Direct interaction of STAT4 with the IL-12 receptor

Signal transduction by interleukin-12 (IL-12) requires phosphorylation and activation of STAT4 Direct interaction of the SH2 domain of STAT4 with a ph osphotyrosine residue in the IL-12 receptor has been proposed to be require d for the subsequent STAT4 phosphorylation. The IL-12 receptor beta 2 subun it contains three tyrosine residues in its cytoplasmic domain. To test the hypothesis that one of these tyrosines is involved in binding STAT4, phosph opeptides were synthesized according to the amino acid sequences surroundin g each of these tyrosine residues. Only the phosphopeptide containing pTyr8 00 strongly bound to STAT4 in a cell-free binding assay. When this phosphop eptide was introduced into TALL-104 cells, it blocked IL-12-induced STAT4 p hosphorylation by competing with the IL-12 receptor for binding to STAT4. A series of alanine replacements was performed in this phosphopeptide to elu cidate which amino acids surrounding the pTyr800 residue are critical for S TAT4 binding. To summarize, the site on the IL-12 receptor which binds STAT 4 can be described as -T-X-X-G-pY(800)-L-, where the core G-pY(800)-L motif is critical for the binding; the threonine at the pY-4 position has only a minor contribution and X represents amino acids not critical for the bindi ng. These results demonstrate that only a small region of the IL-12 recepto r is critically involved in binding STAT4 and suggest the feasibility that small molecule inhibitors could be identified which interfere with IL-12 si gnal transduction for treatment of autoimmune diseases. (C) 1999 Academic P ress.