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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045985-10
Application #
6329725
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Goldman, Stephen
Project Start
1991-07-01
Project End
2002-11-30
Budget Start
2000-12-01
Budget End
2002-11-30
Support Year
10
Fiscal Year
2001
Total Cost
$232,531
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
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Baek, K J; Kang, S; Damron, D et al. (2001) Phospholipase Cdelta1 is a guanine nucleotide exchanging factor for transglutaminase II (Galpha h) and promotes alpha 1B-adrenoreceptor-mediated GTP binding and intracellular calcium release. J Biol Chem 276:5591-7
Hwang, K C; Gray, C D; Sivasubramanian, N et al. (1995) Interaction site of GTP binding Gh (transglutaminase II) with phospholipase C. J Biol Chem 270:27058-62
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Das, T; Baek, K J; Gray, C et al. (1993) Evidence that the Gh protein is a signal mediator from alpha 1-adrenoceptor to a phospholipase C. II. Purification and characterization of a Gh-coupled 69-kDa phospholipase C and reconstitution of alpha 1-adrenoceptor, Gh family, and phospholipase C. J Biol Chem 268:27398-405
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Im, M J; Gray, C; Rim, A J (1992) Characterization of a phospholipase C activity regulated by the purified Gh in reconstitution systems. J Biol Chem 267:8887-94
Ramarao, C S; Denker, J M; Perez, D M et al. (1992) Genomic organization and expression of the human alpha 1B-adrenergic receptor. J Biol Chem 267:21936-45
Perez, D M; Piascik, M T; Graham, R M (1991) Solution-phase library screening for the identification of rare clones: isolation of an alpha 1D-adrenergic receptor cDNA. Mol Pharmacol 40:876-83