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Originally published In Press as doi:10.1074/jbc.M006073200 on January 5, 2001

J. Biol. Chem., Vol. 276, Issue 14, 11246-11251, April 6, 2001
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Characterization of a Phospholipase C beta 2-Binding Site Near the Amino-terminal Coiled-coil of G Protein beta gamma Subunits*

Daniel M. Yoshikawa, Karen Bresciano, Mamata Hatwar, and Alan V. SmrckaDagger

From the Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

In previous work (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148-7154), we showed that overlapping peptides, N20K (Asn564-Lys583) and E20K (Glu574-Lys593), from the catalytic domain of phospholipase C (PLC) beta 2 block Gbeta gamma -dependent activation of PLC beta 2. The peptides could also be directly cross-linked to beta gamma subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to Gbeta 1 was inhibited by PLC beta 2 but not by alpha i1(GDP), indicating that the peptide-binding site on beta 1 represents a binding site for PLC beta 2 that does not overlap with the alpha i1-binding site. Here we identify the site of peptide cross-linking and thereby define a site for PLC beta 2 interaction with beta  subunits. Each of the 14 cysteine residues in beta 1 were altered to alanine. The ability of the PLC beta 2-derived peptide to cross-link to each beta gamma mutant was then analyzed to identify the reactive sulfhydryl moiety on the beta  subunit required for the cross-linking reaction. We find that C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding to a region near cysteine 25 of beta 1 which is located in the amino-terminal coiled-coil region of beta 1 and identifies a PLC-binding site distinct from the alpha  subunit interaction site.

* This work was supported by Grant GM 53536 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-0892; Fax: 716-273-2652; E-mail: Alan_Smrcka@URMC.rochester.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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