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)