THE PLECKSTRIN HOMOLOGY DOMAIN IS THE PRINCIPLE LINK BETWEEN INSULIN-RECEPTOR AND IRS-1

Interaction domains located in the NH2 terminus of IRS-1 mediate its r ecognition by the insulin receptor. Alignment of IRS-1 and IRS-S revea ls two homology regions: the IH1(PH) contains a pleckstrin homology (P H) domain, and the IH2(PTB) contains a phosphotyrosine binding (PTB) d omain. A third region in IRS-1 called SAIN was proposed to contain ano ther functional PTB domain. Peptide competition experiments demonstrat ed that the IH2(PTB) in IRS-2, like the corresponding domain in IRS-1, binds directly to peptides containing NPXY motifs. In contrast, these peptides do not bind to IH1(PH) or the SAIN regions. In 32D cells the IH1(PH) was essential for insulin-stimulated tyrosine phosphorylation of IRS-1 and insulin-stimulated phosphatidylinositol 3-kinase activit y and p70(s6k) phosphorylation. In contrast, the IH2(PTB) and the SAIN regions were not required for these insulin actions; however, the IH2 (PTB) improved the coupling between IRS-1 and the insulin receptor. Ov erexpression of the insulin receptor in 32D(IR) cells increased IRS-1 tyrosine phosphorylation and mediated insulin-stimulated DNA synthesis . The sensitivity of these responses was partially reduced by deletion of either the IH1(PH) or the IH2(PTB) and significantly reduced when both regions were deleted together. Thus, the PH and PTB domains equal ly couple IRS-1 to high levels of insulin receptor normally expressed in most cells, whereas at low levels of insulin receptors the PTB doma in is inefficient and the PH domain is essential for a productive inte raction.