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(0)ALPHA

We have used a luciferase reporter gene under the transcriptional cont rol of a cAMP response element (CRE) to monitor the effects of G-prote in alpha subunits on cAMP-regulated gene expression and to examine mus carinic acetylcholine receptor (mAChR) functional coupling to G-protei ns. Expression in JEG-3 cells of a mutationally activated G(i) alpha-2 in which glutamine 205 is replaced with leucine (Q205L) decreased for skolin-stimulated expression from the CRE-luciferase gene by up to 75% . Similarly, mutation of glycine 43 (corresponding to glycine 12 in p2 1(ras)) to valine decreased forskolin-stimulated expression from the C RE-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 al so able to couple the m4 mAChR receptor to inhibition of AC. The amino -terminal myristoylation site was removed from wild type G(i) alpha-2 and Q205L G(i) alpha-2 by changing glycine 2 to alanine (G2A). G(i) al pha-2 with the G2A and Q205L mutations was unable to decrease forskoli n stimulation of CRE-mediated luciferase activity. Furthermore, G2A G( i) alpha-2 was unable to couple the m4 mAChR to inhibition of AC. Thus , myristoylation is required both for the function of constitutively a ctive Q205L G(i) alpha-2 and for receptor-mediated activation of wild type G(i) alpha-2.