curves.

A simple model is introduced to investigate the stability of a sedimenting entanglement. The sedimenting entanglement is represented by a sedimenting sieve. Solvent can pass through it, but single-chain molecules that flow into it become entangled and their flow decreases or, if permanent entanglements form, ceases entirely. With this model we are able to find the conditions under which the mass of a sedimenting entanglement remains constant, grows or decays to a stable value, grows beyond limit, or decays to the mass of a single chain. The theory is applied to the sedimentation of small concentrations of large chain molecules in solutions of small chain molecules in solutions of small chain molecules for the case in which the entanglements are long-lived. Equations are derived which, (1) give the stable entanglement mass as a function of rotor speed and concentration and, (2) for a given concentration predict the rotor speed at which the entanglement mass grows without limit. Numerical results for small concentrations of T2 DNA sedimenting in solutions of T7 DNA are presented.

We are analysing the initial molecular events stimulated by the high-affinity receptor for IgE, FcεRI. Earlier studies have shown that the first response when the receptor-bound IgE interacts with a multivalent antigen is a transphosphorylation of receptor tyrosines, induced by the approximation of two or more receptors by a constitutively associated src-family kinase (Lyn). The amount of weakly associated kinase regulates the intensity of the response. Several aspects are being analyzed: (i) the sites on Lyn and the receptor that account for the constitutive interaction; (ii) how the intrinsic affinity of a ligand for the receptor-bound IgE influences the responses; and (iii) the mechanism(s) by which low-affinity ligands can act as antagonists. In the latter studies, mast cell responses were followed by monitoring the phosphorylation of tyrosines on several proteins and secretion. At equivalent levels of receptor phosphorylation, a ligand with high affinity stimulated vigorous phosphorylation of downstream components, whereas a low-affinity ligand was unable to stimulate phosphorylation of the same components effectively. Cells stimulated with a mixture of high- and low-affinity ligands, under a protocol where simple displacement of one by the other was prevented, remarkably showed that excess low-affinity ligand inhibited the phosphorylation as well as degranulation by the high-affinity ligand. This antagonism results from a competition for the limiting amount of the constitutive initiating kinase. Related receptors that depend on recruitment of initiating kinases may be subject to similar regulatory mechanisms.

We use the mathematical theory of plaque qrowth to determine if there is merit in performing a hemolytic plaque assay in the presence of an external electric field. In particular, we study the effects of an electric field on the transport of antibodies secreted by a single lymphocyte and on the size and shape of the plaques they produce. Our results indicate that in the presence of an applied electric field: (1) The mobility of the antibodies produced by the antibody forming cell can be determined from the plaque shape. (In the electric field the plaques are no longer circular, but cigar shaped.) (2) By changing the magnitude or direction of the applied electric field more than one plaque can be generated by a single AFC. Thus changes in mobility or the rate of antibody secretion can be assayed. (3) Plaques will reach a steady state size; for good emitters (cells that secrete antibodies at a high rate or that secrete high affinity antibodies) this steady state will be achieved rapidly. Equations are given which describe both the temporal development and steady state plaque size and shape. From the equations, computer generated plots of plaques produced by typical antibody forming cells are presented. These plots are then used to show how pictures of plaques formed in an electric field can be analyzed to determine the antibody mobility.

We present a model for the control of immunoglobulin E (IgE)-mediated histamine release from human basophils. We suggest that there is a calcium gating factor which interacts with crosslinked IgE to form a short-lived open calcium channel. After formation of the channel the activated gating factor rapidly decays to an inactive form. It is the loss of the active gating factor which causes the basophil to desensitize nonspecifically. We propose that the crosslinked IgE molecules are deactivated by a mechanism, such as endocytosis or shedding, which is independent of the mechanism which inactivates the calcium gating factor. This loss of functional IgE leads to specific desensitization. The mathematical formulation of the model explains the relationship of specific and nonspecific desensitization to the amount of specific IgE on the basophil surface; explains why there are two types of antigen excess inhibition; explains the relationship between antigen excess inhibition and desensitization; explains why, for a fixed antigen concentration, increasing the concentration of cell surface IgE increases histamine release until an optimal concentration is reached, then decreases histamine release; predicts the effects that changing the external calcium will have on the dose response curve; and predicts that increasing the amount of specific IgE on the cell surface will cause the dose response curve to undergo a transition from a curve with a single maximum to a curve with two maxima.