Regulatory interactions between the amino terminus of G-protein betagamma subunits and the catalytic domain of phospholipase Cbeta2.
We previously identified a 10-amino acid region from the Y domain of phospholipase Cbeta2 (PLCbeta2) that associates with G-protein betagamma 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 Gbetagamma (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 angstroms of Cys(25). A mutant within the amino terminus of the Gbeta subunit, Gbeta(1)(23-27)gamma(2), activated PLCbeta2 more effectively than wild type, with no significant change in the EC(50), indicating that this region is directly involved in the catalytic regulation of PLCbeta2. 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 Gbeta(1)(23-27)gamma(2)-dependent activation of PLC, suggesting a second N20K binding site. Competition analysis with a peptide that binds to the Galpha subunit switch II binding surface of Gbetagamma indicates a second N20K binding site at this surface. Furthermore, mutations to the N20K region within the Y-domain of full-length PLCbeta2 inhibited Gbetagamma-dependent regulation of the enzyme, providing further evidence for aGbetagamma binding site within the catalytic domain of PLCbeta2. The data support a model with two modes of PLC binding to Gbetagamma through the catalytic domain, where interactions with the amino-terminal coiled-coil domain are inhibitory, and interactions with the Galpha subunit switch II binding surface are stimulatory.
Bonacci
Tabetha M
TM
Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, New York 14642, USA,
Ghosh
Mousumi
M
Malik
Sundeep
S
Smrcka
Alan V
AV
D000244:Adenosine Diphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D020134:Catalytic Domain; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry*; D006801:Humans; D007313:Insecta; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D054799:Phospholipase C beta; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism
D003432:Cross-Linking Reagents; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D010455:Peptides; D000244:Adenosine Diphosphate; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D003545:Cysteine