Abstract MCB-9316997 Riedel We will use the insulin receptor as a bait to identify the unknown proteins with which it interacts and to define the roles of these interactions in insulin signaling. We will employ the two-hybrid system as a sensitive and systematic molecular genetic screening strategy in yeast which exploits selection for amino acid prototrophy as a result of bait-library interaction and permits the screening of the full complexity of large mammalian expression libraries. In Preliminary studies we have demonstrated that the two-hybrid system can detect the interaction between the insulin receptor and its known substrate IRS-1 as well as two novel IRS-1- interaction proteins in vivo which also associate with IRS-1 in vitro. Based on these results we will systematically identify and isolate the coding sequences of all proteins that directly interact with the insulin receptor and that are detectable with this sensitive strategy. In particular, we will prepare various fusion plasmids with catalytic insulin receptor domains as baits and screen two mammalian cDNA expression libraries for interacting expression products. Positive clones will be isolated, purified and confirmed by genetic tests, and cDNA inserts encoding interacting proteins will be sequenced and compared with protein sequence databases for motifs which may suggest potential functions. cDNAs lacking sequence homology to known proteins will be used as probes to identify and isolate complete protein coding sequences. Novel interacting proteins and known proteins identified with this strategy will be tested for their role in insulin signaling by biochemical analysis including cDNA expression, co-purification, in vitro binding, co- immunoprecipitation, cross-phosphorylation assays, and by other approaches, which will be specifically designed for the candidate proteins once they have been identified. The knowledge gained is expected to further elucidate the molecular mechanisms involved in insulin signaling and peptide hormone action in general. *** One of the most thoroughly studied models of peptide hormone action is insulin with its receptor, both of which are key elements in signaling pathways in virtually any mammalian cell. Despite great progress in the elucidation of the structure and function of the insulin receptor, many of the detailed mechanisms, the sequence of events and the components involved in the transmission of the insulin signal from the cell surface to the nucleus remain unclear. While a number of direct insulin receptor substrates are suggested by the complex cellular responses to insulin only one, IRS-1 has been characterized in detail at the molecular level. Part of the reason has been the lack of systematic and efficient strategies to identify other unknown direct insulin receptor targets by molecular cloning techniques. Using a new, simple and systematic molecular genetic strategy, the two-hybrid system, we have demonstrated that protein interactions involving the insulin receptor and its substrate IRS-1 can be detected and that IRS-1 interacting target protein sequences can be identified and isolated. With this approach we will screen mammalian cDNA libraries for all cellular targets of the insulin receptor that are detectable. The screen is expected to identify and isolate sequences of known proteins with a previously unknown role in insulin action as well as novel cellular targets of the insulin receptor. Once they are identified these components will be characterized by biochemical analysis for their role in the insulin signaling pathway. This strategy is expected to identify the elements in the insulin signaling pathway stepwise to the final destination in the cell nucleus. The elucidation of these steps should critically contribute to our understanding of the molecular mechanisms involved in peptide hormone action. %%%
