ACTIVATION OF PHOSPHOLIPASE C-BETA-2 MUTANTS BY G-PROTEIN ALPHA(Q)-SUBUNIT AND BETA-GAMMA-SUBUNITS

The beta- but not the gamma- and delta-type isozymes of inositol phosp holipid-specific phospholipase C (PLC) are activated by G protein alph a(q) and betagamma subunits. The beta-type PLC isozymes differ from ot her isozymes in that they contain a long carboxyl-terminal region down stream of the Y catalytic domain and a region rich in acidic amino aci ds between the two separated X and Y catalytic domains. To determine t he sites on PLC-beta2 that participate in the interaction of the enzym e with alpha(q) and betagamma subunits, we introduced specific truncat ions and substitutions in the PLC-beta2 cDNA at positions correspondin g to the carboxyl-terminal and acidic amino acid-rich regions, respect ively. After transient expression of these cDNA clones in CV-1 cells, the mutant enzymes were partially purified and their capacity to be ac tivated by alpha(q) and betagamma subunits determined. Substitution of glutamine residues for three or all seven of a stretch of consecutive glutamic acids in the acidic domain of PLC-beta2 affected neither alp ha(q)- nor betagamma-dependent activation significantly. Carboxyl-term inal truncation to residue Gly-934 or to residue Ala-867 resulted in e nzymes that were activated by betagamma but not by alpha(q). This resu lt suggests that the carboxyl-terminal region of PLC-beta2 is required for activation by alpha(q) and that betagamma subunits interact with a different region of the enzyme. Thus, alpha(q) and betagamma subunit s may independently modulate a single PLC-beta2 molecule concurrently.