The insulin receptor is a alpha2beta2 tetrameric protein having an extracellular alpha subunit with an insulin binding domain and a transmembrane beta subunit with an intracellular tyrosine kinase domain. We propose to study these functional domains of the insulin receptor, and potential mechanisms of insulin receptor transmembrane signalling. We will investigate: 1) the location of the insulin binding site in the alpha subunit; 2) the role of tyrosine kinase activity in the beta subunit; and 3) post receptor signalling mechanisms via serine kinase activation. First we will continue our studies of the insulin binding site in the high cysteine domain of the receptor alpha subunit. By employing """"""""site specific"""""""" mutagenesis and a panel of insulin analogs we will test our recent hypotheses that sequence 240-250 is an insulin binding site. Next we will """"""""domain swap"""""""" regions of the insulin receptor alpha subunit with analogous regions of the IGF-I receptor alpha subunit. Also we will map monoclonal antibody binding sites on the alpha subunit. Second we will investigate which effects of insulin require tyrosine kinase activation by studying insulin receptor beta subunit mutants. We have preliminary data in rat HTC cells that a triple tyrosine autophosphorylation mutant (tyrosines 1158, 1162, 1163) does not undergo receptor autophosphorylation and has no detectable tyrosine kinase activity, but signals regulation of amino acid transport. We plan to express this mutant in mouse 3T3 cells where we can measure a spectrum of biological functions. We will also study the effects of insulin and ATP on receptor beta subunit conformation under conditions where receptor kinase is not activated. We will also continue our studies with insulin receptor monoclonal antibodies that are either insulin agonists or antagonists. Fab fragments will be prepared to test the hypothesis that bivalency and receptor aggregation are important for antibody (and perhaps insulin) action. Third we will investigate intermediate serine/threonine kinase(s) involved in insulin action. As a model we will investigate the kinase cascade leading to the phosphorylation of ribosome protein S6. Intermediate kinases to be investigated include: insulin receptor associated serine kinase (IRSK), multifunctional protein kinase (Fa), and microtubule associated protein kinase (MAP kinase). These studies should provide important information therefore as to the various mechanisms leading to insulin action.
