Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase.
Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA-). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA+ or PKA- cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA- cells. The m2 receptor was more sensitive to agonist-dependent internalization, and its rate of internalization was faster in PKA- cells than it was in PKA+ cells. Treatment of PKA+ cells with 8-(4-chlorophenylthio)-cAMP or forskolin did not result in internalization of either m1 or m2 receptors and did not alter the extent of agonist-dependent internalization of m2. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA+ and PKA- cells was accompanied by desensitization of functional responses. Exposure of PKA+ cells to 10(-7) M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 +/- 9% decrease in the number of m1 receptors on the cell surface. However, treatment of PKA- cells expressing the m1 receptor did not result in internalization, suggesting that PKA was required for some aspect of PMA-dependent internalization.(ABSTRACT TRUNCATED AT 250 WORDS)
Scherer
N M
NM
Department of Pharmacology, University of Washington, Seattle 98195.
Nathanson
N M
NM
D000230:Adenocarcinoma / Q000473:pathology; D000310:Adrenal Gland Neoplasms / Q000473:pathology; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D003208:Concanavalin A / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D051379:Mice; D019832:N-Methylscopolamine; D010703:Phorbol Esters / Q000494:pharmacology; D010862:Pilocarpine / Q000494:pharmacology; D011494:Protein Kinases / Q000172:deficiency / Q000502:physiology*; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000145:classification / Q000187:drug effects* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012602:Scopolamine Derivatives / Q000378:metabolism; D015290:Second Messenger Systems / Q000187:drug effects*; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects
D010703:Phorbol Esters; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D012602:Scopolamine Derivatives; D013873:Thionucleotides; D010862:Pilocarpine; D003208:Concanavalin A; D005576:Colforsin; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D011813:Quinuclidinyl Benzilate; D000242:Cyclic AMP; D011494:Protein Kinases; D019832:N-Methylscopolamine