Identification of a Second Chain of the IL-10 Receptor Complex. Studying the IFN-[alpha] and IFN-[gamma] receptor complexes, Kotenko, Krause, Izotova, Pollack and Wu focused on the orphan receptor, CRFB4, from this family of receptors as noted above. Based on our previous observation that Tyk2 tyrosine kinase associates with the CRFB4 intracellular domain, we proposed that CRFB4 could be the second chain of the IL-10 receptor complex. Only one chain, that for ligand binding (IL-10R1), was previously identified. We demonstrated that, although human IL-10 binds to the human IL-10R1 chain expressed in hamster cells, it does not induce signal transduction. However, the coexpression of CRFB4 together with the IL-10R1 chain renders hamster cells sensitive to IL-10. The IL-10:CRFB4 complex was detected by crosslinking to labeled IL-10. In addition, the IL-10R1 chain was coimmunoprecipitated with anti-CRF antibody when peripheral blood mononuclear cells were treated with IL-10. These results demonstrated that the CRFB4 chain is part of the IL-10 receptor signaling complex. Thus, the CRFB4 chain, which we designated as the IL-10R2 chain, serves as an accessory chain essential for the initiation of IL-10 induced signal transduction events.
Paradigm for Cytokine Class II Receptor Complexes. Based on our studies of the IFN-[gamma] and IL-10 receptor complexes we observed specific features of this family of receptors. The binding of a ligand to the ligand binding subunit causes homodimerization of the receptor subunit, one, IFN-[gamma]R1, however, is not sufficient for initiation of a signal transduction cascade. The presence of an additional accessory subunit, IFN-[gamma]R1, in the ligand-receptor complex is required for signaling. The function of this second receptor chain, which we designated the helper receptor, is to bring an additional tyrosine kinase activity to the receptor complex. Any Jak family member can provide this function. Thus, the helper receptor does not determine the specificity of signaling and is specific only for cytokine binding.
Identification of Amino Acid Residues that Recruit STAT2 to the Hu-IFN-[alpha]R2a Receptor Chain. The type I IFN family members (IFN-[alpha], IFN-[beta], IFN-[omega], and IFN-[chi]) are involved in a variety of physiological responses. They induce antiviral and antiproliferative activities; stimulation of cytotoxic activity of lymphocytes, natural killer cells, and macrophages; modulation of cellular differentiation; stimulation of MHC class I antigens and other surface markers. Type I IFNs activate the Jak-Stat signal transduction pathway. After the ligand binding, Tyk2 and Jak1 kinases are activated, leading to activation of Stat1 and Stat2, latent transcriptional factors. After phosphorylation they form the active transcriptional complex ISGF-3 (IFN-stimulated gene factor-3) through the association of the Stat1/Stat2 heterodimer with the p48 protein. So far, the detailed activation mechanism of Stat2 by the type I IFN receptor complex is still unclear. Unlike other Stats that have been shown to interact with corresponding receptors through the highly specific interaction between Stat SH2 domains and the phosphorylated tyrosine motifs within the intracellular domain of the receptors, Stat2 was shown to associate with the IFN-[alpha]R2c chain through its N-terminal domain. Ge and Kotenko are in the process of identifying the amino acid residues, of the IFN-[alpha]R2 chain that are responsible for interacting with and activating Stat2. In order to avoid the cross species activity of human type I IFNs on hamster cells, the Hu-IFN-[gamma]R1/[alpha]R2c hybrid derivatives were expressed in CHO derived Q21 cells that carry Hu-IFN-[gamma]R2. Thus, Hu-IFN-[gamma], which is not active on hamster cells, was used to activate signal transduction through the chimeric receptor complex. The obtained cell lines were used to study IFN--induced MHC class I expression and Stat1 activation. The results so far indicate that the recruitment site for Stat2 on the IFN-[alpha]2Rc intracellular domain is localized within the C-terminal quarter of the IFN-[alpha]2Rc intracellular domain.