Recombinant interleukin-2 analogs. Dynamic probes for receptor structure.

Interleukin-2 (IL-2) and its receptor complex have become one of the most studied members of a growing family of protein hormones characterized by structural similarities in both ligands and their receptors. Structure-function studies of IL-2 have been complicated by the multimeric nature of its receptor. Two receptor subunits (55- and 75-kDa type I cell surface proteins) can participate to form the high affinity binding site. Although the IL-2 is apparently unique in some respects, similar subunit cooperativity has now been shown to be a common feature for other members of this receptor family. The availability of cell lines expressing the individual IL-2 receptor subunits has allowed detailed analysis of subunit binding characteristics. Results regarding the relationship of molecular recognition at each subunit to the mechanism of ligand binding at the high affinity site, however, have led to different interpretations. In this study we have employed previously prepared C-terminal IL-2 mutant proteins to examine receptor binding at all three classes using a variety of equilibrium and kinetic techniques. These results indicate that the high affinity IL-2 receptor complex includes the p55/p75 heterodimer prior to IL-2 binding and that both receptor subunits participate simultaneously in ligand capture.     

Kinetic analysis of ligand binding to interleukin-2 receptor complexes created on an optical biosensor surface.

The interleukin-2 receptor (IL-2R) is composed of at least three cell surface subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma c. On activated T-cells, the alpha- and beta-subunits exist as a preformed heterodimer that simultaneously captures the IL-2 ligand as the initial event in formation of the signaling complex. We used BIAcore to compare the binding of IL-2 to biosensor surfaces containing either the alpha-subunit, the beta-subunit, or both subunits together. The receptor ectodomains were immobilized in an oriented fashion on the dextran matrix through unique solvent-exposed thiols. Equilibrium analysis of the binding data established IL-2 dissociation constants for the individual alpha- and beta-subunits of 37 and 480 nM, respectively. Surfaces with both subunits immobilized, however, contained a receptor site of much higher affinity, suggesting the ligand was bound in a ternary complex with the alpha- and beta-subunits, similar to that reported for the pseudo-high-affinity receptor on cells. Because the binding responses had the additional complexity of being mass transport limited, obtaining accurate estimates for the kinetic rate constants required global fitting of the data sets from multiple surface densities of the receptors. A detailed kinetic analysis indicated that the higher-affinity binding sites detected on surfaces containing both alpha- and beta-subunits resulted from capture of IL-2 by a preformed complex of these subunits. Therefore, the biosensor analysis closely mimicked the recognition properties reported for these subunits on the cell surface, providing a convenient and powerful tool to assess the structure-function relationships of this and other multiple subunit receptor systems.     

Possible mechanism for the alpha subunit of the interleukin-2 receptor (CD25) to influence interleukin-2 receptor signal transduction

The receptors for interleukin 2 (IL-2) and interleukin 15 (IL-15) in T cells share the IL-2R beta subunit (CD122) and gamma(C) subunit but have private alpha subunits. Despite utilizing the same receptor chains known to be necessary and sufficient to transduce IL-2 signals the two cytokines manifest different cellular effects. It is commonly held that the alpha subunit of the IL-2R (CD25) is involved solely in the generation of a high affinity receptor complex. This is questioned by the development of autoimmune diseases in instances where the expression of CD25 is absent. The timely expression of CD25 in the thymus has been linked with clonal deletion. Evidence from peripheral T cells indicates that survival signals arising from the intermediate affinity IL-2R (lacking CD25) do not require the activation of Janus kinase 3 (Jak3) but do require the presence of the membrane proximal region of the gamma(C) chain. This particular signalling pathway is not observed in the high affinity receptor complex where Jak3 is activated. Recent data point to CD25 having a surface distribution consistent with it being localized within membrane microdomains. Here we suggest that in the absence of CD25 expression, IL-2R activation occurs within the soluble membrane fraction. This membrane environment and the absence of CD25 promotes Jak3 independent signal transduction and induction of antiapoptotic mechanisms. T cell antigen receptor (TCR) signalling leads to the induction of CD25 expression, which localizes to membrane microdomains. There is a dynamic pre-association of CD25 and CD122 leading to the loose association of the heterodimer with membrane microdomains. High affinity IL-2R signalling in the context of CD25 and the microdomain environment is characterized by Jak3 activation. The relative levels of high to intermediate affinity receptor signalling determines whether a cell proliferates or undergoes activation induced cell death dependent upon cell status.     

The murine interleukin 2 receptor. Irreversible cross-linking of radiolabeled interleukin 2 to high affinity interleukin 2 receptors reveals a noncovalently associated subunit

The murine interleukin 2 (IL-2) receptor is a 55- to 60-kDa glycoprotein (p58) that binds IL-2 at a high and low affinity. In this investigation, we have identified sublines of EL4 that vary in their capacity to express high affinity IL-2 receptors after transfection of the IL-2 receptor cDNA. These and other cell populations were used to determine whether unique membrane molecules were specifically associated with the high affinity IL-2 receptor. Irreversible chemical cross-linking of [125I]IL-2 to only high affinity IL-2 receptors resulted in detection of IL-2 cross-linked to p58 as a 70- to 75-kDa band and other complexes of 90 to 95 kDa, 115 kDa, 150 kDa, 170 to 190 kDa, and 245 kDa. Antibodies specific for p58 resulted in precipitation of each of these complexes. However, disruption of noncovalent interactions prior to immunoprecipitation resulted in an inability to detect the material at 90 to 95 kDa. Therefore, we conclude that this complex most likely represented IL-2 cross-linked to a 75- to 80-kDa subunit that was noncovalently associated with p58. The other complexes greater than 150 kDa may represent these subunits cross-linked to each other. The detection of all the cross-linked complexes larger than 75 kDa appeared to be directly related to formation of high affinity IL-2 receptors because IL-2 was cross-linked only to p58 for three cell lines that exclusively expressed low affinity IL-2 receptors. Thus, high affinity murine IL-2 receptors are comprised of at least one alpha (p58)- and beta (p75)-subunit. Our data also raise the possibility of a more complex subunit structure.     

Characterization of the heterodimeric complex of human IL-2 receptor alpha.beta chains reconstituted in a mouse fibroblast cell line, L929

The receptor for IL-2 has been known to exist in three forms on the basis of their affinities to IL-2: high, intermediate, and low affinity forms. Two IL-2R components have been identified as IL-2R alpha (p55, Tac Ag) and IL-2R beta (p70-75) chains, both bind IL-2 with low and intermediate affinities, respectively. Recently, we cloned human IL-2R beta chain cDNA and demonstrated that the cDNA product binds IL-2 with intermediate affinity and forms high affinity IL-2R with coexpressed IL-2R alpha chain in a human T cell line, Jurkat. In this study, we report the establishment of the mouse fibroblast transformants expressing either the IL-2R beta chain alone or both the IL-2R alpha and IL-2R beta chains. In contrast to lymphoid cells, significant IL-2 binding was not detected in the transformants expressing the IL-2R beta chain alone at IL-2 concentrations (50 pM to 10 nM) generally utilized. Nonetheless, the transformants expressing both IL-2R alpha and IL-2R beta chains displayed two forms of the IL-2R with high and low affinities to IL-2. However, neither IL-2 internalization nor signal transduction via the high affinity IL-2R complex were observed in the L929 transformants. Those findings suggest that the interaction of the IL-2R beta chain with the IL-2R alpha chain occurs in the absence of additional lymphoid specific component(s) to form high affinity IL-2R, but that this interaction is insufficient for IL-2 internalization and signal transduction just as observed in lymphoid cells. The experimental approach described here may allow further dissection of the molecular architecture of the IL-2R complex in the ligand binding, internalization, and signal transduction.     

Subunit structure of the high and low affinity human interleukin-15 receptors

Radio-iodinated cytokines and monoclonal antibodies directed at the IL-2R beta- and gamma-chains were used to analyze the structure of the cell-surface IL-15 and IL-2 receptors expressed by the human lymphoma cell clone YT-2C2. YT-2C2 cells are IL-2R alpha negative and express IL-2R gamma (15,000 molecules/cell) in excess of IL-2R beta (11,000 molecules/cell). Accordingly, they display a number of beta/gamma complexes of intermediate affinity for IL-2 and IL-15 which is equivalent to the number of beta-chains. Both cytokines compete for binding to this beta/gamma complex. There are about 800 high affinity IL-15 receptors, suggesting the presence of a similar number of IL-15R alpha-chains. Within the common intermediate affinity beta/gamma-complex, the anti-beta-chain A41 mAb defines an epitope which is similarly engaged in IL-2 and IL-15 binding, whereas the anti-beta-chain 284 mAb defines an epitope which does not display similar interaction with either cytokines. Thus, although IL-2 and IL-15 compete for binding to this beta/gamma-complex, they do not use similar binding areas. Cross-linking and immunoprecipitation experiments have shown that the high affinity IL-15 receptors comprises IL-2R beta/gamma, in association with IL-15R alpha and that the three chains can be efficiently cross-linked to IL-15 and co-immunoprecipitated. Contrary to the intermediate affinity situation, high affinity IL-15 binding and subunit cross-linking were not affected by excess amounts of IL-2, A41 or 284 mAb, suggesting that when engaged in the IL-15 high affinity complex, the beta- and gamma-chains adopt different conformations, at least with respect to IL-15 binding. Finally, we provide evidence for the participation of a novel 35 kDa component within the high affinity structure. This component is immunoprecipitated with anti-IL-2R gamma mAb but not with anti-IL-2R beta mAb and might correspond to a truncated form of IL-2R gamma-chain.     

Binding of interleukin 2 to its 75-kDa intermediate affinity receptor is sufficient to activate Na+/H+ exchange

High affinity interleukin 2 (IL-2) binding sites are composed of two IL-2-binding molecules: one of 55 kDa, commonly called TaC, and another of 75 kDa. In the absence of the other IL-2-binding molecule, the 55-kDa molecule binds IL-2 with a relatively low affinity and the 75 kDa molecule binds IL-2 with an intermediate affinity. One of the earliest events following interaction of IL-2 with its receptor on the surface of cells is an increase in intracellular pH due to activation of the Na+/H+ antiport. In contrast to IL-2-induced proliferation of human IL-2-sensitive T cells, interaction of IL-2 with a low affinity binding site was sufficient to activate the Na+/H+ antiport. By determining the effect of IL-2 on cytosolic pH in cells that express one of the two IL-2-binding molecules in the absence of the other IL-2-binding molecule, we have demonstrated that interaction of IL-2 with the 75 kDa IL-2-binding molecule is sufficient to activate the Na+/H+ antiport and thus induce cytosolic alkalinization. This indicates that the 75-kDa IL-2-binding molecule, in the absence of the 55-kDa IL-2-binding molecule, forms a functional receptor that can transduce an activation signal across the cell membrane.     

Receptor binding and internalization of mouse interleukin-2 derivatives that are partial agonists.

Mouse interleukin-2 (mIL-2) mutant proteins with subunit-specific receptor binding defects have been previously described. Some of these mutant proteins are unable to trigger a maximum proliferative response of T cells. In this study, mIL-2 and mIL-2 mutant proteins were labeled with 32P, and their association and dissociation kinetics with the high affinity IL-2 receptor (IL-2R) were investigated. A mIL-2 mutant protein with a partial defect in binding to the low affinity component of IL-2R had a slower on-rate than mIL-2. On the other hand, a mIL-2 antagonist with a binding defect to the intermediate affinity component of IL-2R had a normal on-rate, whereas its off-rate at 37 degrees C was faster than mIL-2. This fast off-rate at physiological temperature interfered with mIL-2 internalization. When three mIL-2 partial agonists, each inducing a different maximal response, were examined, no difference was found between their dissociation rates or their internalization properties. The significance of these findings for the function of each receptor subunit in the IL-2R complex, as well as for the mechanism of activation of the receptor, is discussed.     

Putative gamma-subunit of the IL-2 receptor is detected in low, intermediate, and high affinity IL-2 receptor-bearing cells

Three forms of the IL-2R have been reported: low, intermediate, and high affinity. Each form correlates with the expression of two different chains: p55 (alpha-subunit) and p75 (beta-subunit). We report here a putative new subunit of the IL-2R, termed p95-110 or gamma-subunit. This new subunit has a molecular mass of 95 to 110 kDa and is expressed in low, intermediate, and high affinity IL-2R-bearing cells. We propose that p95-110 is the partner of p75 in the formation of intermediate affinity IL-2R, inasmuch as neither p75 nor p95-110 alone can bind IL-2.     

Heterodimerization of a functional GABAB receptor is mediated by parallel coiled-coil alpha-helices.

A detailed understanding of GABAB receptor assembly is an important issue in view of its role as attractive target for treatment of epilepsy, anxiety, depression, cognitive defects, and nociceptive disorders. Heteromerization of GABAB-R1 and GABAB-R2 subunits is a prerequisite for the formation of a functional GABAB receptor. Each individual subunit contains one stretch of approximately 30 amino acid residues within its intracellular C-terminal domain that mediates heteromer formation. To investigate the mechanism of the GABAB-R1/GABAB-R2 interaction and to assess the subunit stoichiometry of the complex, recombinant polypeptide chain fragments containing the heteromerization site were produced by heterologous gene expression in Escherichia coli. When mixed in equimolar amounts, these peptides preferentially formed parallel coiled-coil heterodimers under physiological buffer conditions. This demonstrates that the short C-terminal regions are sufficient to determine the specificity of interaction between GABAB receptor subunits. In contrast, isolated GABAB-R1 peptides folded into relatively unstable homodimers, whereas GABAB-R2 peptides were largely unstructured. Together with the data reported in the literature, the results presented here indicate that the functional GABAB receptor is a heterodimer assembled by parallel coiled-coil alpha-helices.     

Interleukin-11 signals through the formation of a hexameric receptor complex

Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. IL-11 has been shown to induce gp130-dependent signaling through the formation of a high affinity complex with the IL-11 receptor (IL-11R) and gp130. Site-directed mutagenesis studies have identified three distinct receptor binding sites of IL-11, which enable it to form this high affinity receptor complex. Here we present data from immunoprecipitation experiments, using differentially tagged forms of ligand and soluble receptor components, which show that multiple copies of IL-11, IL-11R, and gp130 are present in the receptor complex. Furthermore, it is demonstrated that sites II and III of IL-11 are independent gp130 binding epitopes and that both are essential for gp130 dimerization. We also show that a stable high affinity complex of IL-11, IL-11R, and gp130 can be resolved by nondenaturing polyacrylamide gel electrophoresis, and its composition verified by second dimension denaturing polyacrylamide gel electrophoresis. Results indicate that the three receptor binding sites of IL-11 and the Ig-like domain of gp130 are all essential for this stable receptor complex to be formed. We therefore propose that IL-11 forms a hexameric receptor complex composed of two molecules each of IL-11, IL-11R, and gp130.     

Molecular cloning of the guinea-pig IL-5 receptor alpha and beta subunits and reconstitution of a high affinity receptor

The functional IL-5 receptor is a heteromeric complex consisting of an alpha and beta subunit. The cloning, sequencing and expression of guinea-pig IL-5Ralpha and beta subunits is described. The guinea-pig IL-5Ralpha subunit cDNA encodes a protein of M(r)47 kDa, which is 72 and 66% homologous to the human and murine orthologs, respectively. Three guinea-pig IL-5Rbeta subunit cDNA clones were isolated, which differ in the N-terminus and are 56-64% homologous to the human and murine IL-5Rbeta subunits. Expressing human IL-5Ralphabeta and guinea-pig IL-5Ralphabeta(1)in the baculovirus-insect cell system resulted in recombinant receptors which bound hIL-5 with high affinity (K(d)=0.19 and 0.11 nM, respectively). Expressing just gpIL-5Ralpha was not sufficient to demonstrate binding. This contrasts with the human receptor, where hIL-5Ralpha alone can bind hIL-5 with high affinity. gpIL-5Ralphabeta(1)bound both hIL-5 and mIL-5 with comparable affinity (K(i)=0.10 and 0.06 nM), similar to that seen with hIL-5Ralphabeta. Thus, both the heteromeric hIL-5R and gpIL-5Ralphabeta(1)can bind multiple IL-5 orthologs with high affinity whereas the murine IL-5R is selective for the murine ligand.     

The 75,000-dalton interleukin-2 receptor transmits a signal for the activation of a tyrosine protein kinase

The high-affinity receptor for interleukin-2 (IL-2) is composed of two distinct subunits with molecular weights of 55,000 and 75,000 (p55 and p75). While the presence of the high-affinity receptor requires the simultaneous expression of p55 and p75, these subunits can also be expressed independently, resulting in IL-2 receptors with low and intermediate affinities, respectively. IL-2 can induce proliferation in cells expressing either the intermediate affinity p75 receptor or the p55.p75 high-affinity complex, suggesting that p75 is responsible for signal transduction. We have previously shown that signal transduction by the high-affinity IL-2 receptor involves the activation of a tyrosine protein kinase. In order to evaluate the role of p75 in the activation of this kinase we assessed the ability of IL-2 to induce the activation of a tyrosine protein kinase in the human leukemic cell lines Hut 78 and YT. These cells express p75 as the predominant IL-2 receptor. IL-2-dependent tyrosine phosphorylation was observed in both cell lines and the concentrations of IL-2 needed to stimulate this phosphorylation were similar to that required for binding to the p75 receptor. Antibodies that inhibit binding of IL-2 to p55 had no effect on the IL-2-induced tyrosine phosphorylations in YT cells, while antibodies that block the binding of IL-2 to p75 completely inhibited the phosphorylations. These results demonstrate that the signaling capacity for the IL-2-induced tyrosine phosphorylation resides in the p75 receptor.     

A fusion protein of the gp130 and interleukin-6Ralpha ligand-binding domains acts as a potent interleukin-6 inhibitor

Interleukin (IL)-6 is involved in the maintenance and progression of several diseases such as multiple myeloma, rheumatoid arthritis, or osteoporosis. The present work aims at the development of an IL-6 inhibitor for the use in anti-cytokine therapies. The IL-6 receptor is composed of two different subunits, an alpha-subunit (IL-6Ralpha) that binds IL-6 with low affinity and a beta-subunit (gp130) that binds the IL-6.IL-6Ralpha complex with high affinity and as a result triggers intracellular signaling. In its soluble form, gp130 is a natural antagonist that neutralizes IL-6.soluble IL-6Ralpha complexes. It was our strategy to appropriately fuse the two receptor subunit fragments involved in IL-6 receptor complex formation to bind IL-6 with high affinity and to antagonize its effects. The ligand-binding domains of gp130 (D1-D2-D3) and IL-6Ralpha (D2-D3) were connected using three different linkers. The resulting constructs were expressed in stably transfected insect cells and tested for their ability to inhibit IL-6 activity in several in vitro systems. All fusion proteins were strong inhibitors of IL-6 signaling and abrogated IL-6-induced phosphorylation of STAT3, proliferation of transfected Ba/F3 cells, and induction of acute-phase protein synthesis. As intended, the fused receptors were much more effective than the separately expressed soluble receptor proteins. The fusion protein strategy presented here can also be applied to other cytokines that signal via receptors composed of two different subunits to design new potent inhibitors for anti-cytokine therapies.     

Condensin but not cohesin SMC heterodimer induces DNA reannealing through protein-protein assembly

Condensin and cohesin are chromosomal protein complexes required for chromosome condensation and sister chromatid cohesion, respectively. They commonly contain the SMC (structural maintenance of chromosomes) subunits consisting of a long coiled-coil with the terminal globular domains and the central hinge. Condensin and cohesin holo-complexes contain three and two non-SMC subunits, respectively. In this study, DNA interaction with cohesin and condensin complexes purified from fission yeast was investigated. The DNA reannealing activity is strong for condensin SMC heterodimer but weak for holo-condensin, whereas no annealing activity is found for cohesin heterodimer SMC and Rad21-bound heterotrimer complexes. One set of globular domains of the same condensin SMC is essential for the DNA reannealing activity. In addition, the coiled-coil and hinge region of another SMC are needed. Atomic force microscopy discloses the molecular events of DNA reannealing. SMC assembly that occurs on reannealing DNA seems to be a necessary intermediary step. SMC is eliminated from the completed double-stranded DNA. The ability of heterodimeric SMC to reanneal DNA may be regulated in vivo possibly through the non-SMC heterotrimeric complex.     

Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP: fused receptor domains act as high affinity cytokine-binding proteins

Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6Ralpha and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFPIL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6Ralpha, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6.IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors.     

IL-4 inhibits the expression of high affinity IL-2 receptors on monoclonal human B cells

In the presence of anti-mu antibodies (anti-microAb), monoclonal B lymphocytes from patients suffering from B type chronic lymphocytic leukemia (B-CLL) can respond to IL-2. In contrast to the effect it exerts on normal B cells, IL-4 does not promote DNA synthesis by B-CLL lymphocytes. Rather this interleukin inhibits the response to IL-2 in all patients' cells that responded to this interleukin. We thus examined whether IL-4 would modulate the number and/or the affinity of IL-2 receptors. A 3-day activation of cells by anti-microAb induced a few hundred high affinity IL-2 receptors (HA-IL-2R) on B-CLL cell surface, as determined by Scatchard analysis. Treatment of cells with IL-4 caused a marked decrease in the number of HA-IL-2R without interfering with the binding ability of IL-2. In contrast with this profound suppressive effect, IL-4 did not down-regulate the expression of each chain, alpha and beta (p55 and p75, respectively), of the HA-IL-2R heterodimer. In fact, the expression of alpha and beta induced by anti-microAb was enhanced by IL-4. Altogether, IL-4 exerts a critical influence on the function and the configuration of HA-IL-2R without inhibiting the expression of two subunits, alpha and beta.     

Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells

We have used site-directed insertion and point mutagenesis in an attempt to increase the cytotoxic potency and receptor-binding affinity of the diphtheria-toxin-related interleukin-2 (IL-2) fusion toxins. Previous studies have demonstrated that both the DAB486-IL-2 and DAB389-IL-2 forms of the fusion toxin consist of three functional domains: the N-terminal fragment-A-associated ADP-ribosyltransferase, the hydrophobic-membrane-associating domains, and the C-terminal receptor-binding domain of human IL-2. By insertion mutagenesis we have increased the apparent flexibility of the polypeptide chain between the membrane-associating domains and the receptor-binding domain of this fusion toxin. In comparison to DAB486-IL-2, the cytotoxic potency of the insertion mutants was increased by approximately 17-fold for high-affinity IL-2-receptor-bearing cell lines in vitro. Moreover, competitive displacement experiments using [125I]rIL-2 demonstrate that the increase in cytotoxic potency correlates with an increase in receptor-binding affinity for both the high and intermediate forms of the IL-2 receptor.     

Transforming growth factor-beta (TGF-beta) binding to the extracellular domain of the type II TGF-beta receptor: receptor capture on a biosensor surface using a new coiled-coil capture system demonstrates that avidity contributes significantly to high affinity binding

Mature TGF-beta isoforms, which are covalent dimers, signal by binding to three types of cell surface receptors, the type I, II and III TGF-beta receptors. A complex composed of the TGF-beta ligand and the type I and II receptors is required for signaling. The type II receptor is responsible for recruiting TGF-beta into the heteromeric ligand/type I receptor/type II receptor complex. The purpose of this study was to test for the extent that avidity contributes to receptor affinity. Using a surface plasmon resonance (SPR)-based biosensor (the BIACORE), we captured the extracellular domain of the type II receptor (TbetaRIIED) at the biosensor surface in an oriented and stable manner by using a de novo designed coiled-coil (E/K coil) heterodimerizing system. We characterized the kinetics of binding of three TGF-beta isoforms to this immobilized TbetaRIIED. The results demonstrate that the stoichiometry of TGF-beta binding to TbetaRIIED was one dimeric ligand to two receptors. All three TGF-beta isoforms had rapid and similar association rates, but different dissociation rates, which resulted in the equilibrium dissociation constants being approximately 5pM for the TGF-beta1 and -beta3 isoforms, and 5nM for the TGF-beta2 isoform. Since these apparent affinities are at least four orders of magnitude higher than those determined when TGF-beta was immobilized, and are close to those determined for TbetaRII at the cell surface, we suggest that avidity contributes significantly to high affinity receptor binding both at the biosensor and cell surfaces. Finally, we demonstrated that the coiled-coil immobilization approach does not require the purification of the captured protein, making it an attractive tool for the rapid study of any protein-protein interaction.     

Receptor binding properties of four-helix-bundle growth factors deduced from electrostatic analysis

Hormones of the hematopoietin class mediate signal transduction by binding to specific transmembrane receptors. Structural data show that the human growth hormone (hGH) forms a complex with a homodimeric receptor and that hGH is a member of a class of hematopoietins possessing an antiparallel 4-α-helix bundle fold. Mutagenesis experiments suggest that electrostatic interactions may have an important influence on hormone-receptor recognition. In order to examine the specificity of hormone-receptor complexation, an analysis was made of the electrostatic potentials of hGH, interleukin-2 (IL-2), interleukin-4 (IL-4), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the hGH and IL-4 receptors. The binding surfaces of hGH and its receptor, and of IL-4 and its receptor, show complementary electrostatic potentials. The potentials of the hGH and its receptor display approximately 2-fold rotational symmetry because the receptor subunits are identical. In contrast, the potentials of GM-CSF and IL-2 lack such symmetry, consistent with their known high affinity for hetero-oligomeric receptors. Analysis of the electrostatic potentials supports a recently proposed hetero-oligomeric model for a high-affinity IL-4 receptor and suggests a possible new receptor binding mode for G-CSF; it also provides valuable information for guiding structural and mutagenesis studies of signal-transducing proteins and their receptors.