Abstract
Abstract: :
Purpose: Recently we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated through in vivo light activation of the insulin receptor ß-subunit [Rajala et al JBC 277:43319-43326 (2002)]. In this study we have cloned the 41-kDa cytoplasmic region of the retinal insulin receptor (IRß) and studied its interaction with p85 subunit of PI3K and insulin receptor substrate-1 (IRS-1). Methods: We used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to study the interaction of IRß with p85 and IRS-1. Full-length p85, and p85-N (SH2) domain and its mutant form (R358A) were expressed as glutathione S-transferase (GST) fusions proteins, and their ability to interact with IRß was determined by GST pull-down assays. Receptor activation was measured in retinal organ cultures through GST pull-down assays. IRß C-terminal tail (1293-1343) was expressed, phosphorylated, and incubated with bovine rod outer segment membranes, and the interaction with p85 determined by pull-down assay. Results: We demonstrate that p85 forms a specific complex with IRß when both are expressed as hybrid proteins in yeast cells. This interaction is strictly dependent upon receptor tyrosine kinase (TK) activity, since p85 shows no interaction with a kinase-inactive receptor. Substitution of Y1322F and M1325P in IRß did not affect the intrinsic TK activity, but abolished p85 binding to IRß. These results confirm that p85 binds to Y1322 in IRß. Substitution of Y960F and Y1316F did not affect the binding of IRß, whereas substitution of Y960F abolished binding of IRS-1 to IRß. Receptor activation experiments indicate the direct interaction of IRß with p85, which does not require IRS-1. Expression and phosphorylation of the C-terminal YTHM motif of IRß also showed direct interaction with p85 independent of IRS-1. Conclusions: This is the first in vivo study demonstrating the direct interaction of p85 subunit of PI3K to IRß.
Keywords: second messengers • signal transduction • protein structure/function