Abstract
Interaction domains located in the NH2 terminus of IRS-1 mediate its recognition by the insulin receptor. Alignment of IRS-1 and IRS-2 reveals two homology regions: the IH1(PH) contains a pleckstrin homology (PH) domain, and the IH2(PTB) contains a phosphotyrosine binding (PTB) domain. A third region in IRS-1 called SAIN was proposed to contain another functional PTB domain. Peptide competition experiments demonstrated 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 activity 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. Overexpression of the insulin receptor in 32DIR 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 equally couple IRS-1 to high levels of insulin receptor normally expressed in most cells, whereas at low levels of insulin receptors the PTB domain is inefficient and the PH domain is essential for a productive interaction.
Highlights
During stimulation of cells with growth factors or cytokines, specific cell surface receptors are activated by dimerization, which stimulates the phosphorylation of tyrosine residues on the intracellular domain of the receptor itself or in tightly associated subunits
The IH1PH was shown previously to mediate the interaction of the insulin receptor with IRS-1, whereas peptide binding experiments revealed that the IH2PTB contains a functional PTB domain [16, 17]
Several years ago we proposed that Tyr960 in the intracellular juxtamembrane region of the insulin receptor was essential for IRS-1 phosphorylation and normal biological responses during insulin stimulation [15]
Summary
During stimulation of cells with growth factors or cytokines, specific cell surface receptors are activated by dimerization, which stimulates the phosphorylation of tyrosine residues on the intracellular domain of the receptor itself or in tightly associated subunits. The second conserved region was called the IH2PTB, reflecting a weak similarity to putative phosphotyrosine-binding (PTB) domains in Shc and other proteins [5]. The IH1PH was shown previously to mediate the interaction of the insulin receptor with IRS-1, whereas peptide binding experiments revealed that the IH2PTB contains a functional PTB domain [16, 17]. These regions are distinct from the so-called SAIN region, which contains a weak sequence similarity to the PTB domain of Shc and was proposed to function as a PTB domain.
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