RESCUE AND ACTIVATION OF A BINDING DEFICIENT INSULIN-RECEPTOR - EVIDENCE FOR INTERMOLECULAR TRANSPHOSPHORYLATION

Binding of insulin to the a subunit of the insulin receptor (IR) leads to autophosphorylation of the beta subunit. The reaction proceeds as intramolecular transphosphorylation between alpha beta half-receptors of the heterotetrameric receptor dimer (alpha(2) beta(2)). Since IRs a re mobile in the plane of the plasma membrane, it is also possible tha t transphosphorylation may occur between adjacent holoreceptors (alpha (2) beta(2)) by an intermolecular reaction. To address this question, we cotransfected NIH-3T3 cells with two IR cDNA constructs: a truncate d but functionally normal IR lacking the C-terminal 43 amino acids (De lta 43) and a full-length Leu(323) mutant receptor that is expressed o n the cell surface but that does not bind insulin. A clonal cell line was selected from cells cotransfected with a 1/5 ratio of Delta 43 cDN A/Leu(323) cDNA. The two homodimers (Leu(323) and Delta 43) were expre ssed without detectable formation of hybrid receptors. By using specif ic antibodies, we demonstrate that in cells coexpressing both homodime rs, the Leu(323) mutant receptor was phosphorylated in vivo by the Del ta 43 IR in an insulin-dependent manner. However, when the Leu(323) mu tant receptor was expressed alone, no phosphorylation was detected. In addition, we demonstrate the association of the phosphorylated Leu(32 3) mutant receptor with insulin receptor substrate-1 and with phosphat idylinositol 3-kinase. These findings indicate that insulin binding is not required for phosphorylation of the Leu(323) mutant receptor, tha t the phosphorylation of the Leu(323) mutant receptor occurs by an int ermolecular transphosphorylation mechanism, and, finally, that the Leu (323) mutant receptor, once phosphorylated, can associate with insulin receptor substrate-1 and phosphatidylinositol 3-kinase.