TRANSMEMBRANE ACTIVATION OF THE INSULIN RECEPTOR KINASE

The overall objectives of this research proposal are focused at understanding the molecular mechanisms by which extracellular insulin binding to the alpha subunit of the insulin receptor results in the transmembrane activation of the intracellular beta subunit protein kinase domain. Initially we plan to use the purified human placental insulin receptor to examine the role of subunit-subunit interaction in the insulin-dependent activation of the insulin receptor beta subunit autophosphorylation and exogenous substrate protein kinase activities. This will be accomplished by the careful analyses of the functional properties, both insulin binding and protein kinase activities, of an isolated alpha beta heterodimeric insulin receptor complex from the purified alpha2 beta2 heterotetrameric complex. These alpha beta heterodimeric complexes will then be used to determine the functional symmetry of the alpha2 beta2 heterotetrameric complex in terms of the spatial relationship between the insulin binding site(s) and the beta subunit autophosphorylation as well as ATP binding sites. These studies will then be extended to determine if one intracellular beta subunit can function as a competitive inhibitor for the exogenous protein kinase activity of the other beta subunit in an analogous fashion to the regulatory subunit of the cAMP- dependent kinase. Recent preliminary results from our laboratory have suggested that ATP may be an allosteric regulator of the insulin receptor protein kinase activity. This will be evaluated by examining the kinetic properties of ATP and various ATP analogous (i.e: AMPPNP) on the insulin receptor protein kinase activity as well as in direct substrate and nuleotide binding experiments. Similar studies will then be performed on the isolated alpha beta heterodimeric complexes which presumably should contain only one insulin an one ATP binding site. We also plan to directly determine which reduced cysteine residues are critical for the insulin receptor protein kinase activity and which may be responsible for the activation of the insulin receptor protein kinase by the previously predicted insulin induced intramolecular disulfide-sulfhydryl rearrangement. These studies will employ (3H)NEM labeling followed by proteolytic peptide mapping and microsequencing. Further, these studies will be extended to determine which of the possible disulfide bonds are responsible for the covalent association between the alpha and beta subunits as well as between the alpha beta heterodimeric complexes that are required for the formation of the mature alpha2 beta2 complex.