MUTATIONAL ANALYSIS OF PHOSPHOLIPASE C-BETA(2) IDENTIFICATION OF REGIONS REQUIRED FOR MEMBRANE ASSOCIATION AND STIMULATION BY GUANINE-NUCLEOTIDE-BINDING PROTEIN BETA-GAMMA-SUBUNITS

Members of the beta isozyme subfamily of the phosphoinositide-specific phospholipases C (PLCbeta) have recently been shown to be stimulated by both guanine-nucleotide-binding protein alpha and betagamma subunits. The alpha subunits of the G(q) class activate PLCbeta isozymes in the order of PLCbeta1 greater-than-or-equal-to PLCbeta3 much greater than PLCbeta2, which is different from the order of PLCbeta3 > PLCbeta2 > PLCbeta1 for betagamma subunit stimulation. The C-terminal region of PLCbeta1, in particular the sequence between Thr903 and Leu1142, has been shown to be involved in interacting with activated alpha(q) subunits and to contain a region required for efficient membrane association of PLCbeta1 [Park, D., Jhon, D.-Y., Lee, C.-W., Ryu, S. H. & Rhee, S. G. (1993) J. Biol. Chem. 268, 3710-3714, and Wu, D., Jiang, H., Katz, A. & Simon, M. 1. (1993) J. Biol. Chem. 268,3704-3709]. To examine the structure-function relationships of a PLCbeta isozyme highly sensitive to betagamma subunit stimulation, we have altered the cDNA of PLCbeta2 by site-directed mutagenesis and have examined the effects of these structural alterations on the functional properties of the mutant polypeptides. The results show that the C-terminal region of PLCbeta2 downstream of Phe818, which corresponds to Tyr816 of PLCbeta1, contains a region essential for membrane association, but is required neither for the interaction of PLCbeta2 with Ca2+ and the phospholipid substrate, nor for betagamma subunit stimulation of PLCbeta2. These data suggest that PLCbeta isozymes are activated by alpha(q) and betagamma subunits via distinct domains.