PATHWAYS OF INSULIN AND IGFI RECEPTOR SIGNALING

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Much progress has been made in recent years toward an understanding of the molecular mechanisms by which receptors with intrinsic tyrosine kinase activity mediate their cellular effects. These advances have come predominantly from the identification and cloning of cytoplasmic proteins which interact with the activated receptor tyrosine kinase (RTK) and transduce signals from the receptor to the interior of the cell. The insulin receptor (IR) and insulin-like growth factor I receptor (IGFIR) represent a subclass of RTKs which mediate a variety of effects in responsive tissues including regulation of cellular mitosis, metabolism and differentiation. Although a number of well-characterized mitogenic signaling pathways have been shown to emanate from these receptors, the nature of the metabolic and differentiation-related signaling is less clear. It is of great importance that we have a full understanding of the pathways which mediate signaling by these receptors. Such information will allow insight into diseases such as Type II diabetes which is characterized by insulin resistance and certain neoplastic diseases in which IGFI has been implicated. In this proposal we will ask the following fundamental questions regarding insulin and IGFI signaling, (1) What are the intracellular targets and signaling pathways responsible for mediating the diverse effects of the IR and IGFIR in skeletal muscle, an insulin- and IGFI- responsive tissue?, (2) How do signaling proteins acting through the structurally similar IR and IGFIR regulate such distinct cellular effects as mitogenesis, metabolism and differentiation?, and (3) What is the molecular nature of the novel (non-SH2) phosphotyrosine-dependent interaction of SHC and IRS-1 with the IR and IGFIR which we have identified? To begin to address these questions, we have developed a novel approach with which to rapidly identify and clone cDNAs whose protein products interact directly with the cytoplasmic domains of these receptors. We have successfully adapted the "two-hybrid" assay of protein:protein interaction in the yeast Saccharomyces cerevisiae to demonstrate and characterize the interaction of the IR and IGFIR with several proteins known to be involved in insulin and IGFI signaling including IRS-1, p85, and SHC. This assay has allowed the identification of a novel homologous motif within SHC and IRS-1 which mediates the phosphotyrosine-dependent interaction with the NPEY motif of the IR and IGFIR. In addition, we have demonstrated the utility of the two-hybrid assay in the identification and cloning of known and novel interacting proteins from a cDNA library. This new approach should allow a more complete understanding of the fundamental molecular mechanisms by which these important receptors function.