Epitope-tagged Gq alpha subunits: expression of GTPase-deficient alpha subunits persistently stimulates phosphatidylinositol-specific phospholipase C but not mitogen-activated protein kinase activity regulated by the M1 muscarinic acetylcholine receptor.
Gq is the heterotrimeric guanine nucleotide-binding protein that activates the beta isoforms of phosphatidyl-inositol-specific phospholipase C (PI-PLC). The Gq alpha-subunit polypeptide (alpha qa) was N-terminally modified by addition of a 9-aa sequence, YPYDVPDYA. Placement of the 9-aa epitope tag at the N terminus allowed expression of functional alpha q polypeptides and selective identification of plasmid-expressed wild-type and mutant G-protein alpha subunits. Mutation of glutamine-209 to leucine in the N-terminally epitope-tagged alpha q (N(epi) alpha qQ209L) inhibited GTPase activity and persistently activated PI-PLC, resulting in high steady-state levels of inositol phosphates. The elevated levels of inositol phosphates resulting from N(epi) alpha qQ209L expression were similar to those obtained with carbachol activation of the M1 muscarinic acetylcholine receptor. The Gq-coupled M1 receptor, which stimulates PI-PLC activity, and phorbol esters, acting via protein kinase C, activate the cytoplasmic mitogen-activated protein kinase in COS cells. However, the constitutive activation of PI-PLC enzymatic activity resulting from expression of GTPase-deficient alpha q was unable to persistently activate this kinase. The results indicate that persistent PI-PLC activation is insufficient to sustain the stimulation of a cytoplasmic serine/threonine protein kinase regulated by Gq-coupled receptor signal-transduction pathways.
Qian
N X
NX
Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206.
Winitz
S
S
Johnson
G L
GL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006224:Cricetinae; D004789:Enzyme Activation; D000939:Epitopes / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D000939:Epitopes; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate; D011494:Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D013755:Tetradecanoylphorbol Acetate