AGONIST ACTIVATION OF TRANSFECTED HUMAN M1-MUSCARINIC ACETYLCHOLINE-RECEPTORS IN CHO CELLS RESULTS IN DOWN-REGULATION OF BOTH THE RECEPTOR AND THE ALPHA SUBUNIT OF THE G-PROTEIN GQ

CHO cells stably transfected with cDNA encoding the human MI muscarinic acetylcholine (HM1) receptor were treated with the cholinergic agonist carbachol at various concentrations for differing times. Levels of the HM1 receptor and of a range of G-proteins were subsequently measured. Carbachol treatment of the transfected cells caused a substantial down-regulation of cellular levels of the alpha subunit of G(q) (G(q)alpha), but did not significantly alter cellular levels of the a subunits of G(s) or G(i)2. A small decrease in levels of G-protein beta-subunit was also produced. Parallel assessment of agonist-induced down-regulation of the HM1 receptor demonstrated that it was lost in concert with the G-protein. Similar concentrations of carbachol (5 muM) were required to produce half-maximal stimulation of inositol phosphate generation and loss of each of the HM1 receptor and G(q)alpha, and half-maximal losses of both receptor and G(q)alpha were produced by 3 h of treatment with 1 mM-carbachol. By contrast, treatment of the non-transfected parental CHO cells, which do not express detectable levels of the receptor, with carbachol had no effect on cellular G(q)alpha levels. Concurrent treatment of the HM1-expressing CHO cells with carbachol and cycloheximide indicated that suppression of protein synthesis de novo did not mimic the effect of carbachol, and hence even complete inhibition of transcription of the G(q)alpha gene and/or translation of pre-existing G(q)alpha mRNA could not account for the agonist-induced effect. We have previously noted that cellular levels of both G(s)alpha [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093] and the alpha subunits of the pertussis-toxin-sensitive G-proteins G(i)1, G(i)2 and G(i)3 [Green, Johnson and Milligan (1990) J. Biol. Chem. 265, 5206-5210] can be regulated in certain cell systems by agonist activation of receptors expected to interact with these G-proteins. These results demonstrate that the same is true of G(q)alpha and suggest that agonist-induced co-ordinate loss of receptors and associated G-proteins may be a more common feature than has been appreciated to date.