Characterization of Gq family G proteins GL1 alpha (G14 alpha), GL2 alpha (G11 alpha), and Gq alpha expressed in the baculovirus-insect cell system.
The alpha subunits of Gq family G proteins, GL1 alpha (G14 alpha), GL2 alpha(G11 alpha), and Gq alpha were expressed with G protein beta 1 and gamma 2 subunits in insect cells using a baculovirus system. The trimeric forms of G proteins, GL1 (GL1 alpha beta gamma), GL2 (GL2 alpha beta gamma), and Gq (Gq alpha beta gamma), were solubilized by 1% sodium cholate and purified by sequential chromatography on three kinds of columns. GL1, GL2, and Gq activated phospholipase C-beta purified from bovine brain in the presence of aluminum fluoride to the same extent. Muscarinic acetylcholine receptor m1 subtype stimulated the guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to GL1, GL2, and Gq in the presence of similar concentrations of carbamylcholine. When m1 receptor, G protein, and phospholipase C-beta were reconstituted in lipid vesicles, each subtype of Gq family G proteins mediated the activation of phospholipase C-beta by carbamylcholine in the presence of either 1 microM GTP gamma S or 1 mM GTP. Phospholipase C-beta stimulated the GTPase activity of GL1, GL2, and Gq in the presence of m1 receptor and carbamylcholine but did not stimulate the GTPase activity of GO. Protein kinase C phosphorylated m1 receptor and phospholipase C-beta, but the phosphorylation did not significantly affect the ability of the m1 receptor to stimulate phospholipase C-beta in the reconstitution system of purified proteins.
Nakamura
F
F
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
Kato
M
M
Kameyama
K
K
Nukada
T
T
Haga
T
T
Kato
H
H
Takenawa
T
T
Kikkawa
U
U
D000818:Animals; D016367:Baculoviridae; D001921:Brain / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D005822:Genetic Vectors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D011976:Receptors, Muscarinic / Q000096:biosynthesis / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D018411:Spodoptera; D014162:Transfection; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D046911:Macromolecular Substances; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002217:Carbachol; D017886:Durapatite; D010738:Type C Phospholipases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins