Sequestration of muscarinic acetylcholine receptor m2 subtypes. Facilitation by G protein-coupled receptor kinase (GRK2) and attenuation by a dominant-negative mutant of GRK2 (original) (raw)
Related papers
Journal of Biological Chemistry, 1998
Internalization and down-regulation of human muscarinic acetylcholine m2 receptors (hm2 receptors) and a hm2 receptor mutant lacking a central part of the third intracellular loop (I3-del m2 receptor) were examined in Chinese hamster ovary (CHO-K1) cells stably expressing these receptors and G protein-coupled receptor kinase 2 (GRK2). Agonist-induced internalization of up to 80-90% of hm2 receptors was demonstrated by measuring loss of [ 3 H]N-methylscopolamine binding sites from the cell surface, and transfer of [ 3 H]quinuclidinyl benzilate binding sites from the plasma membrane into the lightvesicle fractions separated by sucrose density gradient centrifugation. Additionally, translocation of hm2 receptors with endocytic vesicles were visualized by immunofluorescence confocal microscopy. Agonist-induced down-regulation of up to 60-70% of hm2 receptors was demonstrated by determining the loss of [ 3 H]quinuclidinyl benzilate binding sites in the cells. The half-time (t1 ⁄2) of internalization and down-regulation in the presence of 10 ؊4 M carbamylcholine was estimated to be 9.5 min and 2.3 h, respectively. The rates of both internalization and down-regulation of hm2 receptors in the presence of 10 ؊6 M or lower concentrations of carbamylcholine were markedly increased by coexpression of GRK2. Agonist-induced internalization of I3-del m2 receptors was barely detectable upon incubation of cells for 1 h, but agonist-induced down-regulation of up to 40-50% of I3-del m2 receptors occurred upon incubation with 10 ؊4 M carbamylcholine for 16 h. However, the rate of down-regulation was lower compared with wild type receptors (t1 ⁄2 ؍ 9.9 versus 2.3 h). These results indicate that rapid internalization of hm2 receptors is facilitated by their phosphorylation with GRK2 and does not occur in the absence of the third intracellular loop, but downregulation of hm2 receptors may occur through both GRK2-facilitating pathway and third intracellular loopindependent pathways.
Immunochemical Studies of the Muscarinic Acetylcholine Receptor
Journal of Receptors and Signal Transduction, 1987
Binding studies with the radiolabeled muscarinic antagonists dexetimide, quinuclidinyl benzilate and Nmethylscopolamine showed that the human embryonic lung fibroblast CCLl37 possesses approximately 2 x lo5 muscarinic receptors/cell, i. e. 2.1 pmol/mg membrane protein. These receptors showed a marked stereoselectivity towards dexetimide and levetimide and only low affinity for another antagonist, pirenzepine. The muscarinic agonist carbamylcholine inhibited forskolin-stimulated adenylate cyclase and induced phosphatidylinositide turnover in the intact cells. Both effects were inhibited by the muscarinic antagonist atropine. Affinity labeling with tritiated propylbenzylcholine mustard revealed a protein of 72 kDa. Finally, down-regulation of the membrane receptors following prolonged treatment with the agonist carbamylcholine was assessed by means of the hydrophilic antagonist N-methylscopolamine.
European Journal of Biochemistry, 1994
A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by P-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating P-adrenergic-receptor-kinase-lmediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), Go and G,*, displayed guaninenucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [3H]quinuclidinylbenzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-0-[35S]thiotriphosphate (['5S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The K, values of carbamoylcholine effects on [3H]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by P-adrenergic-receptor kinase-l did not affect m2 interaction with G proteins as assessed by the binding of [3H]qunuclidinyl benzilate or ['jS ]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of P-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by P-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles. GTP-binding-protein-coupled receptors (G-protein-coupled receptors) such as rhodopsin, P adrenergic receptors and the muscarinic acetylcholine receptors m2 subtype (m2 receptors) are phosphorylated by G-protein-coupled receptor kinases in a light-dependent or agonist-dependent manner [l-31. At least six different G-protein-coupled receptor kinases have been cloned, and they are classified into three subgroups, rhodopsin kinase, P-adrenergic-receptor kinases
Journal of Biological Chemistry, 1996
Human muscarinic acetylcholine receptor m1 subtypes (m1 receptors) were expressed in and purified from insect Sf9 cells and then subjected to phosphorylation by G protein-coupled receptor kinase 2 (GRK2) expressed in and purified from Sf9 cells and by protein kinase C purified from rat brain (a mixture of ␣, , and ␥ types, PKC). The m1 receptor was phosphorylated by either GRK2 or PKC in an agonist-dependent or independent manner, respectively. G protein ␥ subunits stimulated the phosphorylation by GRK2 but did not affect the phosphorylation by PKC. The number of incorporated phosphates was 4.6 and 2.8 mol/mol of receptor for phosphorylation by GRK2 and PKC, respectively. The number of incorporated phosphates was 7.5 mol/mol receptor for phosphorylation by GRK2 followed by PKC, but was 5.8 mol/mol of receptor for the phosphorylation by PKC followed by GRK2. Major sites phosphorylated by GRK2 and PKC were located in the third intracellular loop and the carboxyl-terminal tail, respectively. These results indicate that GRK2 and PKC phosphorylate different sites of m1 receptors and that the phosphorylation by PKC partially inhibits the phosphorylation by GRK2, probably by affecting activation of GRK2 by agonist-bound receptors.
Activation by G protein βγ subunits of β-adrenergic and muscarinic receptor kinase
Journal of Biological Chemistry
We have shown previously that GTP-binding regulatory protein (G protein) By subunits stimulate the agonist-or light-dependent phosphorylation of muscarinic acetylcholine receptors (mAChRs) and rhodopsin by a protein kinase partially purified from porcine brain (mAChR kinase) but not the phosphorylation of rhodopsin by rhodopsin kinase (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267,2222-2227). We report here that the mAChR kinase phosphorylates &adrenergic receptors (8-ARs) purified from bovine lung in an agonist-dependent manner, and the phosphorylation is also stimulated by G protein & subunits. We also report that recombinant &adrenergic receptor kinase 1 (B-ARKl) expressed in COS-7 cells phosphorylates mAChRs (human m2 subtype) and rhodopsin in an agonist-or light-dependent manner, respectively, and that this phosphorylation is stimulated by G protein j3-y subunits. By contrast, the By subunits do not stimulate the phosphorylation of mAChRs or rhodopsin by a 8-ARK1 mutant lacking a part of the carboxyl-terminal region which is present in &ARKS but not in rhodopsin kinase. These results indicate that the &ARK1 is the same as or very similar to the mAChR kinase but is distinguished from the rhodopsin kinase with respect to activation by the /3-y subunits and that the extra carboxyl-terminal sequence in &ARKS is required for the stimulation by the 8-y subunits. Rhodopsin and @-adrenergic receptors (P-ARs)' are known to be phosphorylated in a light-or agonist-dependent manner by rhodopsin kinase and @-adrenergic receptor kinase (@-ARK), respectively, and the phosphorylation is thought to be involved in their homologous desensitization (1-3). cDNAs for @-ARK1 and 2 (4, 5) and rhodopsin kinase (6) have been cloned and shown to be similar to each other except for an extra region encoding 127 or 128 amino acid residues in the carboxyl terminus of @-ARKS. The agonist-dependent phosphorylation of muscarinic acetylcholine receptors (mAChRs) has been demonstrated by
Inhibition of acetylcholine muscarinic M(1) receptor function 2000
1 MT-7 (1 ± 30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M 1 receptor, inhibited the acetylcholine (ACh)-stimulated [ 35 S]-guanosine-5'-O-(3-thio)triphosphate ([ 35 S]-GTPgS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M 1 receptor subtype. 2 MT-7 failed to aect the ACh-stimulated [ 35 S]-GTPgS binding in membranes of CHO cells expressing either the M 2 , M 3 or M 4 receptor subtype. 3 In N1E-115 neuroblastoma cells endogenously expressing the M 1 and M 4 receptor subtypes, MT-7 (0.3 ± 3.0 nM) inhibited the carbachol (CCh)-stimulated inositol phosphates accumulation, but failed to aect the CCh-induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38-stimulated cyclic AMP accumulation. 4 In both CHO/M 1 and N1E-115 cells the MT-7 inhibition consisted in a decrease of the maximal agonist eect with minimal changes in the agonist EC 50 value. 5 In CHO/M 1 cell membranes, MT-7 (0.05 ± 25 nM) reduced the speci®c binding of 0.05, 1.0 and 15 nM [ 3 H]-N-methylscopolamine ([ 3 H]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nM) decreased the dissociation rate of [ 3 H]-NMS by about 5 fold.
Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors
Scientific Reports
Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M 2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.
Differential agonist-induced regulation of human M2 and M3 muscarinic receptors
Biochemical Pharmacology, 2003
We have compared the regulation of M 2 and M 3 muscarinic receptors heterologously expressed in HEK-293 cells upon long-term exposure towards the agonist carbachol. Carbachol time-and concentration-dependently reduced M 2 receptor density with a maximum reduction of about 60%. Treatment with 1 mM carbachol for 24 hr was accompanied by desensitisation of carbachol-induced Ca 2þ elevations (maximum response reduced by 70%) but not by alterations in the expression of various G-protein a-subunits. Consistently, heterologous desensitisation of Ca 2þ elevations by the purinergic receptor agonist ATP or by sphingosine-1-phosphate was not detected. In contrast, carbachol time-and concentration-dependently up-regulated M 3 receptors with maximum increases to about 350% of control values. The up-regulation was fully blocked by cycloheximide indicating that it was dependent on protein synthesis. Concomitant with the up-regulation of the M 3 receptor was a reduction in the expression of the a-subunit of G q/11. The net effect of these two opposite regulatory mechanisms was a lack of alteration of carbachol-stimulated Ca 2þ elevation. However, the reduction of G q/11 was accompanied by a heterologous desensitisation of Ca 2þ elevations by ATP and sphingosine-1-phosphate. Levels of M 2 and M 3 receptor mRNA as assessed by real-time PCR were not significantly altered by carbachol exposure for either receptor, suggesting that alterations of mRNA stability did not contribute to the observed changes in receptor number. We conclude that M 2 and M 3 receptor expression within the same cell undergoes differential agonist-induced regulation being accompanied by distinct regulation of G-protein expression leading to differential effects on signal transduction by other receptor systems.