A ROLE FOR THE INSULIN-INTERLEUKIN (IL)-4 RECEPTOR MOTIF OF THE IL-4 RECEPTOR ALPHA-CHAIN IN REGULATING ACTIVATION OF THE INSULIN-RECEPTOR SUBSTRATE 2 AND SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 8 PATHWAYS - ANALYSIS BY MUTAGENESIS

The interleukin (IL)-4 receptor alpha-chain (IL-4R alpha) contains a s equence motif ((488)PLVIAGNPAYRSFSD) termed the insulin IL-4 receptor motif (I4R motif). Mutation of the central Tyr(497) to Phe blocks the tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1) an d diminishes proliferation in response to IL-4. Recent data suggest th at the I4R motif encodes binding sites for several protein tyrosine bi nding (PTB) domain-containing proteins such as IRS1 and Shc and potent ially for the Src homology 2 domain of signal transducer and activator of transcription 6 (STAT6). To analyze the function of the I4R motif in regulating IL-4 signaling, we changed conserved residues upstream a nd downstream of the central Tyr to Ala in the human IL-4R alpha. We a nalyzed the ability of these constructs to signal the tyrosine phospho rylation of IRS2 and STAT6, the induction of DNA binding activity, and CD23 induction in response to human IL-4 (huIL-4) in transfected M12. 4.1 cells. Mutagenesis of residues downstream of Tyr(497), such as Arg (498) or Phe(500), to Ala had no effect on any of these responses, sug gesting that the I4R motif may not be important for functional Src hom ology 2 domain interactions. However, mutagenesis of Pro(488) to Ala ( P488A) greatly diminished the tyrosine phosphorylation of IRS2 and abo lished tyrosine phosphorylation of STAT6, induction of DNA binding act ivity, and CD23 induction in response to huIL-4. By contrast, a P488G mutant signaled these responses to huIL-4. Mutagenesis of hydrophobic amino acids previously shown to contact the PTB domain of IRS1, Leu(48 9) or Ile(491), to Ala had only minimal effects on responses to huIL-4 . However, changing both Leu(498) and Ile(491) to Ala greatly diminish ed the tyrosine phosphorylation of IRS2 and abolished STAT6 activation . Taken together, these results indicate the important role of the I4R motif in regulating IRS docking and suggest that I4R docking to a PTB domain-containing protein regulates activation of the STAT6 pathway.