Expression and in vitro properties of guinea pig IL-5: comparison to human and murine orthologs

Interleukin-5 (IL-5) is a key mediator of eosinophilic inflammation. The biological role of this cytokine in an allergic airway inflammatory response has been widely demonstrated in guinea pigs, yet the interaction of guinea pig IL-5 (gpIL-5) with its receptor has not been studied. Experiments were performed to quantitate the interaction of gpIL-5 with gpIL-5r and to compare this affinity with that of hIL-5 and mIL-5 and their cognate receptors. The cross-species affinity and agonist efficacy were evaluated to see if gpIL-5r had a restricted species reactivity (as is the case with mIL-5r) or did not distinguish between IL-5 orthologs (similar to hIL-5r). gpIL-5 was cloned using mRNA isolated from cells obtained by bronchoalveolar lavage. Recombinant gpIL-5 was expressed in T. ni insect cells and purified from spent media. Binding assays were performed using insect cells expressing hIL-5ralphabeta or gpIL-5ralphabeta1 as previously described (Cytokine, 12:858-866, 2000) or using B13 cells which express mIL-5r. The agonist potency and efficacy properties of each IL-5 ortholog were evaluated by quantitating the proliferative response of human TF-1 cells and murine B13 cells. gpIL-5 bound with high affinity to recombinant gpIL-5r as demonstrated by displacing [125I]hIL-5 (Ki = 160 pM). gpIL-5 also bound to hIL-5r with high affinity (Ki = 750 pM). hIL-5 and mIL-5 showed similar, high-affinity binding profiles to both gpIL-5r and hIL-5r. In contrast, gpIL-5 and hIL-5 did not bind to the mIL-5r as demonstrated by an inability to displace [125I]mIL-5, even at 1000-fold molar excess. These differences in affinity for IL-5r orthologs correlated with bioassay results: human TF-1 cells showed roughly comparable proliferative responses to guinea pig, human and murine IL-5 whereas murine B13 cells showed a strong preference for murine over guinea pig and human IL-5 (EC50 = 1.9, 2200 and 720 pM, respectively). Recombinant gpIL-5 binds to the gpIL-5r with high affinity, similar to that seen with the human ligand-receptor pair. gpIL-5r and hIL-5r do not distinguish between the three IL-5 orthologs whereas mIL-5r has restricted specificity for its cognate ligand.     

Expression cloning of the human IL-3 receptor cDNA reveals a shared beta subunit for the human IL-3 and GM-CSF receptors.

A cDNA for a human interleukin-3 (hIL-3) binding protein has been isolated by a novel expression cloning strategy: a cDNA library was coexpressed with the cDNA for the beta subunit of human granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (hGMR beta) in COS7 cells and screened by binding of 125I-labeled IL-3. The cloned cDNA (DUK-1) encodes a mature protein of 70 kd, which belongs to the cytokine receptor family and which alone binds hIL-3 with extremely low affinity (Kd = 120 +/- 60 nM). A high affinity IL-3-binding site (Kd = 140 +/- 30 pM) was reconstituted by coexpressing the DUK-1 protein and hGMR beta, indicating that hIL-3R and hGMR share the beta subunit. Therefore, we designated DUK-1 as the alpha subunit of the hIL-3R. As in human hematopoietic cells, hIL-3 and hGM-CSF complete for binding in fibroblasts expressing the cDNAs for hIL-3R alpha, GMR alpha, and the common beta subunit, indicating that different alpha subunits compete for a common beta subunit.     

Analysis of the role of the interleukin-2 receptor gamma chain in ligand binding

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.     

Ligand-independent homomeric and heteromeric complexes between interleukin-2 or -9 receptor subunits and the gamma chain

Signaling via interleukin-2 (IL-2) and interleukin-9 receptors (IL-2R and IL-9R) involves heteromeric interactions between specific interleukin receptor subunits, which bind Janus kinase 1 (JAK1) and the JAK3 binding common gamma chain (gamma c). The potential existence and roles of homomeric and heteromeric complexes before ligand binding and their modulation by ligand and JAK3 are unclear. Using computerized antibody-mediated immunofluorescence co-patching of epitope-tagged receptors at the surface of live cells, we demonstrate that IL-2Rbeta, IL-9Ralpha, and gamma c each display a significant fraction of ligand-independent homomeric complexes (24-28% co-patching), whereas control co-patching levels with unrelated receptors are very low (7%). Heteromeric complex formation of IL2-Rbeta or IL-9Ralpha with gamma c is also observed in the absence of ligand (15-30%). Ligand binding increases this hetero-oligomerization 2-fold but does not affect homo-oligomerization. Co-expression of IL-2Ralpha does not affect the hetero-oligomerization of IL-2Rbeta and gamma c. Recruitment of gamma c into heterocomplexes is partly at the expense of its homo-oligomerization, suggesting that a functional role of the latter may be to keep the receptors inactive in the absence of ligand. At the same time, the preformed complexes between gamma c and IL-2Rbeta or IL-9Ralpha promote signaling by the JAK3 A572V mutant without ligand, supporting a pathophysiological role for the constitutive oligomerization in triggering ligand-independent activation of JAK3 (and perhaps other JAK mutants) mutants identified in several human cancers.     

Kinetic analysis of the interleukin-13 receptor complex

Interleukin (IL)-13 is a key cytokine associated with the asthmatic phenotype. It signals via its cognate receptor, a complex of IL-13 receptor alpha1 chain (IL-13Ralpha1) with IL-4Ralpha; however, a second protein, IL-13Ralpha2, also binds IL-13. To determine the binding contributions of the individual components of the IL-13 receptor to IL-13, we have employed surface plasmon resonance and equilibrium binding assays to investigate the ligand binding characteristics of shIL-13Ralpha1, shIL-13Ralpha2, and IL-4Ralpha. shIL-13Ralpha1 bound IL-13 with moderate affinity (K(D) = 37.8 +/- 1.8 nm, n = 10), whereas no binding was observed for hIL-4Ralpha. In contrast, shIL-13Ralpha2 produced a high affinity interaction with IL-13 (K(D) = 2.49 +/- 0.94 nm n = 10). IL-13Ralpha2 exhibited the binding characteristics of a negative regulator with a fast association rate and an exceptional slow dissociation rate. Although IL-13 interacted weakly with IL-4Ralpha on its own (K(D) > 50 microm), the presence of hIL-4Ralpha significantly increased the affinity of shIL-13Ralpha1 for IL-13 but had no effect on the binding affinity of IL-13Ralpha2. Detailed kinetic analyses of the binding properties of the heteromeric complexes suggested a sequential mechanism for the binding of IL-13 to its signaling receptor, in which IL-13 first binds to IL-13Ralpha1 and this then recruits IL-4Ralpha to stabilize a high affinity interaction.     

Solution assembly of the pseudo-high affinity and intermediate affinity interleukin-2 receptor complexes

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.     

Detailed analysis of the IL-5-IL-5R alpha interaction: characterization of crucial residues on the ligand and the receptor.

The receptor for interleukin-5 (IL-5) is composed of two different subunits. The IL-5 receptor alpha (IL-5R alpha) is required for ligand-specific binding while association with the beta-chain results in increased binding affinity. Murine IL-5 (mIL-5) has similar activity on human and murine cells, whereas human IL-5 (hIL-5) has marginal activity on murine cells. We found that the combined substitution of K84 and N108 on hIL-5 by their respective murine counterpart yields a molecule which is as potent as mIL-5 for growth stimulation of a murine cell line. Since the unidirectional species specificity is due only to the interaction with the IL-5R alpha subunit, we have used chimeric IL-5R alpha molecules to define regions of hIL-5R alpha involved in species-specific hIL-5 ligand binding. We found that this property is largely determined by the NH2-terminal module of hIL-5R alpha, and detailed analysis defined D56 and to a lesser extent E58 as important for binding. Moreover, two additional residues, D55 and Y57, were identified by alanine scanning mutagenesis within the same region. Based on the observed homology between the NH2-terminal module and the membrane proximal (WSXWS-containing) module of hIL-5R alpha we located this stretch of four amino acid residues (D55, D56, Y57 and E58) in the loop region that connects the C and D beta-strands on the proposed tertiary structure of the NH2-terminal module.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversal of some viral IL-6 electrostatic properties compared to IL-6 contributes to a loss of alpha receptor component recruitment

Human interleukin-6 (hIL-6) is a pleiotropic mediator of activation and proliferation across a large number of different cell types. Human herpesvirus-8 (HHV-8) has been associated with classical and AIDS-related Kaposi's sarcoma (KS). HHV-8 encodes viral IL-6 (vIL-6), a functional homolog of human interleukin-6, that promotes the growth of KS and of some lymphoma cells. Signaling induced by human IL-6 requires recruitment of the glycoprotein gp130, which acts as the signal transducing chain, and of IL-6Ralpha, which is necessary for cognate recognition and high affinity receptor complex formation. In contrast, the formation of a functional complex between vIL-6 and gp130 does not require the presence of IL-6Ralpha. The physico-chemical properties of vIL-6 have been analyzed and compared to those of hIL-6 and of the receptor chains, gp130 and IL-6Ralpha. Interaction sites on vIL-6 involve more hydrophobic residues than those of hIL-6. The electrostatic fields induced by vIL-6 and IL-6Ralpha are repulsive and prevent interaction between vIL-6 and IL-6Ralpha, whereas the electrostatic field induced by hIL-6 steers the complex formation with IL-6Ralpha. Subsequently, electrostatic binding free energy in the vIL-6/IL-6Ralpha complex is destabilizing, whereas it is stabilizing in the complex comprising hIL-6. These properties result from charge reversals between viral and human IL-6, an unusual phenomenon of amino acid substitutions within a homologous protein family. This suggests a selection pressure for vIL-6 to by-pass the IL-6Ralpha control of host defense against virus infection. This selection pressure has yielded the reversal of electrostatic properties of vIL-6 when compared to hIL-6.     

Oligomerization of IL-2Ralpha

Interleukin (IL) 2 receptor subunit alpha (IL-2Ralpha) increases the affinity of the IL-2 receptor complex while hetero-association of IL-2Rbeta and gamma(c) chains initiates a proliferative signal. We show here that IL-2Ralpha is necessary for receptor clustering required for augmentation of IL-2 signalling. Cells expressing chimeras incorporating the extracellular domain of IL-2Ralpha demonstrated IL-2 independent homo-association of the IL-2Ralpha chimera. Singly or co-transfected IL-2Rbeta and gamma(c) chimeras showed no spontaneous or IL-2-inducible oligomerization. Co-transfection of IL-2Ralpha and IL-2Rbeta (+/- gamma(c)) chimeras diminished spontaneous IL-2Ralpha chimera oligomerization and permitted IL-2-inducible hetero-oligomerization of receptor components. Homo-association of IL-2Ralpha was also demonstrated by fluorescence resonance energy transfer (FRET). The spontaneous homo-oligomerization property of IL-2Ralpha required the membrane proximal region of the receptor (exon 6) by deletion analysis; the IL-2 inducible oligomerization property of IL-2Ralpha required the second "sushi" domain (exon 4). This work provides insight into the mechanics of this complex receptor system and to other receptor complexes in the immune system that send signals by clustering receptor subunits.     

Engineering human interleukin-6 to obtain variants with strongly enhanced bioactivity.

Interleukin-6 (IL-6) triggers the formation of a high affinity receptor complex with the ligand binding subunit IL-6Ralpha and the signal transducing chain gp130. Since the intracytoplasmic region of the IL-6Ralpha does not contribute to signaling, soluble forms of the extracytoplasmic domain (sIL-6Ralpha), potentiate IL-6 bioactivity and induce a cytokine-responsive status in cells expressing gp130 only. This observation, together with the detection of high levels of circulating soluble human IL-6Ralpha (shIL-6Ralpha) in sera, suggests that the hIL-6-shIL-6Ralpha complex is an alternative form of the cytokine. Here we describe the generation of human IL-6 (hIL-6) variants with strongly enhanced shIL-6Ralpha binding activity and bioactivity. Homology modeling and site-directed mutagenesis of hIL-6 suggested that the binding interface for hIL-6Ralpha is constituted by the C-terminal portion of the D-helix and residues contained in the AB loop. Four libraries of hIL-6 mutants were generated by each time fully randomizing four different amino acids in the predicted AB loop. These libraries were displayed monovalently on filamentous phage surface and sorted separately for binding to immobilized shIL-6Ralpha. Mutants were selected which, when expressed as soluble proteins, showed a 10- to 40-fold improvement in shIL-6Ralpha binding; a further increase (up to 70-fold) was achieved by combining variants isolated from different libraries. Interestingly, high affinity hIL-6 variants show strongly enhanced bioactivity on cells expressing gp13O in the presence of shIL-6Ralpha at concentrations similar to those normally found in human sera.     

Identification of regions within the third FnIII-like domain of the IL-5Ralpha involved in IL-5 interaction

Previously, two binding sites for interleukin 5 (IL-5) were identified on the IL-5 receptor alpha chain (IL-5Ralpha). They are located within the CD loop of the first fibronectin type III (FnIII)-like domain and the EF loop of the second FnIII-like domain. The first binding site was identified by exploiting the different abilities of human IL-5Ralpha (hIL-5Ralpha) and mouse IL-5Ralpha (mIL-5Ralpha) to bind hIL-5. Here we show that ovine IL-5 (oIL-5) has the ability to activate the hIL-5Ralpha but not the mIL-5Ralpha. By using chimeras of the mIL-5Ralpha and hIL-5Ralpha we demonstrate that residues within the first and third FnIII-like domains of mIL-5Ralpha are responsible for this lack of activity. Furthermore, mutation of residues on hIL-5Ralpha to mIL-5Ralpha within the predicted DE and FG loop regions of the third FnIII domain reduces oIL-5 activity. These results show that regions of the third FnIII domain of IL-5Ralpha are involved in binding, in addition to the regions in domains one and two of the IL-5Ralpha that were identified in an earlier study.     

Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor.

Murine interleukin-5 (IL-5) binds to its receptor with high and low affinity. It has been shown that the high affinity IL-5 receptor (IL-5-R) is composed of at least two membrane protein subunits and is responsible for IL-5-mediated signal transduction. One subunit of the high affinity IL-5-R is a 60 kDa membrane protein (p60 IL-5-R) whose cDNA was isolated using the anti-IL-5-R monoclonal antibody (mAb), H7. This subunit alone binds IL-5 with low affinity. The second subunit does not bind IL-5 by itself, and is expressed not only on IL-5-dependent cell lines but also on an IL-3-dependent cell line, FDC-P1. Expression of the p60 IL-5-R cDNA in FDC-P1 cells, which do not bind IL-5, reconstituted the high affinity IL-5-R. We have characterized the second subunit of the IL-5-R by using another anti-IL-5-R mAb, R52.120, and the anti-IL-3-R mAb, anti-Aic-2. The anti-Aic-2 mAb down-regulated binding of IL-5 to an IL-5-dependent cell line, Y16. Both R52.120 and anti-Aic-2 mAbs recognized membrane proteins of 130-140 kDa expressed on FDC-P1 and Y16 cells. The R52.120 mAb recognized both murine IL-3-R (AIC2A) and its homologue (AIC2B) expressed on L cells transfected with suitable cDNAs. The high affinity IL-5-R was reconstituted on an L cell transfectant co-expressing AIC2B and p60 IL-5-R, whereas only the low affinity IL-5-R was detected on a transfectant co-expressing AIC2A and p60 IL-5-R.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-5 receptor subunit oligomerization and rearrangement revealed by fluorescence resonance energy transfer imaging

Interleukin (IL)-5 exerts hematopoietic functions through binding to the IL-5 receptor subunits, alpha and betac. Specific assembly steps of full-length subunits as they occur in cell membranes, ultimately leading to receptor activation, are not well understood. We tracked the oligomerization of IL-5 receptor subunits using fluorescence resonance energy transfer (FRET) imaging. Full-length IL-5Ralpha and betac were expressed in Phoenix cells as chimeric proteins fused to enhanced cyan or yellow fluorescent protein (CFP or YFP, respectively). A time- and dose-dependent increase in FRET signal between IL-5Ralpha-CFP and betac-YFP was observed in response to IL-5, indicative of heteromeric receptor alpha-betac subunit interaction. This response was inhibited by AF17121, a peptide antagonist of IL-5Ralpha. Substantial FRET signals with betac-CFP and betac-YFP co-expressed in the absence of IL-5Ralpha demonstrated that betac subunits exist as preformed homo-oligomers. IL-5 had no effect on this betac-alone FRET signal. Interestingly, the addition of IL-5 to cells co-expressing betac-CFP, betac-YFP, and nontagged IL-5Ralpha led to further increase in FRET efficiency. Observation of preformed betac oligomers fits with the view that this form can lead to rapid cellular responses upon IL-5 stimulation. The IL-5-induced effects on betac assembly in the presence of nontagged IL-5Ralpha provide direct evidence that IL-5 can cause higher order rearrangements of betac homo-oligomers. These results suggest that IL-5 and perhaps other betac cytokines (IL-3 and granulocyte/macrophage colony-stimulating factor) trigger cellular responses by the sequential binding of cytokine ligand to the specificity receptor (subunit alpha), followed by binding of the ligand-subunit alpha complex to, and consequent rearrangement of, a ground state form of betac oligomers.     

Human IL-Rbeta chains form IL-2 binding homodimers

Two types of functional interleukin-2 receptor (IL-2Ralpha/IL-2Rbeta/gammac and IL-2Rbeta/gammac) have already been characterized in humans. Here we describe a new form consisting of IL-2Rbeta/beta homodimers that assemble spontaneously in the absence of gammac. Co-transfection of COS-7 cells with constructs expressing IL-2Rbeta chains tagged with either HA or MYC sequences results in the formation of IL-2Rbeta:HA/IL-2Rbeta:MYC complexes detectable by coimmunoprecipitation. The formation of these IL-2Rbeta:HA/IL-2Rbeta:MYC dimers is also observed in the absence of IL-2. Moreover, in COS cells expressing chimeras of IL-2Rbeta fused to fluorescence reporters such as IL-2Rbeta:ECFP and IL-2Rbeta:EYFP, we also observed specific FRET at the surface of living cells, as expected for dimer formation. Transiently transfected COS-7 cells expressing IL-2Rbeta bind 125I-labeled IL-2 (homodimers, Kd = 1nM) as cells expressing both IL-2Rbeta and gammac chains (heterodimers, Kd = 1 nM). IL-2Rbeta/IL-2Rbeta could represent either a decoy receptor or a new form of IL-2R involved in signaling when gammac expression is low.     

A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit,IL-23R

IL-23 is a heterodimeric cytokine composed of the IL-12p40 "soluble receptor" subunit and a novel cytokine-like subunit related to IL-12p35, termed p19. Human and mouse IL-23 exhibit some activities similar to IL-12, but differ in their capacities to stimulate particular populations of memory T cells. Like IL-12, IL-23 binds to the IL-12R subunit IL-12Rbeta1. However, it does not use IL-12Rbeta2. In this study, we identify a novel member of the hemopoietin receptor family as a subunit of the receptor for IL-23, "IL-23R." IL-23R pairs with IL-12Rbeta1 to confer IL-23 responsiveness on cells expressing both subunits. Human IL-23, but not IL-12, exhibits detectable affinity for human IL-23R. Anti-IL-12Rbeta1 and anti-IL-23R Abs block IL-23 responses of an NK cell line and Ba/F3 cells expressing the two receptor chains. IL-23 activates the same Jak-stat signaling molecules as IL-12: Jak2, Tyk2, and stat1, -3, -4, and -5, but stat4 activation is substantially weaker and different DNA-binding stat complexes form in response to IL-23 compared with IL-12. IL-23R associates constitutively with Jak2 and in a ligand-dependent manner with stat3. The ability of cells to respond to IL-23 or IL-12 correlates with expression of IL-23R or IL-12Rbeta2, respectively. The human IL-23R gene is on human chromosome 1 within 150 kb of IL-12Rbeta2.     

JAK1 N-terminus binds to conserved Box 1 and Box 2 motifs of cytokine receptor common beta subunit but signal activation requires JAK1 C-terminus

The human interleukin-3 receptor (hIL-3R) consists of a unique alpha subunit (hIL-3Ralpha) and a common beta subunit (betac). Binding of IL-3 to IL-3R activates Janus kinases JAK1 and JAK2. Our previously study showed that JAK2 and JAK1 were constitutively associated with the hIL-3Ralpha and betac subunits, respectively. In this study, we further demonstrate that JAK2 binds to the intracellular domain of hIL-3Ralpha and JAK1 binds to the Box 1 and Box 2 motifs of betac using GST-hIL-3R fusion proteins in pull-down assays. JAK1 mutational analysis revealed that its JH7-3 domains bound directly to the Box 1 and Box 2 motifs of betac. We further examined the role of JAK1 JH7-3 domains in JAK1 and JAK2-mediated signaling using the CDJAKs fusion proteins, which consisted of a CD16 extracellular domain, a CD7 transmembrane domain, and either JAK1 (CDJAK1), JAK2 (CDJAK2), or JAK1-JH7-3 domains (CDJAK1-JH7-3) as intracellular domains. Anti-CD16 antibody crosslinking of wild type fusion proteins CDJAK1 with CDJAK2 could mimic IL-3 signaling, however, the crosslinking of fusion proteins CDJAK1-JH7-3 with CDJAK2 failed to activate downstream proteins. These results suggest that the JAK1-JH7-3 domains are required for betac interaction and abolish wild type JAK1 and JAK2-mediated signaling.     

Inhibiting protein-protein interactions: a model for antagonist design

Protein-protein interactions (PPI) are a ubiquitous mode of transmitting signals in cells and tissues. We are testing a stepwise, generic, structure-driven approach for finding low molecular weight inhibitors of protein-protein interactions. The approach requires development of a high-affinity, single chain antibody directed specifically against the interaction surface of one of the proteins to obtain structural information on the interface. To this end, we developed a single chain antibody (sc1E3) against hIL-1beta that exhibited the equivalent affinity of the soluble IL-1 receptor type I (sIL-1R) for hIL-1beta and competitively blocked the sIL-1R from binding to the cytokine. The antibody proved to be more specific for hIL-1beta than the sIL-1R in that it failed to bind to either murine IL-1beta or human/murine IL-1alpha proteins. Additionally, failure of sc1E3 to bind to several hIL-1beta mutant proteins, altered at receptor site B, indicated that the antibody interacted preferentially with this site. This, coupled with other surface plasmon resonance and isothermal titration calorimetry measurements, shows that sc1E3 can achieve comparable affinity of binding hIL-1beta as the receptor through interactions at a smaller interface. This stable single chain antibody based heterodimer has simplified the complexity of the IL-1/IL-1R PPI system and will facilitate the design of the low molecular weight inhibitors of this interaction.     

Signaling domains of the interleukin 2 receptor

Interleukin (IL-)2 and its receptor (IL-2R) constitute one of the most extensively studied cytokine receptor systems. IL-2 is produced primarily by activated T cells and is involved in early T cell activation as well as in maintaining homeostatic immune responses that prevent autoimmunity. This review focuses on molecular signaling pathways triggered by the IL-2/IL-2R complex, with an emphasis on how the IL-2R physically translates its interaction with IL-2 into a coherent biological outcome. The IL-2R is composed of three subunits, IL-2Ralpha, IL-2Rbeta and gammac. Although IL-2Ralpha is an important affinity modulator that is essential for proper responses in vivo, it does not contribute to signaling due a short cytoplasmic tail. In contrast, IL-2Rbeta and gammac together are necessary and sufficient for effective signal transduction, and they serve physically to connect the receptor complex to cytoplasmic signaling intermediates. Despite an absolute requirement for gammac in signaling, the majority of known pathways physically link to the receptor via IL-2Rbeta, generally through phosphorylated cytoplasmic tyrosine residues. This review highlights work performed both in cultured cells and in vivo that defines the functional contributions of specific receptor subdomains-and, by inference, the specific signaling pathways that they activate-to IL-2-dependent biological activities.     

Immunoreceptor tyrosine-based inhibitory motif of the IL-4 receptor associates with SH2-containing phosphatases and regulates IL-4-induced proliferation.

Immunoreceptor tyrosine-based inhibitory motifs (ITIM) have been implicated in the negative modulation of immunoreceptor signaling pathways. The IL-4R alpha-chain (IL-4Ralpha) contains a putative ITIM in the carboxyl terminal. To determine the role of ITIM in the IL-4 signaling pathway, we ablated the ITIM of IL-4Ralpha by deletion and site-directed mutagenesis and stably expressed the wild-type (WT) and mutant hIL-4Ralpha in 32D/insulin receptor substrate-2 (IRS-2) cells. Strikingly, 32D/IRS-2 cells expressing mutant human (h)IL-4Ralpha were hyperproliferative in response to IL-4 compared with cells expressing WT hIL-4Ralpha. Enhanced tyrosine phosphorylation of Stat6, but not IRS-2, induced by hIL-4 was observed in cells expressing mutant Y713F. Using peptides corresponding to the ITIM of hIL-4Ralpha, we demonstrate that tyrosine-phosphorylated peptides, but not their nonphosphorylated counterparts, coprecipitate SH2-containing tyrosine phosphatase-1, SH2-containing tyrosine phosphatase-2, and SH2-containing inositol 5'-phosphatase. The in vivo association of SH2-containing inositol 5'-phosphatase with IL-4Ralpha was verified by coimmunoprecipitation with anti-IL-4Ralpha Abs. These results demonstrate a functional role for ITIM in the regulation of IL-4-induced proliferation.