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-binding protein G(q) class of alpha subunits could activate phospholipase C (PLC) beta2. Similar experiments also demonstrated that Gbeta1gamma1, Gbeta1gamma5, and Gbeta2gamma5 could activate the beta2 isoform of PLC but not the beta1 isoform, while Gbeta2gamma1 did not activate PLC beta2. To determine which portions of PLC beta2 are required for activation by Gbetagamma or Galpha, a number of PLC beta2 deletion mutants 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 required for activation by Gbetagamma and the C-terminal region as the segment containing amino acid sequences required for activation by Galpha. Furthermore, we found that coexpression of Galpha16 and Gbeta1gamma1 but not Gbeta1gamma5 in COS-7 cells was able to synergistically activate recombinant PLC beta2. We suggest that Galpha16 may act together with free Gbeta1gamma1 to activate PLC beta2, while Galpha16 may form heterotrimeric complexes with Gbeta1gamma5 and be stabilized in an inactive form. We conclude that the regions of PLC beta2 required for activation by Gbetagamma and Galpha are physically separate and that the nature of the Gbeta subunit may play a role in determining the relative specificity of the Gbetagamma complex for effector activation while the nature of the Ggamma subunit isoform may be important for determining the affinity of the Gbetagamma complex for specific Galpha proteins.