Somatostatin (SRIF) is a neuropeptide which acts as a hormone, a neurotransmitter or an autocrine regulator to inhibit secretion and/or proliferation in endocrine, exocrine, neuronal and tumor cells. SRIF has been implicated in numerous physiological processes ranging from growth to memory as well as in several disorders including endocrine cancers and Alzheimer's disease. The biological actions of this peptide are initiated by binding to plasma membrane receptors which are coupled to effector enzymes and ion channels via o proteins. Recently five related SRIF receptor (sstr) genes have been identified by molecular cloning. These sstr subtypes are believed to serve unique biological roles by virtue of their tissue specific expression and particular biochemical properties. However, we do not understand the function of any individual sstr in its normal cellular milieu. Such studies have been difficult because SRIF- responsive tissues and cell lines often express multiple sstr's and, in the absence of subtype specific SRIF analogs, the actions of individual receptors cannot be identified. Moreover, sstr function depends on cellular environment so that the behavior of a receptor expressed in heterologous cells does not necessarily mimic that of endogenous receptors. This proposal has two overall goals: (l) characterize the signal transduction mechanisms activated by sstr subtypes, and (2) elucidate the mechanisms involved in the regulation of sstr's. In the last award period, we developed several receptor antibodies each of which immunoprecipitates the corresponding sstr subtype specifically. Moreover, we developed conditions such that the G-proteins coupled to sstr's in membranes remain receptor associated in the immunoprecipitate. We propose to characterize the G proteins coupled to each sstr subtype in different target cells by immunoprecipitating each receptor and identifying the associated G protein subunits with G protein specific antisera. We will therefore determine how cellular milieu affects the specificity of sstr-G protein coupling. Further, we will use biochemical and electrophysiological techniques to identify the effectors regulated by individual receptor subtypes in different target cells. We also propose to examine the mechanisms involved in sstr desensitization and regulation. Some SRIF responses are rapidly desensitized whereas others are not. Moreover, we have shown that sstr's are subject to chronic regulation by several different hormones. We propose to determine how specific hormonal treatments alter the expression of different sstr subtype mRNAs and proteins. In addition, we will examine the role of receptor phosphorylation in SRIF desensitization. Together these studies will elucidate the cellular mechanisms by which SRIF induces its biological actions and will identify the molecular basis for hormone induced changes in cellular responsiveness to SRIF.
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