Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes

To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.     

Analysis of the interaction between human interleukin-5 and the soluble domain of its receptor using a surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used to study the interaction of human interleukin-5 (hIL5) with its receptor. IL5 is a major growth factor in the production and activation of eosinophilis. The receptor for IL5 is composed of two subunits, α and β. The α subunit provides the specificity for IL5 and consist of an extracellular soluble domain, a single transmembrane region and a cytoplasmic tail. We expressed the soluble domain of the human IL5 receptor α subunit (shIL5Rα) and human IL5 (hIL5) in Drosophila. Both hIL5 and shIL5Rα were immobilized separately through amine groups onto the carboxylated dextran layer of sensor chips of the BIAcore? (Pharmacia) SPR biosensor after N-hydroxysuccinimide/carbodiimide activation of the chip surface. Interactions were measured for the complementary macromolecule, either shIL5Rα or hIL5, in solution. Kinetics of binding of soluble analyst to immobilized ligand were measured and from this the association rate constant, dissociation rate constant and equilibrium dissociation constant (K_d) were derived. With immobilized shIL5Rα and soluble hIL5, the measured K_d was 2 nM . A similar value was obtained by titration calorimetry. The K_d for Drosophila expressed receptor and IL5 is higher than the values reported for proteins expressed in different systems, likely due to differences in the methods of interaction analysis used for differences in protein glycosylation. Receptor-IL5 binding was relatively pH independent between pH 6.5 and 9.5. Outside this range the dissociation rate increased with compressibility little increased in association rate. The values obtained for the interaction of hIL5 and shIL5Rα were found to depend on which component was immobilized; the K_d was 5.5 nM with immobilized hIL5 and soluble shIL5Rα. The SPR biosensor provides a unified methodology to measure the interaction properties of shIL5Rα and hIL5 derivatives, mutants and mimetic as well as to evaluate potential antagonists of the receptor-cytokine interaction.     

Peptide insertions in domain 4 of hbeta(c), the shared signalling receptor subunit for GM-CSF, IL3 and IL5, induce ligand-independent activation

A mutant form of the common beta-subunit of the GM-CSF, interleukin-3 (IL3) and IL5 receptors is activated by a 37 residue duplicated segment which includes the WSXWS motif and an adjacent, highly conserved, aliphatic/basic element. Haemopoietic expression of this mutant, hbeta(c)FIDelta, in mice leads to myeloproliferative disease. To examine the mechanism of activation of this mutant we targetted the two conserved motifs in each repeat for mutagenesis. Here we show that this mutant exhibits constitutive activity in BaF-B03 cells in the presence of mouse or human GM-CSF receptor alpha-subunit (GMRalpha) and this activity is disrupted by mutations of the conserved motifs in the first repeat. In the presence of these mutations the receptor reverts to an alternative conformation which retains responsiveness to human IL3 in a CTLL cell line co-expressing the human IL3 receptor alpha-subunit (hIL3Ralpha). Remarkably, the activated conformation is maintained in the presence of substitutions, deletions or replacement of the second repeat. This suggests that activation occurs due to insertion of extra sequence after the WSXWS motif and is not dependent on the length or specific sequence of the insertion. Thus hbeta(c) displays an ability to fold into functional receptor conformations given insertion of up to 37 residues in the membrane-proximal region. Constitutive activation most likely results from a specific conformational change which alters a dormant, inactive receptor complex, permitting functional association with GMRalpha and ligand-independent mitogenic signalling.     

Sp1 represses IL-2 receptor alpha chain gene expression

Sp1 is a DNA-binding protein that acts as a positive regulator of eukaryotic gene expression. The interleukin-2 receptor alpha chain (IL2R alpha) gene 5' regulatory region contains a single Sp1 consensus motif that overlaps a CArG box capable of binding serum response factor (SRF). The CArG box has previously been shown to be important for IL2R alpha gene expression. In this study, the results of competition experiments suggest that Sp1 and SRF compete for binding to the CArG region. Site-directed mutagenesis and transient transfection assays indicate that the IL2R alpha gene Sp1 serves the unusual role of repressing gene expression, most likely by competing for binding of nuclear factor(s) to the CArG box.     

Three residues in the common beta chain of the human GM-CSF, IL-3 and IL-5 receptors are essential for GM-CSF and IL-5 but not IL-3 high affinity binding and interact with Glu21 of GM-CSF.

The beta subunit (beta c) of the receptors for human granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is essential for high affinity ligand-binding and signal transduction. An important feature of this subunit is its common nature, being able to interact with GM-CSF, IL-3 and IL-5. Analogous common subunits have also been identified in other receptor systems including gp130 and the IL-2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site-directed mutagenesis and binding assays with radiolabelled GM-CSF, IL-3 and IL-5 to identify residues in the beta c subunit involved in affinity conversion for each ligand. Alanine substitutions in the region Tyr365-Ile368 in beta c showed that Tyr365, His367 and Ile368 were required for GM-CSF and IL-5 high affinity binding, whereas Glu366 was unimportant. In contrast, alanine substitutions of these residues only marginally reduced the conversion of IL-3 binding to high affinity by beta c. To identify likely contact points in GM-CSF involved in binding to the 365-368 beta c region we used the GM-CSF mutant eco E21R which is unable to interact with wild-type beta c whilst retaining full GM-CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chains Y365A, H367A and I368A than to those composed of wild-type beta c or mutant E366A. These results (i) identify the residues Tyr365, His367 and Ile368 as critical for affinity conversion by beta c, (ii) show that high affinity binding of GM-CSF and IL-5 can be dissociated from IL-3 and (iii) suggest that Tyr365, His367 and Ile368 in beta c interact with Glu21 of GM-CSF.     

A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.     

Human interleukin-3 (IL-3) induces disulfide-linked IL-3 receptor alpha- and beta-chain heterodimerization, which is required for receptor activation but not high-affinity binding.

The human interleukin-3 receptor (IL-3R) is a heterodimer that comprises an IL-3 specific alpha chain (IL-3R alpha) and a common beta chain (beta C) that is shared with the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5. These receptors belong to the cytokine receptor superfamily, but they are structurally and functionally more related to each other and thus make up a distinct subfamily. Although activation of the normal receptor occurs only in the presence of ligand, the underlying mechanisms are not known. We show here that human IL-3 induces heterodimerization of IL-3R alpha and beta c and that disulfide linkage of these chains is involved in receptor activation but not high-affinity binding. Monoclonal antibodies (MAb) to IL-3R alpha and beta c were developed which immunoprecipitated, in the absence of IL-3, the respective chains from cells labelled with 125I on the cell surface. However, in the presence of IL-3, each MAb immunoprecipitated both IL-3R alpha and beta c. IL-3-induced receptor dimers were disulfide and nondisulfide linked and were dependent on IL-3 interacting with both IL-3R alpha and beta c. In the presence of IL-3 and under nonreducing conditions, MAb to either IL-3R alpha or beta c immunoprecipitated complexes with apparent molecular weights of 215,000 and 245,000 and IL-3R alpha and beta c monomers. Preincubation with iodoacetamide prevented the formation of the two high-molecular-weight complexes without affecting noncovalent dimer formation or high-affinity IL-3 binding. Two-dimensional gel electrophoresis and Western blotting (immunoblotting) demonstrated the presence of both IL-3R alpha and beta c in the disulfide-linked complexes. IL-3 could also be coimmunoprecipitated with anti-IL-3R alpha or anti-beta c MAB, but it was not covalently attached to the receptor. Following IL-3 stimulation, only the disulfide-linked heterodimers exhibited reactivity with antiphosphotyrosine antibodies, with beta c but not IL-3R alpha being the phosphorylated species. A model of IL-3R activation is proposed which may be also applicable to the related GM-CSF and IL-5 receptors.     

Identification of residues involved in binding of IL5 to betacom using betaIL3 and betacom chimeras

In mice there are two forms of the beta chain used in the IL3 receptor system, betacom and betaIL3. betacom is used by the IL3, IL5 and GM-CSF receptors whereas betaIL3 is only used in the IL3 receptor. In this work an assay was developed to identify residues of beta1L3 that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the betaIL3 B'-C' loop were replaced with betacom sequence a form of betaIL3 was produced that was able to respond to IL5. This region is also responsible for IL3 binding to betaIL3 in the absence of alpha chain. It is therefore an important structural motif of betacom and betaIL3 responsible for both ligand interaction and specificity.     

Slow-dissociation effect of common signaling subunit beta c on IL5 and GM-CSF receptor assembly

Receptor activation by IL5 and GM-CSF is a sequential process that depends on their interaction with a cytokine-specific subunit alpha and recruitment of a common signaling subunit beta (betac). In order to elucidate the assembly dynamics of these receptor subunits, we performed kinetic interaction analysis of the cytokine-receptor complex formation by a surface plasmon resonance biosensor. Using the extracellular domains of receptor fused with C-terminal V5-tag, we developed an assay method to co-anchor alpha and betac subunits on the biosensor surface. We demonstrated that dissociation of the cytokine-receptor complexes was slower when both subunits were co-anchored on the biosensor surface than when alpha subunit alone was anchored. The slow-dissociation effect of betac had a similar impact on GM-CSF receptor stabilization to that of IL5. The effects were abolished by alanine replacement of either Tyr18 or Tyr344 residue in betac, which together constitute key parts of a cytokine binding epitope. The data argue that betac plays an important role in preventing the ligand-receptor complexes from rapidly dissociating. This slow-dissociation effect of betac explains how, when multiple betac cytokine receptor alpha subunits are present on the same cell surface, selective betac usage can be controlled by sequestration in stabilized cytokine-alpha-betac complexes.     

Regulation of the human interleukin-2 receptor alpha chain promoter: activation of a nonfunctional promoter by the transactivator gene of HTLV-I

We have characterized regulatory regions of the human IL-2 receptor alpha chain (IL2R alpha) promoter. 5' deletion constructs extending to -327 directed CAT expression in HTLV-I-infected T cells, which express IL2R alpha constitutively, and in Jurkat cells, which express IL2R alpha only after induction. Deletions to -267 and -265 were active only in HTLV-I-transformed T cells, but their activity in Jurkat cells was restored by cotransfection of a construct expressing the HTLV-I transactivator protein (tat-I). However, HTLV-I-infected human osteosarcoma cells do not express IL2R alpha-CAT constructs. Thus cell-type-specific factors are required for IL2R alpha expression, and direct or indirect interaction(s) between tat-I and a specific region of the IL2R alpha promoter may cause altered regulation. Tat-I also augments IL2-CAT expression under some conditions, suggesting possible autocrine or paracrine mechanisms for HTLV-I-induced leukemogenesis.     

Coiled coil miniprotein randomization on phage leads to charge pattern mimicry of the receptor recognition determinant of interleukin 5

Phage display was used to identify sequences that mimic structural determinants in interleukin5 (IL5) for IL5 receptor recognition. A coiled coil stem loop (CCSL) miniprotein scaffold library was constructed with its turn region randomized and panned for binding variants against human IL5 receptor alpha chain (IL5Ralpha). Competition enzyme-linked immunosorbent assays identified CCSL-phage selectants for which binding to IL5Ralpha was competed by IL5. The most frequently selected and IL5-competed CCSL-phage contain charged residues Arg and Glu in their turn sequences, in this regard resembling a beta strand sequence in the 'CD turn' region, of IL5, that has been proposed to present a key determinant for IL5 receptor alpha chain recognition. The most dominant CCSL-phage selectant sequence, PVEGRV, contains a negative/positive charge pattern similar to that seen in the original CD turn. To test the relatedness of CCSL-phage selectant sequences to the IL5 receptor recognition epitope, PVEGRV was grafted into the sequence 87--92 of a monomeric IL5. The resulting IL5 variant, [(87)PVEGRV(92)]GM1, was able to bind to IL5Ralpha in biosensor assays, to elicit TF-1 cell proliferation and to induce STAT5 phosphorylation in TF-1 cells. The results help discern sequence patterns in the IL5 CD turn region which are key in driving receptor recognition and demonstrate the utility of CCSL miniprotein scaffold phage display to identify local IL5 mimetic sequence arrangements that may ultimately lead to IL5 antagonists.     

The beta common chain (beta c) of the granulocyte macrophage-colony stimulating factor, interleukin-3 and interleukin-5 receptors

The hematopoietic cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin (IL)-5 and IL-3 utilise a common receptor signalling molecule, the beta common chain (beta c). This shared receptor component explains, in part the overlapping actions of these cytokines. Mice lacking beta c have a low circulating eosinophil level, have impaired eosinophilic responses to parasitic infection and develop lung disease analogous to human pulmonary alveolar proteinosis (PAP). Surprisingly however, mature hematopoietic cell function is relatively intact, although all GM-CSF-mediated mature cell responses, including glucose transport are absent. Intriguing observations suggesting altered susceptibility to some infectious agents and amelioration of responses to inflammatory stimuli, require further clarification.     

Epitope randomization redefines the functional role of glutamic acid 110 in interleukin-5 receptor activation

Sequence randomization through functional phage display of single chain human interleukin (IL)-5 was used to investigate the limits of replaceability of the Glu(110) residues that form a part of the receptor-binding epitope. Mutational analysis revealed unexpected affinity for IL-5 receptor alpha chain with variants containing E110W or E110Y. Escherichia coli-expressed Glu(110) variants containing E110W in the otherwise sequence-intact N-terminal half, including a variant with an E110A replacement in the sequence-disabled C-terminal half, were shown by their CD spectra to be folded into secondary structures similar to that of single chain human IL-5 (scIL-5). Biosensor kinetics analysis revealed that (E110W/A5)scIL-5 and (E110W/A6)scIL-5 had receptor alpha chain binding affinities similar to that of (wt/A5)scIL-5. However, (E110W/A6)scIL-5 had a significantly reduced bioactivity in TF-1 cell proliferation compared with both (wt/A5)scIL-5 and (E110W/A5)scIL-5, and this activity reduction was disproportionately greater than the much smaller effect of Glu(110) mutation on receptor binding affinity. The marked and disproportionate decrease in TF-1 proliferation observed with (E110W/A6)scIL-5 suggests a role for Glu(110) in the biological activity mediated by the signal transducing receptor betac subunit of the IL-5 receptor. This is also consistent with the lack of stimulation of JAK2 phosphorylation by the (E110W/A6)scIL-5 mutant in recombinant 293T cells, as compared with the concentration-dependent stimulation seen for scIL-5. The results reveal the dispensability of charge in the Glu(110) locus of IL-5 for receptor alpha chain binding and, in contrast, its heretofore underappreciated importance for receptor activation.     

The interleukin-7 receptor alpha chain transmits distinct signals for proliferation and differentiation during B lymphopoiesis.

The interleukin 7 receptor (IL7R), which contains a unique alpha chain and a gamma chain shared by other cytokine receptors, is indispensable for normal lymphocyte development. The basis for this role is poorly understood. Here we show that the IL7R alpha chain not only causes progenitors to proliferate, but also has a distinct activity in inducing differentiation. First, we identify a single cytoplasmic tyrosine residue in the IL7R alpha chain that is essential for cell cycle entry and proliferation dependent on phosphatidylinositol 3-kinase. We use a mutant alpha chain in which this residue has been altered to reconstitute B lymphopoiesis by retrovirus-mediated gene transfer in cultures of bone marrow from mice deficient in IL7R alpha chain. The mutation abrogates the proliferation of B-lymphocyte progenitors, but reveals a novel function of the alpha chain in promoting immunoglobulin heavy chain gene rearrangement leading to B-cell differentiation. This function is lost (but proliferation sustained) when the cytoplasmic domain of IL7R alpha is replaced by corresponding sequences from the IL2R, despite the similarity on their signalling mechanisms. Thus, the signals which mediate a differentiative function of the IL7R in B lymphopoiesis are specific and distinct from those causing proliferation.     

Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF receptor alpha-chain expression by cytokines: IL-3, IL-5, and GM-CSF down-regulate IL-5 receptor alpha expression with loss of IL-5 responsiveness,but up-regulate IL-3 receptor alpha expression

Our recent data suggested that tissue eosinophils may be relatively insensitive to anti-IL-5 treatment. We examined cross-regulation and functional consequences of modulation of eosinophil cytokine receptor expression by IL-3, IL-5 GM-CSF, and eotaxin. Incubation of eosinophils with IL-3, IL-5, or GM-CSF led to reduced expression of IL-5R alpha, which was sustained for up to 5 days. Eosinophils incubated with IL-5 or IL-3 showed diminished respiratory burst and mitogen-activated protein kinase kinase phosphorylation in response to further IL-5 stimulation. In contrast to these findings, eosinophil expression of IL-3R alpha was increased by IL-3, IL-5, and GM-CSF, whereas GM-CSF receptor alpha was down-regulated by GM-CSF, but was not affected by IL-3 or IL-5. CCR3 expression was down-regulated by IL-3 and was transiently reduced by IL-5 and GM-CSF, but rapidly returned toward baseline. Eotaxin had no effect on receptor expression for IL-3, IL-5, or GM-CSF. Up-regulation of IL-3R alpha by cytokines was prevented by a phosphoinositol 3-kinase inhibitor, whereas this and other signaling inhibitors had no effect on IL-5R alpha down-regulation. These data suggest dynamic and differential regulation of eosinophil receptors for IL-3, IL-5, and GM-CSF by the cytokine ligands. Since these cytokines are thought to be involved in eosinophil development and mobilization from the bone marrow and are present at sites of allergic inflammation, tissue eosinophils may have reduced IL-5R expression and responsiveness, and this may explain the disappointing effect of anti-IL-5 therapy in reducing airway eosinophilia in asthma.     

A novel polymorphism in the 5' promoter region of the human interleukin-4 receptor alpha-chain gene is associated with decreased soluble interleukin-4 receptor protein levels

Interleukin (IL)-4 exerts its biological effects through binding to the IL-4 receptor (IL4R) complex, plays a central role in stimulating B-cell differentiation, and is crucial for the development of T helper 2 cells. Recently, a soluble form of the human IL4R alpha chain (sIL4R alpha), which is produced by alternate mRNA splicing of exon 8, was discovered. sIL4R is thought to play an important role in either enhancing or inhibiting IL-4 signalling. We analyzed the 5' promoter region of the human IL4R alpha-chain gene (IL4RA) of healthy volunteers by DNA sequencing and found three novel single-nucleotide polymorphisms (SNPs; T-890C, T-1914C, C-3223T) and one novel short tandem repeat [(CAAAA)(5-7)-3600]. The two common promoter region SNPs T-1914C and C-3223T as well as six known coding SNPs in the IL4RA gene were genotyped in healthy blood donors by PCR with sequence-specific primers; total sIL4R levels were measured by ELISA. Results revealed a highly significant association of the -3223T variant with lowered sIL4R levels (two-tailed t-test, P=0.0002). Results remained highly significant after Bonferroni adjustment for multiple comparisons (P=0.0017). Moreover, the C-3223T variant was found to be in strong linkage disequilibrium with the extracellular 150V variant (P<0.001), which was recently described to be associated with atopic asthma in a Japanese population. Since this novel IL4RA promoter region SNP is common (allele frequency 29.8%), we conclude that it may be of importance for the genetic regulation of the IL-4 signalling pathway.     

Randomization of the receptor alpha chain recruitment epitope reveals a functional interleukin-5 with charge depletion in the CD loop.

We report the functional phage display of single chain human interleukin-5 (scIL-5) and its use for receptor-binding epitope randomization. Enzyme-linked immunosorbent assays and optical biosensor analyses verified expression of scIL-5 on the phage surface and binding of scIL-5 phage to interleukin-5 receptor alpha chain. Furthermore, an asymmetrically disabled but functional scIL-5 mutant, (wt/A5)scIL-5, was displayed on phage. (wt/A5)scIL-5 was constructed from an N-terminal half containing the original five charged residues (88EERRR92) in the CD loop, including the Glu89 and Arg91 believed key in the alpha chain recognition site, combined with a C-terminal half containing a disabled CD loop sequence (88AAAAA92) missing the key recognition residues. This asymmetric variant was used as a starting point to generate an scIL-5 library in which the intact 88-92 N-terminal CD loop was randomized. From this epitope library, a receptor-binding variant of IL-5 was detected, (SLRGG/A5)scIL-5, in which the only charged residue in the CD loop is an Arg at position 90. Characterization of this variant expressed as a soluble protein in E. coli shows that the IL-5 pharmacophore for receptor alpha chain binding can function with a single positive charge in the CD loop. Charge-depleted CD loop mimetics of IL-5 suggest the importance of charge distribution in functional IL-5 receptor recruitment.     

Asymmetric usage of antagonist charged residues drives interleukin-5 receptor recruitment but is insufficient for receptor activation

The cyclic peptide AF17121 (VDECWRIIASHTWFCAEE) is a library-derived antagonist for human Interleukin-5 receptor alpha (IL5Ralpha). We have previously demonstrated that AF17121 mimics Interleukin-5 (IL5) by binding in a region of IL5Ralpha that overlaps the IL5 binding epitope. In the present study, to explore the functional importance of the amino acid residues of AF17121 required for effective binding to, and antagonism of, IL5Ralpha, each charged residue was subjected to site-directed mutagenesis and examined for IL5Ralpha interaction by using a surface plasmon resonance biosensor. One residue, Arg(6), was found to be essential for receptor antagonism; its replacement with either alanine or lysine completely abolished the interaction between AF17121 and IL5Ralpha. Other charged residues play modulatory roles. One class consists of the N-terminal acidic cluster (Asp(2) and Glu(3)) for which alanine replacement decreased the association rate. A second class consists of His(11) and the C-terminal acidic cluster (Glu(17) and Glu(18)) for which alanine replacement increased the dissociation rate. Binding model analysis of the mutants of the latter class of residues indicated the existence of conformational rearrangement during the interaction. On the basis of these results, we propose a model in which Arg(6) and N-terminal acidic residues drive the encounter complex, while Arg(6), His(11), and C-terminal acidic residues are involved in stabilizing the final complex. These data argue that the charged residues of AF17121 are utilized asymmetrically in the pathway of inhibitor-receptor complex formation to deactivate the receptor function. The results also help focus emerging models for the mechanism by which IL5 activates the IL5Ralpha-betac receptor system.