AGONIST-INDUCED TRANSFER OF THE ALPHA-SUBUNITS OF THE GUANINE-NUCLEOTIDE-BINDING REGULATORY PROTEINS G(Q) AND G(11) AND OF MUSCARINIC M1 ACETYLCHOLINE-RECEPTORS FROM PLASMA-MEMBRANES TO A LIGHT-VESICULAR MEMBRANE-FRACTION

A clone of a Chinese hamster ovary (CHO) cell line expressing high levels of the human muscarinic M1 acetylcholine (Hm1) receptor undergoes a substantial agonist-specific down-regulation of both Hm1 receptors and the alpha subunits of the guanine-nucleotide-binding (G)-proteins G(q) and G(11) which is accompanied by a de sensitization of inositol-phospholipid-specific-phospholipase-C response [Mullaney, I., Dodd, M. W., Buckley, N. J. and Milligan, G. (1993) Biochem. J. 289, 125-231]. To examine early events in this process, the effect of agonist on subcellular distribution of G(q) alpha and G(11)alpha and of Hm1 receptors was assessed after short-term and long-term treatment with carbachol. Short-term (30 min) incubation with carbachol (1 mM) induced a simultaneous transfer of a proportion of both G(q) alpha and G(11)alpha and Hm1 receptors from plasma membranes to distinct light vesicular membranes. The total number of receptors and of G(q) alpha and G(11)alpha in each cell remained unchanged under these conditions. A similar transfer was noted for the G-protein G(s) alpha but not for intrinsic plasma membrane markers. The plasma membrane, as well as light vesicular membrane, pool of G(s) alpha subunit was unaffected by further sustained incubation with carbachol (16 h), whereas Hm1 receptors and both G(q) alpha and G(11)alpha proteins were down-regulated to 25% and 40%, respectively, when compared with untreated cells. Such observations support the idea that down-regulation of both the Hm1 receptor and its associated inositol-phospholipid-specific-phospholipase-C-linked G-proteins is produced by two sequential steps. The first is a transfer of signal-transducing polypeptides from the plasma membrane to a non-plasma membrane light vesicle fraction. The second step is represented by an agonist-specific down-regulation pathway. Both the Hm1 receptor and G(q) alpha/G(11)alpha appear to follow similar sequestration and down-regulation patterns.