Regulation of cAMP-mediated gene transcription by wild type and mutated G-protein alpha subunits. Inhibition of adenylyl cyclase activity by muscarinic receptor-activated and constitutively activated G(o) alpha.
We have used a luciferase reporter gene under the transcriptional control of a cAMP response element (CRE) to monitor the effects of G-protein alpha subunits on cAMP-regulated gene expression and to examine muscarinic acetylcholine receptor (mAChR) functional coupling to G-proteins. Expression in JEG-3 cells of a mutationally activated Gi alpha-2 in which glutamine 205 is replaced with leucine (Q205L) decreased forskolin-stimulated expression from the CRE-luciferase gene by up to 75%. Similarly, mutation of glycine 43 (corresponding to glycine 12 in p21ras) to valine decreased forskolin-stimulated expression from the CRE-luciferase gene by a maximum of 50%, indicating that this mutation activates the G-protein and is potentially oncogenic. Transfection of the activated Q205L G(o) alpha subunit decreased forskolin stimulation of CRE-luciferase expression. Transfected wild type G(o) alpha was also able to couple the m4 mAChR receptor to inhibition of AC. The amino-terminal myristoylation site was removed from wild type Gi alpha-2 and Q205L Gi alpha-2 by changing glycine 2 to alanine (G2A). Gi alpha-2 with the G2A and Q205L mutations was unable to decrease forskolin stimulation of CRE-mediated luciferase activity. Furthermore, G2A Gi alpha-2 was unable to couple the m4 mAChR to inhibition of AC. Thus, myristoylation is required both for the function of constitutively active Q205L Gi alpha-2 and for receptor-mediated activation of wild type Gi alpha-2.
Migeon
J C
JC
Department of Pharmacology, University of Washington, Seattle 98195.
Thomas
S L
SL
Nathanson
N M
NM
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000235:genetics; D009154:Mutation; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D051381:Rats; D011976:Receptors, Muscarinic / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection
D000067956:Adenylyl Cyclase Inhibitors; D009227:Myristic Acids; D011976:Receptors, Muscarinic; D019814:Myristic Acid; D005576:Colforsin; D000242:Cyclic AMP; D008156:Luciferases; D019204:GTP-Binding Proteins