Phosphorylation by Protein Kinase C of the Muscarinic Acetylcholine Receptor

Abstract

Muscarinic acetylcholine receptors purified from porcine cerebrum were phosphorylated by protein kinase C purified from the same tissue. More than 1 mol of phosphate was incorporated per mole of receptor, with both serine and threonine residues being phosphorylated. Neither the degree nor the rate of the phosphorylation was affected by the presence or absence of acetylcholine. GTP-sensitive high-affinity binding with acetylcholine was observed for muscarinic receptors reconstituted with GTP-binding proteins (Gi or Go), irrespective of whether muscarinic receptors or the GTP-binding proteins had been phosphorylated by protein kinase C or not. This indicates that the interaction between purified muscarinic receptors and purified GTP-binding proteins in vitro is not affected by their phosphorylation.

Dual regulation by G proteins of agonist‐dependent phosphorylation of muscarinic acetylcholine receptors

“…Thus, the effective concentrations of carbamylcholine and atropine are essentially the same as those in the absence of G proteins [ 14,161. These apparent & values are comparable to those estimated from the [3H]QNB binding experiments [9,10], confirming that the effects of carbamylcholine and atropine are due to their 'binding to muscarinic receptors. The agonist-dependent phosphorylation of muscarinic receptors by the present kinase preparation was not affected by CAMP or cGMP, did not require the presence of Ca and was inhibited by 0.15 M NaCl or KC1 [16], which are properties common to the &adrenergic receptor kinase [17].…”

“…Muscarinic receptors were purified from porcine atrium [18], reconstituted with G proteins in lipid vesicles [9] and then subjected to phosphorylation with a protein kinase. ylated in the presence of carbamylcholine but not in its absence and were the only major proteins phosphorylated, no phosphorylation of G proteins being detected.…”

“…It is known that both cerebral [7][8][9]21,24] and atria1 110,251 muscarinic receptors reconstituted with one of the three G proteins (Gi, Go, Gn) show high affinity for agonists in the absence of a guanine nucleotide but low affinity in its presence, and that the proportion of guanine nucleotide-sensitive high affinity agonist binding increases with an increase in the G protein concentration. The concentrations of G proteins required for the maximum effect differ depending on the experi-46 mental conditions, being reported to be a only 3-fold excess of cardiac Gi over atria1 receptors by Tota et al [lo], and an SOO-fold excess of Go over cerebral receptors by Florio and Sternweis [24].…”

Characterization of radioligand binding to a transmembrane receptor reconstituted into Lipobeads

“…Each point represents the mean of either three or four experiments (AES.E.M.). Individual estimates of B obsd were adjusted as described previously to obtain the corresponding values of B sp plotted on the y-axis [10] Table 1.…”

“…Reconstitution was carried out essentially as described by Haga et al [10]. The following conditions are for reconstitution on a 2-mL bed volume of Sephadex G-50 (fine); they were scaled up accordingly for larger volumes of reconstitution.…”