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 6 pathways. Analysis by mutagenesis.

The interleukin (IL)-4 receptor alpha-chain (IL-4Ralpha) contains a sequence motif (488PLVIAGNPAYRSFSD) termed the insulin IL-4 receptor motif (I4R motif). Mutation of the central Tyr497 to Phe blocks the tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1) and diminishes proliferation in response to IL-4. Recent data suggest that the I4R motif encodes binding sites for several protein tyrosine binding (PTB) domain-containing proteins such as IRS1 and Shc and potentially 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 and downstream of the central Tyr to Ala in the human IL-4Ralpha. We analyzed the ability of these constructs to signal the tyrosine phosphorylation 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 Tyr497, such as Arg498 or Phe500, to Ala had no effect on any of these responses, suggesting that the I4R motif may not be important for functional Src homology 2 domain interactions. However, mutagenesis of Pro488 to Ala (P488A) greatly diminished the tyrosine phosphorylation of IRS2 and abolished tyrosine phosphorylation of STAT6, induction of DNA binding activity, 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, Leu489 or Ile491, to Ala had only minimal effects on responses to huIL-4. However, changing both Leu498 and Ile491 to Ala greatly diminished 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.