Activation of phospholipase C beta 2 by the alpha and beta gamma subunits of trimeric GTP-binding protein.
Cotransfection assays were used to show that the members of the GTP-binding protein Gq class of alpha subunits could activate phospholipase C (PLC) beta 2. Similar experiments also demonstrated that G beta 1 gamma 1, G beta 1 gamma 5, and G beta 2 gamma 5 could activate the beta 2 isoform of PLC but not the beta 1 isoform, while G beta 2 gamma 1 did not activate PLC beta 2. To determine which portions of PLC beta 2 are required for activation by G beta gamma or G alpha, a number of PLC beta 2 deletion mutants and chimeras composed of various portions of PLC beta 1 and PLC beta 2 were prepared. We identified the N-terminal segment of PLC beta 2 with amino acid sequence extending to the end of the Y box as the region required for activation by G beta gamma and the C-terminal region as the segment containing amino acid sequences required for activation by G alpha. Furthermore, we found that coexpression of G alpha 16 and G beta 1 gamma 1 but not G beta 1 gamma 5 in COS-7 cells was able to synergistically activate recombinant PLC beta 2. We suggest that G alpha 16 may act together with free G beta 1 gamma 1 to activate PLC beta 2, while G alpha 16 may form heterotrimeric complexes with G beta 1 gamma 5 and be stabilized in an inactive form. We conclude that the regions of PLC beta 2 required for activation by G beta gamma and G alpha are physically separate and that the nature of the G beta subunit may play a role in determining the relative specificity of the G beta gamma complex for effector activation while the nature of the G gamma subunit isoform may be important for determining the affinity of the G beta gamma complex for specific G alpha proteins.
Division of Biology, California Institute of Technology, Pasadena 91125.
D
D
Wu
A
A
Katz
M I
MI
Simon
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D016296:Mutagenesis; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D001616:beta-Galactosidase; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase