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R. Rajala, M.D. Chan, M.E. McClellan; Interaction of GRB14 with Retinal Insulin Receptor . Invest. Ophthalmol. Vis. Sci. 2004;45(13):667.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have previously demonstrated the light–dependent tyrosine phosphorylation of insulin receptor in the retina, which leads to the activation of anti–apoptotic signaling molecules. The mechanism of the regulation of the insulin receptor in the retina is not known. Yeast two hybrid screening of a bovine retinal cDNA library with the cytoplasmic domain of retinal insulin receptor (IRß) identified a novel member of the GRB7 (growth factor receptor bound protein 7) gene family, the GRB14. Methods: We used the two–hybrid assay of protein–protein interaction in the yeast Saccharomyces cerevisiae to identify binding partners to IRß from the bovine retinal cDNA library. Deletion and site directed mutagenesis of IRß and GRB14 identified the critical regions involved in the interaction. Glutathione S–transferase (GST) fusions proteins containing various domains of GRB14 were incubated with retina lysates that were stimulated with insulin in retinal organ cultures. IR kinase assays were carried out in the presence and absence of GRB14 with baculovirus expressed IR, employing a polypeptide of IRS–1656–762 that contains the phosphorylation sites of the p85 subunit of phosphoinositide 3–kinase. Results: GRB14 forms a specific complex with the cytoplasmic domain of IRß when both are expressed as hybrid proteins in yeast cells. This interaction is strictly dependent upon receptor tyrosine kinase activity. Western blot analysis with anti–GRB14 antibody on bovine and rat retina lysates indicates the presence of GRB14, confirming the yeast two hybrid assays. Deletion mutagenesis on GRB14 indicated a region between PH (pleckstrin homology) and SH2 (Src homology) domain, PIR domain that binds to IRß at positions Y1146, Y1150 and Y1151. In GST pull–down assays, IRß from insulin–stimulated retinas interacted with full length GRB14, PIR, PIR–SH2 and PIR–SH2 (R466A) mutant, suggesting that the PIR domain is responsible for the interaction. IR catalyzed phosphorylation of IRS–1656–762 was inhibited by the PIR domain. Conclusions: These findings suggest that GRB14 could be a major downstream signaling component of the insulin receptor mediated pathways in the retina.
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