Enhanced degradation of the phosphoinositidase C-linked guanine-nucleotide-binding protein Gq alpha/G11 alpha following activation of the human M1 muscarinic acetylcholine receptor expressed in CHO cells.
Treatment of CHO cells stably expressing the human M1 muscarinic acetylcholine (HM1) receptor with the cholinergic agonist carbachol results in a reduction in cellular levels of Gq alpha/G11 alpha. Half-maximal effects are produced by 3 h, and a new steady state of some 50% of the resting levels of Gq alpha/G11 alpha is subsequently established [Mullaney, Dodd, Buckley and Milligan, (1993) Biochem. J. 289, 125-131]. To analyse the mechanism of this effect, we examined the rate of turnover of Gq alpha/G11 alpha in these HM1-expressing cells in the presence and absence of carbachol (1 mM). In untreated cells the measured removal of 35S-labelled Gq alpha/G11 alpha was adequately described by a monoexponential curve with a half-time (t0.5) of 18.0 +/- 2.2 h. When the cells were treated with carbachol a more complex pattern of Gq alpha/G11 alpha degradation was observed. Upon addition of the agonist, the rate of degradation initially increased markedly (t0.5 = 2.9 +/- 0.2 h). The maintained presence of the agonist was unable, however, to sustain the enhanced rate of degradation. Beyond 8 h of treatment with carbachol, degradation of Gq alpha/G11 alpha returned to a rate close to that observed in untreated cells (t0.5 = 18.5 +/- 1.3 h). Parallel experiments indicated that the effect of carbachol was specific for Gq alpha/G11 alpha, as the t0.5 of Gi2 alpha (approx. 30 h) was not affected by the agonist. Analysis of Gq alpha/G11 alpha mRNA levels by reverse transcriptase/PCR indicated that there was no difference in cells maintained in the absence and presence of carbachol. Such data demonstrate that agonist-induced establishment of a new steady-state level of Gq alpha/G11 alpha results from an initial receptor-mediated enhancement of protein turnover followed by a desensitization of the receptor response.
Mitchell
F M
FM
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Buckley
N J
NJ
Milligan
G
G
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology*; D006224:Cricetinae; D004277:DNA, Single-Stranded; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000235:genetics
D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D002217:Carbachol; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D019204:GTP-Binding Proteins