Regulatory interactions between the amino terminus of G-protein βγ subunits and the catalytic domain of phospholipase cβ2

We previously identified a 10-amino acid region from the Y domain of phospholipase C beta 2 (PLC beta 2) that associates with G-protein beta gamma subunits (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. ( 1998) J. Biol. Chem. 273, 7148-7154). We mapped the site for cross-linking of a synthetic peptide (N20K) corresponding to this Y domain region to Cys(25) within the amino-terminal coiled-coil domain of G beta gamma (Yoshikawa, D. M., Bresciano, K., Hatwar, M., and Smrcka, A. V. ( 2001) J. Biol. Chem. 276, 11246-11251). Here, further experiments with a series of variable length cross-linking agents refined the site of N20K binding to within 4.4-6.7 angstrom of Cys(25). A mutant within the amino terminus of the G beta subunit, G beta(1)(23-27)gamma(2), activated PLC beta 2 more effectively than wild type, with no significant change in the EC50, indicating that this region is directly involved in the catalytic regulation of PLC beta 2. This mutant was deficient in cross- linking to N20K, suggesting that a binding site for the peptide had been eliminated. Surprisingly, N20K could still inhibit G beta 1(23-27)gamma(2)-dependent activation of PLC, suggesting a second N20K binding site. Competition analysis with a peptide that binds to the G alpha subunit switch II binding surface of G beta gamma indicates a second N20K binding site at this surface. Furthermore, mutations to the N20K region within the Y-domain of full-length PLC beta 2 inhibited G beta gamma-dependent regulation of the enzyme, providing further evidence for a G beta gamma binding site within the catalytic domain of PLC beta 2. The data support a model with two modes of PLC binding to G beta gamma through the catalytic domain, where interactions with the amino-terminal coiled-coil domain are inhibitory, and interactions with the G alpha subunit switch II binding surface are stimulatory.