Characterization of a phospholipase C beta2-binding site near the amino terminal coiled-coil of G protein betagamma subunits

doi:10.1074/jbc.M006073200

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Papers In Press, published online ahead of print January 5, 2001
J. Biol. Chem, 10.1074/jbc.M006073200
Submitted on July 10, 2000
Revised on January 4, 2001
Accepted on January 4, 2001

Characterization of a phospholipase C beta2-binding site near the amino terminal coiled-coil of G protein betagamma subunits

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

Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, NY 14642

Corresponding Author: Alan_Smrcka{at}urmc.rochester.edu

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 (N564-K583) and E20K (E574-K593), from the catalytic domain of phospholipase C (PLC) $beta$ 2 block G $beta$ $gamma$ -dependent activation of PLC $beta$ 2. The peptides could also be directly crosslinked to $beta$ $gamma$ subunits with a heterobifunctional crosslinker SMCC. Crosslinking of peptides to G $beta$ 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 crosslinking 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 crosslink to each {beat} $gamma$ mutant was then analyzed to identify the reactive sulfhydryl moiety on the $beta$ subunit required for the crosslinking reaction. We find that C25A was the only mutation that significantly affected peptide crosslinking. This indicates that the peptide is specifically binding to a region near cysteine 25 of $beta$ ₁ which is located in the N terminal coiled coil region of $beta$ ₁ and identifies a PLC-binding site distinct from the $alpha$ subunit interaction site

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				M. Sato, M. J. Cismowski, E. Toyota, A. V. Smrcka, P. A. Lucchesi, W. M. Chilian, and S. M. Lanier Identification of a receptor-independent activator of G protein signaling (AGS8) in ischemic heart and its interaction with Gbeta{gamma} PNAS, January 17, 2006; 103(3): 797 - 802. [Abstract] [Full Text] [PDF]

				S. Chen, F. Lin, and H. E. Hamm RACK1 Binds to a Signal Transfer Region of G{beta}{gamma} and Inhibits Phospholipase C {beta}2 Activation J. Biol. Chem., September 30, 2005; 280(39): 33445 - 33452. [Abstract] [Full Text] [PDF]

				T. M. Bonacci, M. Ghosh, S. Malik, and A. V. Smrcka Regulatory Interactions between the Amino Terminus of G-protein {beta}{gamma} Subunits and the Catalytic Domain of Phospholipase C{beta}2 J. Biol. Chem., March 18, 2005; 280(11): 10174 - 10181. [Abstract] [Full Text] [PDF]

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