Many hormones and neurotransmitters stimulate phospholipase C (PLC), which catalyzes the hydrolysis of phosphatidyl 4,5-bisphosphate to inositol 1, 4, 5-triphosphate (IP3) and diacylglycerol. Both products serve as second messengers, resulting in the release of Ca2+ from intracellular stores and the activation of protein kinase C. The alpha1- adrenoceptor (alpha1-AR) is one of the important initiators of these sequential events that control a variety of sympathetically mediated responses involved in cardiac, metabolic, and central nervous system functions. Over the last two decades, pharmacological and biochemical studies on the alpha1-receptor signal mechanism have revealed that the alpha1a-AR couples to pertussis toxin-sensitive G-protein and stimulates the formation of arachidonic acid via phospholipase A2, and the alpha1b- AR couples to pertussis toxin-insensitive G-protein and stimulates the formation of IP3 via PLC. Recently, cloning and use of corresponding cDNA transfected cells have provided the information that the alpha1-AR subtypes, alpha1a-AR and alpha1c-AR, may stimulate PLC-beta1 through Gq/Gq11. In the case of alpha1b-AR, although in the transfected cells the receptor stimulated PLC-beta1 through Gq family, no specific Gq could yet be found. Thus, at present, transmembrane signaling of alpha1- adrenoceptors is controversial. By focusing on the identification of G- protein and of effector, which are involved in alpha1b-AR signaling pathway, we have isolated 74-kDa GTP-binding protein, which is associated with ~50-kDa protein (holoenzyme was termed at Gh), and a 69-kDa PLC. Reconstituting alpha1b-AR, Gh (Gh7), and 69-kDa PLC into phospholipid vesicles, we have also showed that these three components coupled effectively. Raising antibodies to Gh7alpha and Ghbeta, as well as performing biochemical studies, we have confirmed our functional coupling studies and that the 50-kDa protein is indeed the beta-subunit of Ghalpha that modulates the affinity of the alpha-subunit for guanine nucleotides. Moreover, our studies revealed, for the first time, that existence of a species-specific Gh family which is involved in a1-AR-mediated transmembrane signaling. To better understand the controversial point and to study the coupling mechanism of alpha1-AR system in detail, we propose to clone Ghalpha and GhB as well as to raise site-directed peptide antibodies to Ghalpha and Ghbeta to evaluate the structure- function relationship. We also propose to determine amino acid sequence of the 69-kDa PLC and to raise antibodies against this enzyme. Moreover, to understand GTPase cycle of Gh and role of Ghalpha and Ghbeta in alpha1-adrenergic signal mechanism in detail, we also propose to study involvement of Ghbeta, from receptor level to effector and physiological responses of Gh at the cellular level. These complete studies should provide us with major insights into the signal transduction mechanisms of the alpha1-adrenoceptors, which may also be pertinent to understanding the aspect of the transmembrane signal pathway that involves Gh and the structure-function relationship of this unique large molecular mass Gh family.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045985-07
Application #
2444786
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1991-07-01
Project End
1998-11-30
Budget Start
1997-07-01
Budget End
1998-11-30
Support Year
7
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
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