Identification of an Interferon-γ Receptor α Chain Sequence Required for JAK-1 Binding

Abstract

We have shown previously that a four-amino acid block residing at positions 266-269 (LPKS) in the intracellular domain of the human interferon-gamma (IFN-gamma) receptor alpha chain is critical for IFN-gamma-dependent tyrosine kinase activation and biologic response induction. Herein we show that this sequence is required for the constitutive attachment of the tyrosine kinase JAK-1. Using a vaccinia expression system, a receptor alpha chain-specific monoclonal antibody coprecipitated JAK-1 from cells coexpressing JAK-1 and either (a) wild type IFN-gamma receptor alpha chain, (b) a receptor alpha chain truncation mutant containing only the first 59 intracellular domain amino acids, or (c) a receptor mutant containing alanine substitutions for the functionally irrelevant residues 272-275. In contrast, JAK-1 was not coprecipitated when coexpressed with a receptor alpha chain mutant containing alanine substitutions for the functionally critical residues 266-269 (LPKS). Mutagenesis of the LPKS sequence revealed that Pro-267 is the only residue obligatorily required for receptor function. In addition, Pro-267 is required for JAK-1 binding. These results thus identify a site in the IFN-gamma receptor alpha chain required for constitutive JAK-1 association and establish that this association is critical for IFN-gamma signal transduction.

Two Distinct Domains Within the N-Terminal Region of Janus Kinase 1 Interact with Cytokine Receptors

“…The precise location or motifs that form the Jak1 binding sites in some cytokine receptors have not been elucidated; therefore, their representation is only speculative. The LPKS (20) sequence within the membrane-proximal region of the IFN-␥R␣ appears to be the only region required for the interaction with Jak1 because its deletion completely abrogates binding (Fig. 1).…”

“…A, Schematic representation of the different GST fusion proteins used to study the interaction with Jak1. GST-IFN-␣R␤L, GST-IFN-␥R␣, GST-IL-2R␤, and GST-IL-10R␣ proteins correspond to the entire intracellular domains of IFN-␣R␤L, IFN-␥R␣, IL-2R␤, and IL-10R␣ chains, respectively; GST-IFN-␥R␣s protein encodes the intracellular domain of IFN-␥R␣, starting at aa 271 after the LPKS sequence important for Jak1 binding (20); GST-IL-4R␣-1, GST-IL-4R␣-2, and GST-IL-4R␣-3 fusion proteins encode aa 209 -288, 283-429, and 424 -561 of the IL-4R␣ intracellular domain, respectively; GST-IL-10R␣299 protein encodes the first 39 aa of the IL-10R␣ intracellular domain. B and C, The different GST fusion proteins described in A were used to pull down Jak1 from U-266 cell lysates.…”

“…Box 1 is defined as a proline-rich motif (4), whereas box 2 is characterized as a cluster of hydrophobic amino acids (2). Most single subunit cytokine receptors, such as erythropoietin, growth hormone, and prolactin, as well as some heterodimeric receptors, such as IFN-␥ and GM-CSF/IL-3 and -5 interact with Jak2 through the highly conserved box 1 motif (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”

“…For example, Tyk2 interacts with a domain of IFN-␣R␣, the ␣-chain of the IFN-␣R (also designated as IFNAR1 or IFN-␣R1), which has only distant homology with the box 1 or box 2 motifs (25,26). Jak1 is activated by multichain receptors such as the IL-6 group of cytokines (IL-6, leukemia inhibitory factor, ciliary neurotrophic factor, oncostatin M), IFN-␣, IFN-␥, IL-10, and those cytokine receptors that belong to the IL-2 family (IL-2, IL-4, IL-7, IL-9, and IL-15) (19,20,(27)(28)(29)(30)(31)(32)(33). The Jak1 binding site on cytokine receptors has been explored in only a few cases.…”

“…It is not clear whether a sequence similar to the box 1 or another motif is responsible for the interaction of this kinase with cytokine receptors. For instance, mutational analysis of the L 267 PKS 270 (leucine, proline, lysine, serine) motif of the ␣-chain of the IFN-␥R␣ (also designated as IFNGR1 or IFN-␥R1), which has only very distant similarity with the box 1, revealed that only Pro 268 was important for Jak1 binding (20). In contrast, the main Jak1 binding site on the IFN-␣R␤ L (also designated as IFNAR2 or IFN-␣R2) chain is clearly different from the box 1 (29) and more distant from the transmembrane region than the Jak1 site described for the IFN-␥R␣.…”