Many hormones and neurotransmitters stimulate phospholipase C (PLC) via their specific receptors and GTP-binding regulatory protein (G- proteins), resulting in production of two important second messengers, inositol 1,4,5-triphosphate and diacylglycerol. The alpha1-adrenoceptor (alpha1-AR) is one of the important initiators of these sequential events and controls glycogenolysis and sympathetic nervous system responses, particularly, those involved in arteriolar smooth muscle and cardiac contraction. The alpha1-ARs couple with Gq-family proteins, Gi- like G-proteins, and a new class of G-protein Gh, resulting in stimulation of a variety of phospholipid hydrolyzing enzymes, PLC-beta1, PLA2, PLD, and PLC-delta1. Although cellular functions of the multiple alpha1-AR signaling are not clearly understood, it has been suggested that the alpha1-AR-mediated responses are cell type dependent and differ from species to species. Our goal is to elucidate the role of Gh in alpha1-AR signaling. GH is a heterodimer consisting of greater than or equal to 70 kDa GTP-binding subunit Galphah and approximately 50 kDa its regulatory subunit Gbetah. Galphah accepts the receptor signal and directly interacts with the effector, whereas Gbetah stabilizes GDP- bound Galphah and functions as a counterpart of the receptor. Our recent studies revealed that Galphah is a multifunctional enzyme Known as tissue type transglutaminase (TGase II). Thus, Galphah possesses two important biological functions the GTPase and transglutaminase activities. Although the cellular functions of Galphah should be reevaluated, considering both GTPase and TGase functions, it has implicated that Galphah/TGase II is involved in tumor growth, regulation of cell growth and proliferation, the receptor-mediated endocytosis, and receptor-mediated signaling. On the basis of our findings that Gh is a signal mediator transmitting the receptor signal to PLC, we proposed to study the following three programs: First, sequential activation of Gh-mediated transmembrane signaling has been elucidated, but deactivation mechanism of this pathway has not been clarified at molecular and structural level. Therefore, to further understand GTPase cycle of Gh involving alpha1B-AR and PLC-delta1, we have proposed to clone the regulatory protein Gbetah and study its role in Gh-mediated signaling. Second, the interaction sites of Galphah for Gbetah, including alpha1B-AR and PLC-delta1 will be further determined. Third, the Gh-mediated signaling and cellular functions of Galphah and Gbetah will be studied with the stably transfected cell lines. To dissect the Gh-mediated cellular responses specifically, several negative and positive mutants will be constructed. To aid the research program and refine our previous findings that PLC-delta1 is the effector in the Gh- mediated signaling, we have also proposed to identify the Galphah interaction site(s) on PLC-delta1. Accomplishing the proposed studies that will address the Gh functions from the structural level to the cellular level will provide an insight into understanding of the cellular impact of Gh.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Pharmacology A Study Section (PHRA)
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Cole, Alison E
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Cleveland Clinic Lerner
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