Many hormones and neurotransmitters phospholipase C, which catalyzes the hydrolysis of phosphatidyl-4,5-bisphosphate to inositol-1,4,5-triphosphate and diacylglycerol. Both products serve as second messengers, resulting in the release of Ca2+ from intracellular stores and the activation of protein kinase C. Over the last decade, physiological and biochemical evidence has been emerging that suggests that this hormone-mediated signal pathway in plasma membranes involves a sequential coupling reaction between receptor, GTP-binding regulatory protein, and effector enzyme, phospholipase C. The alpha1-adrenergic receptor is one of the important initial signalling mediators that controls a variety of sympathetically mediated responses involved in cardiac, metabolic, and central nervous system functions. Although alpha1-receptors have been well characterized at the molecular level, the identity of the other important membrane components, the GTP- binding regulatory protein and phospholipase C, is not known. Recently, by focusing on identifying a GTP-binding regulatory protein that couples to alpha1-receptor, we have isolated a 74 kDa GTP-binding protein (termed Gh) that effectively couples to this receptor and very possibly to a phospholipase C. This GTP-binding protein possesses GTPase activity and probably has a subunit (a 50 kDa protein). To understand alpha1-adrenergic receptor transmembrane signalling at the molecular level, we propose to isolate and characterize the effector enzyme, phospholipase C. Thus, knowledge of all three key membrane components will then be available to allow the detailed evaluation of the sequential coupling mechanism of these proteins following their reconstitution into phospholipid vesicles. These studies should provide an important insight into the mechanism of alpha1- adrenergic transmembrane signalling at the molecular level. In many ways, the 74 kDa GTP-binding protein that we have isolated and characterized is very unique in its molecular weight, in its association to the subunit, and in its coupling to phospholipase C. By characterizing this GTP-binding protein in more detail, we also propose to carry out the molecular cloning of the 74 kDa protein and to evaluate the functional role of the subunit (the 50 kDa protein) regarding the regulation of the 74 kDa GTP-binding protein (GDP/GTP exchange) and the overall sequential reaction. The cloning of this protein will also enable us to better understand the alpha1-adrenergic receptor signalling in a different way. Most importantly, the molecular cloning will provide us with information about the total primary structure of the amino acids of Gh. It will also provide a breakthrough for further studies involving the evaluation of the structure-function relationship between proteins related to Gh and the determination of tissue specific expression and regulation of the expression of Gh. These completed studies should provide us with a major insight into the signal transduction mechanism of the alpha1-adrenergic receptor, which may also be pertinent to understanding the aspect of the transmembrane signal pathway that involves the Ca2+-mobilizing receptor system.

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National Institute of General Medical Sciences (NIGMS)
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Pharmacology A Study Section (PHRA)
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Cleveland Clinic Lerner
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