Acidic amino acids flanking phosphorylation sites in the M2 muscarinic receptor regulate receptor phosphorylation, internalization, and interaction with arrestins.

The studies reported here address the molecular events underlying the interactions of arrestins with the M(2) muscarinic acetylcholine receptor (mAChR). In particular, we focused on the role of receptor phosphorylation in this process. Agonist-dependent phosphorylation of the M(2) mAChR can occur at clusters of serines and threonines at positions 286-290 (site P1) or 307-311 (site P2) in the third intracellular loop (Pals-Rylaarsdam, R., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158). Phosphorylation at either P1 or P2 can support agonist-dependent internalization. However, phosphorylation at P2 is required for receptor interaction with arrestins (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682-26389). The present study investigated the role of acidic amino acids between P1 and P2 in regulating receptor phosphorylation, internalization, and receptor/arrestin interactions. Mutation of the acidic amino acids at positions 298-300 (site A1) and/or 304-305 (site A2) to alanines had significant effects on agonist-dependent phosphorylation. P2 was identified as the preferred site of agonist-dependent phosphorylation, and full phosphorylation at P2 required the acidic amino acids at A1 or their neutral counterparts. In contrast, phosphorylation at site P1 was dependent on site A2. In addition, sites A1 and A2 significantly affected the ability of the wild type and P1 and P2 mutant receptors to internalization and to interact with arrestin2. Substitution of asparagine and glutamine for the aspartates and glutamates at sites A1 or A2 did not influence receptor phosphorylation but did influence arrestin interaction with the receptor. We propose that the amino acids at sites A1 and A2 play important roles in agonist-dependent phosphorylation at sites P2 and P1, respectively, and also play an important role in arrestin interactions with the M(2) mAChR.

Arrestin binding to the M(2) muscarinic acetylcholine receptor is precluded by an inhibitory element in the third intracellular loop of the receptor.

Desensitization of G protein-coupled receptors (GPCRs) involves the binding of members of the family of arrestins to the receptors. In the model system involving the visual GPCR rhodopsin, activation and phosphorylation of rhodopsin is thought to convert arrestin from a low to high affinity binding state. Phosphorylation of the M(2) muscarinic acetylcholine receptor (mAChR) has been shown to be required for binding of arrestins 2 and 3 in vitro and for arrestin-enhanced internalization in intact cells (Pals-Rylaarsdam, R., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158). For the M(2) mAChR, arrestin binding requires phosphorylation at multiple serine and threonine residues at amino acids 307-311 in the third intracellular (i3) loop. Here, we have investigated the molecular basis for the requirement of receptor phosphorylation for arrestin binding. Constructs of arrestin 2 that can bind to other GPCRs in a phosphorylation-independent manner were unable to interact with a mutant M(2) mAChR in which the Ser/Thr residues at 307-311 were mutated to alanines. However, although phosphorylation-deficient mutants of the M(2) mAChR that lacked 50-157 amino acids from the i3 loop were unable to undergo agonist-dependent internalization when expressed alone in tsA201 cells, co-expression of arrestin 2 or 3 restored agonist-dependent internalization. Furthermore, a deletion of only 15 amino acids (amino acids 304-319) was sufficient to allow for phosphorylation-independent arrestin-receptor interaction. These results indicate that phosphorylation at residues 307-311 does not appear to be required to activate arrestin into a high affinity binding state. Instead, phosphorylation at residues 307-311 appears to facilitate the removal of an inhibitory constraint that precludes receptor-arrestin association in the absence of receptor phosphorylation.

Internalization of the m2 muscarinic acetylcholine receptor. Arrestin-independent and -dependent pathways.

Recent studies have identified agonist-dependent phosphorylation as a critical event in the rapid uncoupling of the m2 muscarinic cholinergic receptors (mAChR) from G-proteins and sequestration of the receptors away from the cell surface. However, mutant m2 mAChRs were identified that were phosphorylated but unable to desensitize in adenylyl cyclase assays, while they internalized like wild type (WT) mAChRs. We have tested whether these properties might stem from differences in the abilities of the WT and mutant mAChR to bind arrestins, proteins implicated in both receptor/G-protein uncoupling and internalization. We have determined that arrestin binding requires phosphorylation at a cluster of Ser/Thr residues in amino acids 307-311 in the m2 mAChR. A strong correlation was found between the ability of WT and mutant receptors to bind arrestins in vitro or in vivo and to desensitize in adenylyl cyclase assays. However, the phosphorylation-dependent internalization of the m2 mAChR in HEK-tsA201 cells did not require arrestins and did not proceed via clathrin-mediated endocytosis. While the m2 mAChR was able to enter a clathrin- and arrestin-dependent pathway when arrestin 2 or arrestin 3 was significantly overexpressed, the preferred pathway of internalization of WT and certain mutant m2 mAChR in HEK-tsA201 cells did not involve participation of arrestins. The results suggest that the phosphorylation-mediated regulation of the m2 mAChR may involve arrestin-dependent and -independent events.

G protein-coupled receptor kinase GRK2 is a phospholipid-dependent enzyme that can be conditionally activated by G protein betagamma subunits.

G protein-coupled receptor kinases (GRKs) mediate agonist-dependent phosphorylation of G protein-coupled receptors (GPRs) and initiate homologous receptor desensitization. Previously, we reported that charged phospholipids directly interacted with the two GRK isoforms, GRK2 and GKR3, via a pleckstrin homology (PH) domain to regulate GRK activity (DebBurman, S. K., Ptasienski, J., Boetticher, E., Lomasney, J. W., Benovic, J. L., and Hosey, M. M. (1995) J. Biol. Chem. 270: 5742-5747). Here, evidence is provided to support the hypothesis that charged phospholipids are required for agonist-dependent phosphorylation of receptors by GRK2. In the absence of charged phospholipids, the purified human m2 muscarinic acetylcholine receptor (hm2mAChR) reconstituted in pure phosphatidylcholine vesicles or in a noninhibitory detergent was not a substrate for GRK2. However, these receptor preparations were stoichiometrically phosphorylated in an agonist-dependent manner upon addition of charged phospholipids. The known ability of G protein betagamma subunits to stimulate mAChR phosphorylation also was found to be absolutely dependent on the presence of charged phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP2). Phospholipids also regulated GRK-mediated phosphorylation of casein, a nonreceptor-soluble substrate. Among lipids tested, lipid inositol phosphates, PIP2 and phosphatidylinositol 4-monophosphate, were found to be the most potent activators of GRK2 and were the only lipids that regulated GRK2 in a complex biphasic manner. At low micro concentrations, PIP2 activated GRK2 via an interaction with the GRK pleckstrin homology domain; however, at high micro concentrations, PIP2 inhibited GRK2, apparently via another mechanism. PIP2-mediated inhibition could be partly relieved by increasing ATP. The results support the hypothesis that GRK2 is a lipid-dependent protein kinase that requires charged phospholipids for enzyme activation, for regulation by Gbetagamma subunits, and potentially for membrane association.

Lipid-mediated regulation of G protein-coupled receptor kinases 2 and 3.

G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta gamma-subunits of G proteins (G beta gamma) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (beta ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate beta ARK activity in vitro in a manner that is analogous and competitive with G beta gamma. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to beta ARK1, whereas phosphatidylcholine, a lipid that did not modulate beta ARK activity, did not bind to beta ARK1. The lipid regulation of beta ARKs did not occur via a modulation of its autophosphorylation state. PS- and G beta gamma-mediated stimulation of beta ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G beta gamma employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G beta gamma-binding proteins, phosducin and GST-beta ARK-(466-689) fusion protein, suggesting that the G beta gamma-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs.

Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild type and mutant arrestins with rhodopsin, beta 2-adrenergic, and m2 muscarinic cholinergic receptors.

Arrestins play an important role in quenching signal transduction initiated by G protein-coupled receptors. To explore the specificity of arrestin-receptor interaction, we have characterized the ability of various wild-type arrestins to bind to rhodopsin, the beta 2-adrenergic receptor (beta 2AR), and the m2 muscarinic cholinergic receptor (m2 mAChR). Visual arrestin was found to be the most selective arrestin since it discriminated best between the three different receptors tested (highest binding to rhodopsin) as well as between the phosphorylation and activation state of the receptor (> 10-fold higher binding to the phosphorylated light-activated form of rhodopsin compared to any other form of rhodopsin). While beta-arrestin and arrestin 3 were also found to preferentially bind to the phosphorylated activated form of a given receptor, they only modestly discriminated among the three receptors tested. To explore the structural characteristics important in arrestin function, we constructed a series of truncated and chimeric arrestins. Analysis of the binding characteristics of the various mutant arrestins suggests a common molecular mechanism involved in determining receptor binding selectivity. Structural elements that contribute to arrestin binding include: 1) a C-terminal acidic region that serves a regulatory role in controlling arrestin binding selectivity toward the phosphorylated and activated form of a receptor, without directly participating in receptor interaction; 2) a basic N-terminal domain that directly participates in receptor interaction and appears to serve a regulatory role via intramolecular interaction with the C-terminal acidic region; and 3) two centrally localized domains that are directly involved in determining receptor binding specificity and selectivity. A comparative structure-function model of all arrestins and a kinetic model of beta-arrestin and arrestin 3 interaction with receptors are proposed.

Functional effects of protein kinase C-mediated phosphorylation of chick heart muscarinic cholinergic receptors.

Muscarinic cholinergic receptors (mAChR) purified from chick heart were phosphorylated by protein kinase C (PKC) and reconstituted with the purified GTP-binding regulatory protein Go. The effects of PKC phosphorylation on the interaction of mAChR with Go were assessed by monitoring for agonist-stimulated guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) binding to Go, agonist-stimulated GTPase activity of Go, and the capability of Go to induce high affinity agonist binding to mAChR. Both the receptor-stimulated GTP gamma S binding and GTPase activity of Go were markedly diminished as a result of PKC-mediated phosphorylation of the mAChR, whereas the ability of Go to induce high affinity agonist binding to the receptors was unaffected. When mAChR were first reconstituted with Go and then subjected to phosphorylation with PKC, a complete inhibition of the phosphorylation of mAChR by PKC was observed. The inhibitory effect of Go on mAChR phosphorylation was concentration-dependent and was prevented by the presence of GTP gamma S in the reaction mixtures. Taken together, these results indicate that the phosphorylation of mAChR by PKC modulates receptor/G-protein interactions and that the ability of the receptors to act as substrates for PKC may be regulated by receptor/G-protein interactions.

The porcine heart M2 muscarinic receptor: agonist-induced phosphorylation and comparison of properties with the chick heart receptor.

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.

Phosphorylation of the 165-kDa dihydropyridine/phenylalkylamine receptor from skeletal muscle by protein kinase C.

Dihydropyridine-sensitive Ca2+ channels exist in many different types of cells and are believed to be regulated by various protein phosphorylation and dephosphorylation reactions. The present study concerns the phosphorylation of a putative component of dihydropyridine-sensitive Ca2+ channels by the calcium and phospholipid-dependent protein kinase, protein kinase C. A skeletal muscle peptide of 165 kDa, which is known to contain receptors for dihydropyridines, phenylalkylamines, and other Ca2+ channel effectors, was found to be an efficient substrate for protein kinase C when the peptide was phosphorylated in its membrane-bound state. Protein kinase C incorporated 1.5-2.0 mol of phosphate/mol of peptide within 2 min into the 165-kDa peptide in incubations carried out at 37 degrees C. In contrast to the membrane-bound peptide, the purified 165-kDa peptide in detergent solution was phosphorylated to a markedly less extent than its membrane-bound counterpart; less than 0.1 mol of phosphate/mol of peptide was incorporated. Preincubation of the membranes with several types of drugs known to be Ca2+ channel activators or inhibitors had no specific effects on the rate and/or extent of phosphorylation of the 165-kDa peptide by protein kinase C. The phosphorylation of the membrane-bound 165-kDa peptide by protein kinase C was compared to that catalyzed by cAMP-dependent protein kinase and was found to be not additive. Prior phosphorylation of the 165-kDa peptide by cAMP-dependent protein kinase prevented subsequent phosphorylation of the peptide by protein kinase C. Phosphoamino acid analysis indicated that protein kinase C phosphorylated the 165-kDa peptide at both serine and threonine residues. Phosphopeptide mapping experiments showed that protein kinase C phosphorylated one unique site in the 165-kDa peptide, and, in addition, other sites that were phosphorylated by either cAMP-dependent protein kinase or a multifunctional Ca2+/calmodulin-dependent protein kinase. The results suggest that the 165-kDa dihydropyridine/phenylalkylamine receptor could serve as a physiological substrate of protein kinase C in intact cells. It is therefore possible that the regulation of dihydropyridine-sensitive Ca2+ channels by activators of protein kinase C may occur at the level of this peptide.

Correlation of agonist-induced phosphorylation of chick heart muscarinic receptors with receptor desensitization.

We have determined whether the process of agonist-mediated phosphorylation of the muscarinic receptor correlates with the process of muscarinic receptor desensitization in chick cardiac tissue. Exposure of ventricular slices to the agonist carbachol under conditions previously shown to lead to large increases in muscarinic receptor phosphorylation (Kwatra, M. M., and Hosey, M. M. (1986) J. Biol. Chem. 261, 12429-12432) resulted in decreased affinity of the muscarinic receptor for agonists. The agonist oxotremorine mimicked and the antagonist atropine prevented the effects of carbachol on receptor phosphorylation and agonist affinity. The time courses and concentration dependences for agonists to induce phosphorylation of the muscarinic receptor and decreases in agonist affinity were similar. Treatment of chick atria with acetylcholine under conditions which led to receptor phosphorylation resulted in decreased sensitivity of these preparations to the negative inotropic effect of carbachol. Taken together, the results support the concept that phosphorylation of cardiac muscarinic receptors may be related to the process of receptor desensitization. The mechanism by which agonists induce receptor phosphorylation was also investigated. The phosphorylated amino acids formed in response to agonists were serine and threonine. The protein kinase C activator phorbol myristate acetate had no effect on receptor phosphorylation or agonist affinity, nor did it prevent the effects of carbachol on either of these parameters. Receptor phosphorylation also was unaffected by the calmodulin antagonists W-7 and W-13, by elevation of cyclic nucleotides, and by agonists which activate other cardiac receptor systems. The results suggest that the phosphorylation of cardiac muscarinic receptors requires agonist occupancy of the receptor and/or may involve the participation of a selective protein kinase.

Photoaffinity labelling and phosphorylation of a 165 kilodalton peptide associated with dihydropyridine and phenylalkylamine-sensitive calcium channels.

Partially purified fractions of dihydropyridine and phenylalkylamine receptors associated with voltage-dependent calcium channels in rabbit skeletal muscle were found to contain two glycopeptides of similar molecular weight. A peptide of approximately 165 kDa was photoaffinity labelled with an arylazido-phenylalkylamine Ca channel inhibitor and also was phosphorylated with cAMP-dependent protein kinase. Another peptide of 170 kDa could be distinguished from the 165 kDa peptide by peptide mapping and differences in electrophoretic mobility. The results suggest that the 165 kDa peptide contains the sites responsible for regulation of calcium channel activity by calcium channel inhibitors as well as by neurotransmitters that regulate its activity in a cAMP-dependent manner.

Influence of Mg++ on the effect of diltiazem to increase dihydropyridine binding to receptors on Ca++-channels in chick cardiac and skeletal muscle membranes.

The influence of Mg++ on the effect of diltiazem to increase ligand binding to a high-affinity state of dihydropyridine receptors on voltage-dependent Ca-channels has been studied in chick cardiac and skeletal muscle membranes at 25 degrees C. The high-affinity binding of the Ca-channel inhibitors (+)-[3H]PN 200-110 and [3H]nitrendipine to cardiac membranes was depressed markedly by EDTA and restored fully by the addition of free Mg++ (Ptasienski et al., Biochem. Biophys. Res. Commun. 129: 910-917, 1985). Similar results have now been obtained with skeletal muscle membranes. In the presence of EDTA alone, diltiazem, which binds to another receptor on the Ca-channel, increased the high-affinity binding of both ligands to cardiac and skeletal muscle membranes. However, in the presence of added Mg++, diltiazem had smaller or no effects on the binding of these dihydropyridines. Analyses of the data indicated that both Mg++ and diltiazem could increase the maximum binding (Bmax) for these ligands, but the effect of diltiazem was smaller than, and not additive to, that of Mg++. Specific binding of the Ca-channel activator [3H]Bay k 8644 was only observed in assays containing Mg++ in excess of EDTA. The Bmax for [3H]Bay k 8644 in skeletal muscle membranes was less than that for [3H]PN 200-110 and [3H]nitrendipine, whereas with cardiac membranes equal Bmax values were obtained for all ligands. Diltiazem increased the Bmax for [3H]Bay k 8644 in skeletal muscle, but not in cardiac membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

High and low affinity states of the dihydropyridine and phenylalkylamine receptors on the cardiac calcium channel and their interconversion by divalent cations.

Drug receptors associated with Ca2+-channels in isolated chick heart membranes were found to exist in high and low affinity states. When assays were conducted in the presence of EDTA most of the receptors detected with the dihydropyridines (+)[3H]PN 200-110 or [3H]nitrendipine appeared to be in the lower affinity state. Inclusion of either Mg2+ or Ca2+ in the binding reactions resulted in the disappearance of the lower affinity state and the conversion of the receptors to a single high affinity state. Similar results were obtained with the phenylalkylamine derivative [3H]desmethoxyverapamil (D888). The results suggest that both the dihydropyridine and phenylalkylamine receptors on the cardiac Ca2+-channel can exist in interconvertible high and low affinity states in vitro, and that the proportion of receptors in each affinity state can be altered by the absence or presence of divalent cations.

Purification of the cardiac 1,4-dihydropyridine receptor/calcium channel complex.

Chick heart membranes were labelled with [3H]PN 200-110, a 1,4-dihydropyridine calcium channel antagonist. The [3H]PN 200-110 receptors were solubilized with digitonin and purified 600-fold using several chromatographic systems and sucrose density gradient centrifugation. Analyses by gel electrophoresis revealed that the final product was enriched in two peptides with molecular weights of 60,000 and 54,000, and another of 34,000.