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pubmed:abstractText |
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.
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