Evidence for the simultaneous translocation of muscarinic acetylcholine receptor and G protein by carbachol.

Interactions between guanine nucleotide regulatory proteins (G proteins) and muscarinic acetylcholine receptors (mAChRs) were studied in vivo following carbachol treatment. Rat brain homogenates were separated by high speed ultracentrifugation into heavy and light membrane and 300,000 g supernate fractions. The G proteins were partially purified by Sephadex-G200 and heptylamine-Sepharose and the mAChRs by (3,2'-aminobenzhydryloxy)-tropane-(ABT)-affinity chromatographies. Radioligand binding assays showed that acute carbachol induced a biphasic translocation of the mAChRs and G proteins into the light membrane fraction with an initial release at 5-10 min and a second phase at 60 min. Portions of the released mAChRs and the G proteins, were found in the 300,000 g supernates and light membranes and were eluted in the same peak fractions from a Sephadex G-200 column. This dually labelled peak dissociated in the presence of digitonin, suggesting close association between the mAChR and G protein. ABT-affinity chromatography yielded dually labelled mAChR-G protein fractions which eluted as a single radioactive peak on a second ABT column. The partially purified G proteins from these fractions were photoaffinity labelled with 8-azidoguanosine-5'-triphosphate, [gamma-32P]. SDS-PAGE autoradiography revealed the presence of Go alpha and Gi alpha which may be released simultaneously with the mAChRs from the plasma membrane. In addition, 110,000 molecular weight polypeptide was dually labelled by [3H]-PrBCM and [gamma-32P]-8-azido-GTP suggesting the presence of a "mAChR-G protein complex." These findings provide direct evidence for the release of mAChRs and G proteins and a mAChR-G protein complex by agonist occupation of the mAChRs.