Recombinant Gq alpha. Mutational activation and coupling to receptors and phospholipase C.
Gq mediates hormonal stimulation of phosphoinositide-specific phospholipase C (PI-PLC). We mutated the alpha subunit of Gq (alpha q) to replace arginine 183 with cysteine. Mutations that substitute cysteine for the corresponding arginine residues of alpha s and alpha i2 constitutively activate their respective effector pathways, creating the gsp and gip2 oncogenes. Transient expression of alpha q-R183C in COS-7 and HEK-293 cells constitutively activates PI-PLC, but wild type (WT) alpha q does not. This suggests that the mutated arginines in alpha s, alpha i2, and alpha q share a common function in regulating the active state of these proteins and that the alpha q gene may serve as a target for oncogenic mutations in human tumors. In an attempt to develop an assay for receptor stimulation of recombinant alpha q, we co-expressed receptors with alpha q-WT. We found that the alpha 2-adrenoceptor stimulates PI-PLC activation in HEK-293 cells in a fashion that depends completely on co-expression of alpha q-WT. These findings create an experimental model, similar to that provided for alpha s by S49 cyc- cells, that should make it possible to analyze receptor and effector coupling by mutant alpha q against a null background.
Conklin
B R
BR
Department of Pharmacology, University of California, San Francisco 94143.
Chabre
O
O
Wong
Y H
YH
Federman
A D
AD
Bourne
H R
HR
D000067956:Adenylyl Cyclase Inhibitors; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009154:Mutation; D009857:Oncogenes; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010957:Plasmids; D011942:Receptors, Adrenergic, alpha / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000067956:Adenylyl Cyclase Inhibitors; D007295:Inositol Phosphates; D011942:Receptors, Adrenergic, alpha; D011994:Recombinant Proteins; D004247:DNA; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase