Identification of a NPXY Motif in Growth Factor Receptor-Bound Protein 14 (Grb14) and Its Interaction with the Phosphotyrosine-Binding (PTB) Domain of IRS-1

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Abstract
Recently we have shown that insulin fails to induce the phosphorylation of IRS-1 in the retina [Rajala et al. (2004) Biochemistry43, 5637−5650], even though there is widespread expression of IRS-1 throughout the retina. These results suggest the expression of tissue-specific regulators in the retina. Yeast two-hybrid screening of a bovine retinal cDNA library with the cytoplasmic domain of retinal insulin receptor identified a novel member of the Grb7 gene family, Grb14. Phosphorylation prediction software indicated 6 out of 18 tyrosine residues were most likely to be phosphorylated. Out of six tyrosine phosphorylation sites, one of the tyrosine residues in Grb14 is present in a conserved sequence motif, FXNPXY. The NPXY motifs are recognized by proteins containing a domain known as phosphotyrosine-binding (PTB) or phosphotyrosine-interacting domain (PID). The biological function of the PTB domain is to drive recruitment of signaling adapters such as IRS-1 or Shc to NPXpY (pY stands for phosphotyrosine) on activated receptor tyrosine kinases. We have made a novel finding that the PTB domain of IRS-1 binds to the NPXY motif of Grb14 in a phosphorylation-independent manner. In addition, Grb14−IRS-1 complexes are detected in lysates prepared from retina tissues. We suggest that the Grb14 NPXY motif could be acting as a dominant negative for IRS-1 functions in the retina, and this hypothesis is consistent with the recent study that Grb14-deficient mice exhibit enhanced IRS-1 phosphorylation and activation of protein kinase B. This is the first report describing the presence of the NPXY motif in Grb14 and binding of the PTB domain of IRS-1 in a phosphorylation-independent manner.