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.
Highlights
Interferon-␥ (IFN-␥)1 is a potent immunomodulatory cytokine that exerts its pleiotropic biologic effects by interacting with a single high affinity receptor expressed on the surface of most host-derived cells [1,2,3,4,5]
The first was a block of four amino acids (LPKS) situated 13 residues from the transmembrane domain at positions 266 –269 that was required for ligand-induced activation of intracellular tyrosine kinase activity, phosphorylation of the receptor ␣ chain intracellular domain, and induction of IFN-␥-mediated cellular responses
We report the fine mapping of the LPKS sequence and show that the proline residue at position 267 is required for JAK-1-receptor ␣ chain binding and receptor function
Summary
Interferon-␥ (IFN-␥)1 is a potent immunomodulatory cytokine that exerts its pleiotropic biologic effects by interacting with a single high affinity receptor expressed on the surface of most host-derived cells [1,2,3,4,5]. These studies involved replacing specific IFN-␥ receptor amino acids with alanine residues and examining the capacity of the resulting mutant human receptor ␣ chains to confer human IFN-␥ responsiveness to a murine cell line containing the human receptor  chain (SCC16 –5).
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