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, T he 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 solubi lized by 1% sodium cholate and purified by sequential chromatography o n three kinds of columns, G(L1), G(L2), and G(q) activated phospholipa se C-beta purified from bovine brain in the presence of aluminum fluor ide to the same extent, Muscarinic acetylcholine receptor m1 subtype s timulated the guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) bindin g to G(L1), G(L2) and G(q) in the presence of similar concentrations o f carbamylcholine. When m1 receptor, G protein, and phospholipase C-be ta were reconstituted in lipid vesicles, each subtype of G(q) family G proteins mediated the activation of phospholipase C-beta by carbamylc holine in the presence of either 1 mu M GTP gamma S or 1 mM GTP. Phosp holipase 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 stim ulate the GTPase activity of G(o). Protein kinase C phosphorylated m1 receptor and phospholipase C-beta, but the phosphorylation did not sig nificantly affect the ability of the m1 receptor to stimulate phosphol ipase C-beta in the reconstitution system of purified proteins.