That growth hormone (GH) is an important regulator of growth and glucose metabolism has been known for may years, yet the molecular mechanisms of GH action are poorlyunderstood. The goal of this project is to gain insight into signal transduction by GH through examination of its rapid effects on cellular metabolism and the initial interaction of GH with its receptor. The cellular and molecular basis for the early actions of GH will be examined using rat adipocytes and cultured 3T3-F442A adipocytes, which exhibit a rapid (3-5 min) and substantial stimulation of glucose transport. Whether the initial stimulation involves recruitment of transporters from intracellular to plasma membranes, as for insulin, will be examined. The stimulation of transport by GH is unique in being transient, and the question of whether reversal of the initial changes in transport account for the transiency, or involve other mechanisms (e.g. decreased synthesis, increased degradation, or altered activity) will be addressed. To gain insight into the mechanism by which GH transduces the signal from its receptor into altered transport (or other cellular functions), previous studies on phosphorylation of the GH receptor will be extended to determine whether the GH receptor is itself a ligand- activated tyrosine kinase or is a substrate for a separate tyrosine kinase. Purified GH receptors will be examined for tyrosine kinase activity and an ATP binding site. Efforts will be made to separate receptors from kinase activity. The possibility of several categories of GH receptor which differ in their ability to shown GH receptor-associated kinase activity will be addressed by looking for GH promoted receptor phosphorylation and tyrosine kinase activity in various cell types. Gh receptors in 3T3-F442A cells will be compared to the cloned rabbit liver receptor using antibodies and oligonucleotide probes. Finally, the issue of whether GH receptor phosphorylation an contribute to GH action will be assessed. If the GH receptor is a substrate for a separate tyrosine kinase, the kinase will be characterized and isolate, its dependence on bound GH evaluated, and its activity in cells responsive or non-responsive to GH measured. if instead the GH receptor appears to be a tyrosine kinase, its activity will be characterized, potential in vivo substrates will be investigated and changes in receptor phosphorlation will be correlated with changes in responsiveness of cells to GH. These studies will provide greatly needed insight into the molecular mechanisms by which the signal(s) initiated by GH at its receptor may be transduced into alterations in a cellular function.
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