ENHANCED DEGRADATION OF THE PHOSPHOINOSITIDASE C-LINKED GUANINE-NUCLEOTIDE-BINDING PROTEIN G(Q)ALPHA G(II)ALPHA FOLLOWING ACTIVATION OF THEHUMAN M1-MUSCARINIC ACETYLCHOLINE-RECEPTOR EXPRESSED IN CHO CELLS

Treatment of CHO cells stably expressing the human M1 muscarinic acety lcholine (HM1) receptor with the cholinergic agonist carbachol results in a reduction in cellular levels of G(q)alpha/G11alpha. Half-maximal effects are produced by 3 h, and a new steady state of some 50% of th e resting levels of G(q)alpha/G11alpha is subsequently established Mu llaney, Dodd, Buckley and Milligan, (1993) Biochem. J. 289, 125-131). To analyse the mechanism of this effect, we examined the rate of turno ver of G(q)alpha/G11alpha in these HM1-expressing cells in the presenc e and absence of carbachol (1 mM). In untreated cells the measured rem oval of S-35-labelled G(q)alpha/G11alpha 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 G(q)a lpha/G11alpha 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 G(q)alpha/G11alpha 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 G (q)alpha/G11alpha, as the t0.5 of G(i)2alpha (approx. 30 h) was not af fected by the agonist. Analysis of G(q)alpha/G11alpha mRNA levels by r everse transcriptase/PCR indicated that there was no difference in cel ls maintained in the absence and presence of carbachol. Such data demo nstrate that agonist-induced establishment of a new steady-state level of G(q)alpha/G11alpha results from an initial receptor-mediated enhan cement of protein turnover followed by a desensitization of the recept or response.