STRUCTURE DEFINITION OF IGF I RECEPTOR FUNCTIONAL DOMAIN

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Insulin-like growth factor I (IGF I) plays a key role in mediating the growth effects of pituitary growth hormone. In responsive cells, IGF I interacts at the plasma membrane with the IGF I receptor, a heterotetrameric glycoprotein composed of two disulfide-linked alpha-beta dimers. IGF I binding causes the transmembrane activation of the intrinsic catalytic activity of the IGF I receptor, but the mechanism by which this occurs is not yet well understood. In this application, we propose to study the structure and interaction of the functionally-defined domains of the IGF I receptor. To accomplish this, six specific aims are proposed. First, a rigorous analysis of the relationship of IGF I receptor autophosphorylation to activation of kinase activity in the presence and absence of IGF I will be performed. Second, the association of IGF I receptor alpha-beta dimers by disulfide bond formation will be analyzed. Third, the binding domain(s) for IGF I and IGF II in the purified IGF I receptor will be defined. Peptides comprising the IGF I and IGF II binding domains of the IGF I receptor will be identified following affinity cross- linking and cyanogen bromide treatment of the purified IGF I receptor, and anti-peptide antibodies directed against surface-exposed portions of the IGF I receptor will be developed and characterized. Fourth, the sites of IGF I receptor autophosphorylation in vitro and in vivo will be identified. Fifth, the functional consequences of phosphorylation of the IGF I receptor by protein kinase C and the sites at which this modification occurs in vitro and in vivo will be determined. Finally endogenous substrates of IGF I receptor tyrosine kinase activity in cells will be investigated. In these studies, phenylarsine oxide, a trivalent arsenical which has been reported to enhance the detection of endogenous substrates phosphorylated in insulin- and IGF I-stimulated cells, will be employed. The ability of phenylarsine oxide to inhibit IGF I-stimulated biological activities will be examined, and phosphoproteins that accumulate in the presence of IGF I and phenylarsine oxide will be identified. It is anticipated that these studies will lead to a better understanding of signal transduction by the IGF I receptor and its role in human growth.