ACTIVATION OF PHOSPHOLIPASE-C BETA-2 BY THE ALPHA-SUBUNIT AND BETA-GAMMA-SUBUNIT OF TRIMERIC GTP-BINDING PROTEIN

Cotransfection assays were used to show that the members of the GTP-bi nding protein G(q) class of alpha subunits could activate phospholipas e C (PLC) beta2. Similar experiments also demonstrated that Gbeta1gamm a1, Gbeta1gamma5, and Gbeta2gamma5 could activate the beta2 isoform of PLC but not the beta1 isoform, while Gbeta2gamma1 did not activate PL C beta2. To determine which portions of PLC beta2 are required for act ivation by Gbetagamma or Galpha, a number of PLC beta2 deletion mutant s and chimeras composed of various portions of PLC beta1 and PLC beta2 were prepared. We identified the N-terminal segment of PLC beta2 with amino acid sequence extending to the end of the Y box as the region r equired for activation by Gbetagamma and the C-terminal region as the segment containing amino acid sequences required for activation by Gal pha. Furthermore, we found that coexpression of Galpha16 and Gbeta1gam ma1 but not Gbeta1gamma5 in COS-7 cells was able to synergistically ac tivate recombinant PLC beta2. We suggest that Galpha16 may act togethe r with free Gbeta1gamma1 to activate PLC beta2, while Galpha16 may for m heterotrimeric complexes with Gbeta1gamma5 and be stabilized in an i nactive form. We conclude that the regions of PLC beta2 required for a ctivation by Gbetagamma and Galpha are physically separate and that th e nature of the Gbeta subunit may play a role in determining the relat ive specificity of the Gbetagamma complex for effector activation whil e the nature of the Ggamma subunit isoform may be important for determ ining the affinity of the Gbetagamma complex for specific Galpha prote ins.