Interaction of the Retinal Insulin Receptor β-Subunit with the P85 Subunit of Phosphoinositide 3-Kinase

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Abstract
Recently, we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated in vivo through light activation of the insulin receptor β-subunit. In this study, we have cloned the 41 kDa cytoplasmic region of the retinal insulin receptor (IRβ) and used the two-hybrid assay of protein−protein interaction in the yeast Saccharomyces cerevisiae to demonstrate the interaction between the p85 subunit of PI3K and the cytoplasmic region of IRβ. Under conditions where IRβ autophosphorylates, substitution of Y1322F and M1325P in IRβ resulted in the abolition of p85 binding to the IRβ, confirming that the p85 subunit of PI3K binds to Y1322. The binding site for p85 on IRβ was also confirmed in the yeast three-hybrid system. Using the C-terminal region of IRβ (amino acids 1293−1343 encompassing the YHTM motif) as bait and supplying an exogenous tyrosine kinase gene to yeast cells, we determined that the IRβ-pYTHM motif interacts with p85. We also used retinal organ cultures to demonstrate insulin activation of the insulin receptor and subsequent binding of p85, measured through GST pull-down assays with p85 fusion proteins. Further, the Y960F mutant insulin receptor, which does not bind IRS-1, is capable of bringing down PI3K activity from retina lysates. On the other hand, in response to insulin, IRS-2 is able to interact with the p85 subunit of PI3K in the retina. These results suggest that multiple signaling pathways could regulate the PI3K activity and subsequent activation of Akt in the retina.

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