STAT-3 RECRUITMENT BY 2 DISTINCT LIGAND-INDUCED, TYROSINE-PHOSPHORYLATED DOCKING SITES IN THE INTERLEUKIN-10 RECEPTOR INTRACELLULAR DOMAIN

Recent work has shown that IL-10 induces activation of the JAK-STAT si gnaling pathway. To define the mechanism underlying signal transducer and activator of transcription (STAT) protein recruitment to the inter leukin 10 (IL-10) receptor, the STAT proteins activated by IL-10 in di fferent cell populations were first defined using electrophoretic mobi lity shift assays. In all cells tested, IL-10 activated Stat1 and Stat 3 and induced the formation of three distinct DNA binding complexes th at contained different combinations of these two transcription factors . IL-10 also activated Stat5 in Ba/F3 cells that stably expressed the murine IL-10 receptor. Using a structure-function mutagenesis approach , two tyrosine residues (Tyr(427) and Tyr(477)) in the intracellular d omain of the murine IL-10 receptor were found to be redundantly requir ed for receptor function and for activation of Stat3 beat not for Stat 1 car Stat5. Twelve amino acid peptides encompassing either of these t wo tyrosine residues in phosphorylated form coprecipitated Stat3 hut n ot Stat1 and blocked IL-10-induced Stat3 phosphorylation in a cell-fre e system. In contrast, tyrosine-phosphorylated peptides containing Tyr (374) or Tyr(396) did not interact with Stat3 or block Stat3 activatio n. These data demonstrate that Stat3 but not Stat1 or Stat5 is directl y recruited to the ligand-activated IL-10 receptor by binding to speci fic beat redundant receptor intracellular domain sequences containing phosphotyrosine. This study thus supports the concept that utilization of distinct STAT proteins by different cytokine receptors is dependen t on the expression of particular ligand-activatable, tyrosine-contain ing STAT docking sites in receptor intracellular domains.