Agonist-induced transfer of the alpha subunits of the guanine-nucleotide-binding regulatory proteins Gq and G11 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 Gq and G11 which is accompanied by a desensitization of inositol-phospholipid-specific-phospholipase-C response [Mullaney, I., Dodd, M. W., Buckley, N. J. & Milligan, G. (1993) Biochem. J. 289, 125-131]. To examine early events in this process, the effect of agonist on subcellular distribution of Gq alpha and G11 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 Gq alpha and G11 alpha and Hm1 receptors from plasma membranes to distinct light vesicular membranes. The total number of receptors and of Gq alpha and G11 alpha in each cell remained unchanged under these conditions. A similar transfer was noted for the G-protein Gs alpha but not for intrinsic plasma membrane markers. The plasma membrane, as well as light vesicular membrane, pool of Gs alpha subunit was unaffected by further sustained incubation with carbachol (16 h), whereas Hm1 receptors and both Gq alpha and G11 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 Gq alpha/G11 alpha appear to follow similar sequestration and down-regulation patterns.
Svoboda
P
P
Department of Biochemistry, University of Glasgow, Scotland.
Milligan
G
G
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D018721:Muscarinic Agonists; D010042:Ouabain / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D046911:Macromolecular Substances; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D010042:Ouabain; D002217:Carbachol; D000251:Adenosine Triphosphatases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases