Phosphorylation of the porcine atrial muscarinic acetylcholine receptor by cyclic AMP-dependent protein kinase

Abstract

cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, smooth muscle myosin light-chain kinase, and phosphorylase kinase were examined with respect to their ability to phosphorylate porcine atrial muscarinic receptors (mAcChRs). Experiments were performed both in detergent solution and in a reconstituted system containing the mAcChR alone or in the presence of the purified porcine atrial inhibitor guanine nucleotide binding protein (Gi). Only cAMP-dependent protein kinase was capable of phosphorylating the receptor under any of the experimental conditions examined. Phosphorylation of the mAcChR in the detergent-solubilized state resulted in a loss of ligand binding sites that was reversible upon treatment with calcineurin in the presence of calcium and calmodulin. Upon reconstitution, the apparent stoichiometry of phosphorylation was increased by about 15-fold. Carbachol-stimulated covalent incorporation of phosphate was found only in the reconstituted system in the presence of Gi, suggesting that the large agonist-stimulated increase in phosphorylation observed in vivo [Kwatra, M. M., & Hosey, M. M. (1986) J. Biol. Chem. 261, 12429-12432] may in part result from a unique receptor conformation that occurs upon association with this protein. Ligand binding studies indicated that phosphorylation of the mAcChR in the detergent-solubilized or reconstituted state did not affect its interaction with carbachol or L-quinuclidinyl benzilate in vitro. Carbachol-induced stimulation of the GTPase activity of Gi in the reconstituted system was also unaffected by phosphorylation.

Protein kinase regulation of muscarinic receptor signalling in colonic smooth muscle

“…In accordance with published reports (Lai & El-Fakahany, 1986;Abdallah et al, 1990;Lai et al, 1990), our results demonstrate that activation of PKC selectively decreases high-affinity pirenzepine binding sites from the smooth muscle cell surface. Our reasoning that M2 muscarinic receptors on colonic cells are not affected by PKC is supported by the observation that the purified M2 muscarinic receptor from porcine atria is not phosphorylated by purified PKC (Rosenbaum et al, 1987;Haga et al, 1988). The M3 receptors sequestered from the surface of the colonic smooth muscle cell surface following PDBu treatment are not rapidly degraded since [H]-QNB binding is not affected by PDBu.…”

“…The functions of many members of the Gprotein-linked receptor family, including muscarinic receptors, are regulated by receptor phosphorylation. It has been shown that purified Ml muscarinic receptors can be phosphorylated in vitro by both PKC and cyclic AMP-PK and that purified M2 receptors can be phosphorylated by cyclic AMP-PK (Rosenbaum et al, 1987;Haga et al, 1988). In this regard, it is intriguing that phosphorylation of muscarinic receptors shows subtype specificity with Ml and M3 receptor t"I Macmillan Press Ltd, 1993 subtypes being more sensitive to the effects of PKC (Scherer & Nathanson, 1990; Hu et al, 1991).…”

Phosphorylation by Protein Kinase C of the Muscarinic Acetylcholine Receptor

“…We could not detect appreciable phosphorylation by protein kinase C of muscarinic receptors purified from porcine heart, which are supposed to be predominantly composed of one receptor subtype (mAChR I1 or m2) (Kubo et al, 1986ab;Bonner et al, 1987), when the cerebral receptors had been phosphorylated. On the other hand, Rosenbaum et al (1987) reported the phosphorylation by CAMP-dependent protein kinase of atrial muscarinic receptors. We confirmed their observation and also found that the extent of phosphorylation was greater for atrial receptors than for cerebral receptors (data not shown).…”

“…G proteins were shown to be phosphorylated by protein kinase C, although the amount of incorporated phosphate was tl mol/mol of G protein (Katada et al, 1985;Haga et al, 1989). On the other hand, the phosphorylation of muscarinic receptors by protein kinase C has not been reported as far as we know, although muscarinic receptors were shown to be phosphorylated by cyclic AMP (CAMP)-dependent protein kinasc (Ho et al, 1987;Rosenbaum et al, 1987) or unidentified kinases (Kwatra and Hosey, 1986;Burgoyne, 1987;Kwatra et al, 1987). Other G protein-coupled receptors, such as the /3and a,-adrenergic receptors, have been shown to be phosphorylated by protein kinase C (Bouvier et al, 1987;Leeb-Lundberg et al, 1987;Sibley et al, 1987).…”

Cross Talk between Cyclic AMP and the Polyphosphoinositide Signaling Cascade in Iris Sphincter and Other Nonvascular Smooth Muscle

“…The source of trigger 1 rnin . Phosphorylation of the porcine atrial mAChR by PKA did not affect its interaction with CCh or the CCh-induced stimulation of the GTPase activity of Gi in the reconstituted system (29). The return to basal [Ca2+Ii is due to Na+-Ca2+ exchange located at the plasma membrane and to the Ca2+ pumps located at the plasma membrane and the SR (Fig.…”