PURIFICATION AND CHARACTERIZATION OF RECOMBINANT-G(16-ALPHA) FROM SF9 CELLS - ACTIVATION OF PURIFIED PHOSPHOLIPASE-C ISOZYMES BY G-PROTEIN ALPHA-SUBUNITS

A cDNA encoding G16alpha, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16alpha in membrane extracts of Sf9 cells activated phospholipase C-beta1 (PLC-beta1) in the presence of guanosine 5'-[gamma-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16alpha in the cytosolic fraction of Sf9 cells did not stimulate PLC-beta1. Concurrent expression of the G-protein betagamma subunit complex increased the amount of G16alpha. in Sf9 cell membranes. The guanosine 5'-[gamma-thio]triphosphate-activated form of G16alpha was purified from cholate extracts of membranes from cells expressing G16alpha, and the G-protein beta2 and gamma2 subunits. G16alpha activated PLC-beta1, PLC-beta2, and PLC-beta3 in a manner essentially indistinguishable from that of G(qalpha). G16alpha-mediated activation of PLC-beta1 and PLC-beta3 greatly exceeded that of PLC-beta2. G16alpha did not activate PLC-gamma1 or PLC-delta1. Thus, two distantly related members of the G(qalpha) family, G(qalpha) and G16alpha, have the same ability to activate the known isoforms of PLC-beta.