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.
Our studies on the alpha 1-adrenoceptor signaling have demonstrated that the Gh family is a signal mediator. We report here that a 69-kDa phospholipase C (PLC) is the effector in this signal pathway. The enzyme was isolated by dissociating a Gh7-PLC complex which was induced in the bovine liver membranes incubating with (-)-epinephrine and GTP. The enzyme displayed a marked preference hydrolysis for phosphatidylinositol 4,5-bisphosphate over other phosphatidylinositides at micromolar calcium. Reconstitution of PLC with the alpha 1-adrenoceptor and Gh (Gh7) into phospholipid vesicles resulted in a lowered Ca2+ requirement for the substrate hydrolysis in the presence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) when the receptor was activated with the alpha 1-agonist. The formation of inositol phosphate was hormone concentration dependent and reached maximal within 3 min which was faster than the formation in the presence of the alpha 1-antagonist. An Gh7 alpha antibody co-immunoprecipitated 80-85% of phospholipase C activity in the presence of GTP gamma S, but not in the presence of GDP or buffer, showing the association of PLC with the alpha-subunit of Gh family. Thus, our novel approaches to identify the effector involved in the alpha 1-adrenoceptor signaling, as well as the reconstitution studies, substantially demonstrate that the alpha 1-adrenoceptor-mediated transmembrane signaling involves the Gh family and a 69-kDa PLC.
Das
T
T
Department of Cardiovascular Biology, Cleveland Clinic Foundation, Ohio 44195.
Baek
K J
KJ
Gray
C
C
Im
M J
MJ
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D011233:Precipitin Tests; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D018340:Receptors, Adrenergic, alpha-1; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases; D019204:GTP-Binding Proteins