Characterization of a phospholipase C beta 2-binding site near the amino-terminal coiled-coil of G protein beta gamma subunits.
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 (Asn(564)-Lys(583)) and E20K (Glu(574)-Lys(593)), from the catalytic domain of phospholipase C (PLC) beta2 block Gbetagamma-dependent activation of PLC beta2. The peptides could also be directly cross-linked to betagamma subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to Gbeta(1) was inhibited by PLC beta2 but not by alpha(i1)(GDP), indicating that the peptide-binding site on beta(1) represents a binding site for PLC beta2 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 beta2 interaction with beta subunits. Each of the 14 cysteine residues in beta(1) were altered to alanine. The ability of the PLC beta2-derived peptide to cross-link to each betagamma 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.
Yoshikawa
D M
DM
Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Bresciano
K
K
Hatwar
M
M
Smrcka
A V
AV
D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D054799:Phospholipase C beta; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; D007527:Isoenzymes; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D020962:Heterotrimeric GTP-Binding Proteins