Regulatory interactions between the amino terminus of G-protein βγ subunits and the catalytic domain of phospholipase Cβ2

Abstract

We previously identified a 10-amino acid region from the Y domain of phospholipase Cβ2 (PLCβ2) that associates with G-protein βγ 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 Cys25 within the amino-terminal coiled-coil domain of Gβγ (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 Å of Cys25. A mutant within the amino terminus of the Gβ subunit, Gβ1 (23-27)γ2, activated PLCβ2 more effectively than wild type, with no significant change in the EC50, indicating that this region is directly involved in the catalytic regulation of PLCβ2. 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 Gβ1 (23-27) γ2-dependent activation of PLC, suggesting a second N20K binding site. Competition analysis with a peptide that binds to the Gα subunit switch II binding surface of Gβγ indicates a second N20K binding site at this surface. Furthermore, mutations to the N20K region within the Y-domain of full-length PLCβ2 inhibited Gβγ-dependent regulation of the enzyme, providing further evidence for a Gβγ binding site within the catalytic domain of PLCβ2. The data support a model with two modes of PLC binding to Gβγ through the catalytic domain, where interactions with the amino-terminal coiled-coil domain are inhibitory, and interactions with the Gα subunit switch II binding surface are stimulatory. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.