Abstract

Recently we showed that the chick heart muscarinic acetylcholine receptor is a phosphoprotein in intact cells and that treatment with agonists results in a striking increase in receptor phosphorylation [J. Biol. Chem. 261:12429-12432 (1986)]. Furthermore, we showed that the agonist-induced increase in the phosphorylation of chick heart muscarinic receptors correlates with receptor desensitization [J. Biol. Chem. 262:16314-16321 (1987)]. We have now extended studies of receptor phosphorylation to mammalian cardiac muscarinic receptors, in order to test the concept that phosphorylation is of general importance in the regulation of muscarinic receptor function. We have determined that, in intact porcine atria, M2 muscarinic receptors are phosphoproteins and that treatment with the agonist carbachol markedly increases receptor phosphorylation, to 4-6 mol of phosphate/mol of protein. Phosphorylation occurs on serine and threonine residues. Activation of either protein kinase C or cAMP-dependent protein kinase did not mimic the effect of agonists on receptor phosphorylation. These results are very similar to those seen with the chick heart muscarinic receptors. To determine whether the porcine and the chick cardiac muscarinic receptors represent similar or different proteins, we undertook detailed pharmacological studies and, in addition, prepared peptide maps of purified muscarinic receptors from chick heart and porcine atria. Our data show that there are marked differences in the pharmacological properties of the chick and the porcine cardiac muscarinic receptors. The peptide maps of the porcine and chick heart muscarinic receptors are also different, suggesting that muscarinic receptors in chick and porcine cardiac cells differ in their primary structure. Taken together, the data show that porcine and chick cardiac muscarinic receptors possess pharmacological and structural differences, but both receptors undergo agonist-mediated phosphorylation in intact cardiac cells. These data support the possibility that receptor phosphorylation may be of general importance in the regulation of muscarinic receptors.