CHARACTERIZATION OF G(Q) FAMILY G-PROTEINS G(L1)ALPHA(G(14)ALPHA), G(L2)ALPHA(G(11)ALPHA), AND G(Q)ALPHA EXPRESSED IN THE BACULOVIRUS-INSECT CELL SYSTEM

The a subunits of G(q) family G proteins, G(L1)alpha(G(14)alpha), G(L2)alpha(G(11)alpha), and G(q) 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, G(L1) (G(L1)alpha beta gamma), G(L2) (C(L2)alpha beta gamma), and G(q) (G(q) alpha beta gamma), were solubilized by 1% sodium cholate and purified by sequential chromatography on three kinds of columns, G(L1), G(L2), and G(q) 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 G(L1), G(L2) and G(q) 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 G(q) family G proteins mediated the activation of phospholipase C-beta by carbamylcholine in the presence of either 1 mu M GTP gamma S or 1 mM GTP. Phospholipase C-beta stimulated the GTPase activity of G(L1), G(L2), and G(q) in the presence of mi receptor and carbamylcholine but did not stimulate the GTPase activity of G(o). 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.