EPITOPE-TAGGED G(Q) ALPHA-SUBUNITS - EXPRESSION OF GTPASE-DEFICIENT ALPHA-SUBUNITS PERSISTENTLY STIMULATES PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C BUT NOT MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVITY REGULATED BY THE M1 MUSCARINIC ACETYLCHOLINE-RECEPTOR

G(q) is the heterotrimeric guanine nucleotide-binding protein that act ivates the beta isoforms of phosphatidyl-inositol-specific phospholipa se C (PI-PLC). The G(q) alpha-subunit polypeptide (alpha(q)) was N-ter minally modified by addition of a 9-aa sequence, YPYDVPDYA. Placement of the 9-aa epitope tag at the N terminus allowed expression of functi onal alpha(q) polypeptides and selective identification of plasmid-exp ressed wild-type and mutant G-protein alpha subunits. Mutation of glut amine-209 to leucine in the N-terminally epitope-tagged alpha(q) (N(ep i)alpha(q)Q209L) inhibited GTPase activity and persistently activated PI-PLC, resulting in high steady-state levels of inositol phosphates. The elevated levels of inositol phosphates resulting from N(epi)alpha( q)Q209L expression were similar to those obtained with carbachol activ ation of the M1 muscarinic acetylcholine receptor. The G(q)-Coupled M1 receptor, which stimulates PI-PLC activity, and phorbol esters, actin g via protein kinase C, activate the cytoplasmic mitogen-activated pro tein kinase in COS cells. However, the constitutive activation of PI-P LC enzymatic activity resulting from expression of GTPase-deficient al pha(q) was unable to persistently activate this kinase. The results in dicate that persistent PI-PLC activation is insufficient to sustain th e stimulation of a cytoplasmic serine/threonine protein kinase regulat ed by G(q)-coupled receptor signal-transduction pathways.