Structure of the m1 muscarinic acetylcholine receptor gene and its promoter

The m1 receptor is one of five muscarinic receptors that mediate the metabotropic actions of acetylcholine in the nervous system where it is expressed predominantly in the telencephalon and autonomic ganglia. RNase protection, primer extension, and 5'-rapid amplification of cDNA ends analysis of a rat cosmid clone containing the entire m1 gene demonstrated that the rat m1 gene consists of a single 657-base pairs (bp) non-coding exon separated by a 13. 5-kilobase (kb) intron from a 2.54-kb coding exon that contains the entire open reading frame. The splice acceptor for the coding exon starting at -71 bp relative to the adenine of the initiating methionine. This genomic structure is similar to that of the m4 gene (Wood, I. C., Roopra, A., Harrington, C. A., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940 and Wood, I. C., Roopra, A., and Buckley, N. J. (1996) J. Biol. Chem. 271, 14221-14225). Like the m4 gene, the m1 promoter lacks TATA and CAAT consensus motifs, and the first exon and 5'-flanking region are not gc-rich. The 5'-flanking region also contains the consensus regulatory elements Sp-1, NZF-1, AP-1, AP-2, E-box, NFkappaB, and Oct-1. Unike the m4 promoter, there is no evidence of a RE1/NRSE silencer element in the m1 promoter. Deletional analysis and transient transfection assays demonstrates that reporter constructs containing 0.9 kb of 5'-flanking sequence and the first exon are sufficient to drive cell-specific expression of reporter gene in IMR32 neuroblastoma cells while remaining silent in 3T3 fibrobasts.     

Volatile anaesthetics have differential effects on recombinant m1 and m3 muscarinic acetylcholine receptor function

Muscarinic acetylcholine signalling plays major roles in regulation of consciousness, cognitive functioning, pain perception and circulatory homeostasis. Halothane has been shown to inhibit m1 muscarinic signalling. However, no comparative data are available for desflurane, sevoflurane or isoflurane, nor have the anaesthetic effects on the m3 subtype (which is also prominent in the brain) been studied. Therefore, we have investigated the effects of these compounds on isolated m1 and m3 muscarinic receptor function. Defolliculated Xenopus oocytes expressing recombinant m1 or m3 muscarinic or (for comparison) AT1A angiotensin II receptors were voltage clamped, and Ca(2+)-activated Cl- currents (ICl(Ca)) induced by acetyl-beta-methylcholine (Mch) or angiotensin II were measured in the presence of clinically relevant concentrations of halothane, sevoflurane, desflurane or isoflurane. To determine the site of action of the volatile anaesthetics we compared anaesthetic effects on m1, m3 and AT1A receptor function and studied the effects of volatile anaesthetics on signalling induced by intracellular injection of the second messenger IP3. Desflurane had a biphasic effect on m1 signalling, enhancing at a concentration of 0.46 mmol litre-1 but depressing at 0.92 mmol litre-1. A similar, although not significant, trend was observed with m3 signalling. Isoflurane had no effect on m1 signalling, but profoundly inhibited m3 signalling. Sevoflurane depressed the function of m1 and m3 signalling in a dose-dependent manner. Halothane, similar to its known effect on m1 signalling, dose-dependently depressed m3 function. ICl(Ca) induced by intracellular injections of IP3 were unaffected by all four anaesthetics. Similarly, none of the anaesthetics tested affected AT1A signalling. Absence of interference with AT1A signalling and intracellular pathways suggest that the effects of anaesthetics on muscarinic signalling most likely result from interactions with the m1 or m3 receptor molecule. Multiple interaction sites with different affinities may explain the biphasic response to desflurane. Anaesthetic-specific effects on closely related receptor subtypes suggest defined sites of action for volatile anaesthetics on the receptor protein.     

Allosteric modulation of Torpedo nicotinic acetylcholine receptor ion channel activity by noncompetitive agonists

Similar to other neuroreceptors of the vertebrate central nervous system, the nicotinic acetylcholine receptor (nAChR) is subject to modulatory control by allosterically acting ligands. Of particular interest in this regard are allosteric ligands that enhance the sensitivity of the receptor to its natural agonist acetylcholine (ACh), as such ligands could be useful as drugs in diseases associated with impaired nicotinic neurotransmission. Here we discuss the action of a novel class of nAChR ligands which act as allosterically potentiating ligands (APL) on the nicotinic responses induced by ACh and competitive agonists. In addition, APLs also act as noncompetitive agonists of very low efficacy, and as direct blockers of ACh-activated channels. These actions are observed with nAChRs from brain, muscle and electric tissue, and they depend on the structure of the APL and the concentration range applied. We focus here on Torpedo nAChR because (i) the unusual pharmacology of these ligands was first discovered with this system, and (ii) large quantities of this receptor are readily available for biochemical studies.     

Charged amino acids required for signal transduction by the m3 muscarinic acetylcholine receptor

The five muscarinic acetylcholine receptor (mAChR) subtypes, termed m1-m5, transduce agonist signals across the plasma membrane by activating guanine nucleotide binding (G) proteins. The large cytoplasmic domain joining the fifth and sixth transmembrane segments of mAChRs plays a critical role in controlling the specificity of G protein coupling. In this study, we determined which sequences within this domain are required for activation of signaling by the m3 mAChR. By measuring the ability of normal and mutant m3 mAChRs to couple to the G protein pathway leading to activation of phospholipase C and Ca(2+)-dependent chloride currents in RNA-injected Xenopus oocytes, we found that two clusters of charged residues near the fifth and sixth transmembrane segments were required for normal signaling; furthermore, the position of these sequences was critical for their function. Finally, analysis of deletion mutant m3 mAChRs confirmed the importance of these sequences; receptors containing as few as 22 out of 239 amino acids of the cytoplasmic domain were fully active in signaling if they included the critical charged residues. Sequence comparisons suggest that similar charged sequences may be required for signal transduction by many G protein-coupled receptors.     

Two-step binding of green mamba toxin to muscarinic acetylcholine receptor

The mechanism of binding of toxin MT2 from venom of green mamba Dendroaspis angusticeps to muscarinic acetylcholine receptors from rat cerebral cortex was investigated by studying the kinetics of the toxin-receptor interaction. The muscarinic antagonist N-methyl-[3H]scopolamine was used as a 'reporter' ligand. Evidence for a mechanism of toxin-receptor interaction comprising at least two steps was obtained. Such a mechanism increases the potency of the toxin. The first step was fast with no competition between the toxin and the antagonist. The second step was slow with formation of a more stable toxin-receptor complex and inhibition of the antagonist binding. It is proposed that the snake toxin is a muscarinic agonist of slow action.     

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor has revealed a highly-differentiated alpha-helical structure. Lipid-facing residues are distinguished from a patch of residues which selectively stabilise the ground state of the receptor, and from a band of amino acids extending the full length of the helix, which contribute to the active agonist-receptor-G protein complex. The most important residues are strongly conserved in the GPCR superfamily.     

Regulation of IRK3 inward rectifier K+ channel by m1 acetylcholine receptor and intracellular magnesium

Inward rectifier K+ channels control the cell's membrane potential and neuronal excitability. We report that the IRK3 but not the IRK1 inward rectifier K+ channel activity is inhibited by m1 muscarinic acetylcholine receptor. This m1 modulation cannot be accounted for by protein kinase C, Ca2+, or channel phosphorylation, but can be mimicked by Mg2+. Based on quantitative analyses of IRK3 and two different IRK1 mutant channels bestowed with sensitivity to m1 modulation, we suggest that the resting Mg2+ level causes chronic inhibition of IRK3 channels, and m1 receptor stimulation may lead to an increase of cytoplasmic Mg2+ concentration and further channel inhibition, due to the ability of Mg2+ to lead these channels into a prolonged inactivated state.     

M2 muscarinic autoreceptors modulate acetylcholine release in the medial pontine reticular formation

Muscarinic autoreceptors regulate acetylcholine (ACh) release in several brain regions, including the medial pontine reticular formation (mPRF). This study tested the hypothesis that the muscarinic cholinergic receptor mediating mPRF ACh release is the pharmacologically defined M2 subtype. In vivo microdialysis was used to deliver muscarinic cholinergic receptor (MAChR) antagonists to the feline mPRF while simultaneously measuring endogenously released ACh. The lowest concentration of each antagonist that caused a significant increase in mPRF ACh release was determined and defined as the minimum ACh-releasing concentration. Data obtained from 41 mPRF dialysis sites in 10 animals showed that the order of potency (followed by the minimum ACh-releasing concentration) was scopolamine (1 nM) > AF-DX 116 (3 nM) > pirenzepine (300 nM). Comparison of these minimum ACh-releasing concentrations to the known affinities of the antagonists for the five mAChR subtypes is consistent with the conclusion that the autoreceptor regulating mPRF ACh release is the M2 subtype. Considerable evidence supports a role for cholinergic neurotransmission and postsynaptic M2 receptors in the mPRF in regulating levels of arousal. The present data suggest that presynaptic M2 receptors contribute to the regulation of arousal states by modulating mPRF ACh release.     

Desensitization of human muscarinic acetylcholine receptor m2 subtypes is caused by their sequestration/internalization

The most popular pretreatment method of plasma samples for the measurement of ascorbate (AsA) and dehydroascorbate (DHA) has been an acidic deproteinization via metaphosphoric acid or trichloroacetic acid. In general, DHA is absent in plasma samples prepared from human blood in a conventional manner. However, when these plasma samples were subjected to acidic deproteinization, DHA was detected in the acidified sample solutions. In the present study, we demonstrate that the oxidation of AsA to DHA in the solutions was promoted by at least two mechanisms, one involving catalysis by ferric ion released from transferrin, and the other involving catalysis by plasma hemoglobin. In the acidified transferrin solution by trichloroacetic acid, an oxidation of AsA to DHA proceeded with standing time, whereas the oxidation was not observed in that by metaphosphoric acid. This oxidation appeared to be catalyzed by ferric ion released from transferrin. In contrast, plasma hemoglobin functioned as a catalyst for AsA oxidation in both metaphosphoric acid and trichloroacetic acid solutions. Therefore, DHA content in the trichloroacetic acid-treated plasma sample was markedly higher than that in the metaphosphoric acid-treated one. These results suggest that DHA detected in acidified plasma samples is an artifact resulting from AsA oxidation.     

Double mutant cycle analysis of aspartate 69, 97, and 103 to asparagine mutants in the m2 muscarinic acetylcholine receptor

Double mutant cycles provide a method for analyzing the effects of a mutation at a defined position in the protein structure on the properties of an amino acid at a second site. This approach was used to map potential interactions between aspartates 69, 97, and 103 in the m2 muscarinic acetylcholine receptor transmembrane helices 2 and 3. Receptors containing single and double aspartate to asparagine mutants were expressed in Chinese hamster ovary cells and their effects on ligand binding, signal transduction, and thermal stability determined. Analysis of the double mutant cycles showed that the mutations had approximately additive effects on ligand binding, signal transduction, and thermal stability. Ligand binding and thermal inactivation results support the conclusion that aspartate-103 is the ligand amine counterion. Effector coupling properties of the mutant receptors showed that aspartate-103 was also required for signal transduction activity. The mutation of aspartate-69 to asparagine completely eliminated signal transduction by the agonists acetylcholine, carbachol, and pilocarpine but not oxotremorine M, which caused reduced but significant inhibition of adenylyl cyclase and stimulation of phospholipase C. In contrast, adenylyl cyclase stimulation by the asparagine-69 mutant was elicited only by acetylcholine and carbachol but not by oxotremorine M. The variation in agonist-dependent effector coupling properties provides evidence that the asparagine-69 mutant can exist in activated receptor states that are different from the wild-type m2 muscarinic receptor.     

On the unique binding and activating properties of xanomeline at the M1 muscarinic acetylcholine receptor

We investigated the molecular nature of the interaction between the functionally selective M1 muscarinic acetylcholine receptor (mAChR) agonist xanomeline and the human M1 mAChR expressed in Chinese hamster ovary (CHO) cells. In contrast to the non-subtype-selective agonist carbachol, xanomeline demonstrated M1 mAChR binding that was resistant to extensive washout, resulting in a significant reduction in apparent N-[3H]methylscopolamine saturation binding affinity in intact cells. Functional assays, using both M1 mAChR-mediated phosphoinositide hydrolysis and activation of neuronal nitric oxide synthase, confirmed that this persistent binding resulted in elevated basal levels of system activity. Furthermore, this phenomenon could be reversed by the addition of the antagonist atropine. However, pharmacological analysis of the inhibition by atropine of xanomeline-mediated functional responses indicated a possible element of noncompetitive behavior that was not evident in several kinetic and equilibrium binding experimental paradigms. Taken together, our findings indicate for the first time a novel mode of interaction between an mAChR agonist and the M1 mAChR, which may involve unusually avid binding of xanomeline to the receptor. This yields a fraction of added agonist that is retained at the level of the receptor compartment to persistently bind to and activate the receptor subsequent to washout. The results of the current study suggest that elucidation of the mechanism or mechanisms of interaction of xanomeline with the M1 mAChR is particularly important in relation to the potential therapeutic use of this agent in the treatment of Alzheimer's disease.     

Molecular modeling of the interaction of diagnostic radiopharmaceuticals with receptor proteins: m2 antagonist binding to the muscarinic m2 subtype receptor

Models of the m2 muscarinic receptor have been built and acetylcholine and an antagonist of the quinuclidinyl benzilate family docked to the putative active site. We have incorporated aspects of homology, site-directed mutagenesis studies and structure-activity studies of specific lead compounds in the construction of our receptor models with a primary focus on the structure of the binding sites. We have observed a deep pocket binding of 5-BrQNT, suggesting a plausible explanation for the observation that agonists and antagonists do not bind competitively. The results of these computational studies are interpreted within the context of the observed in vitro results. Our goal is to assist in the development of subtype receptor selective radiopharmaceuticals for use in PET and SPECT.     

Mitogen-activated protein kinase-dependent and protein kinase C-dependent pathways link the m1 muscarinic receptor to beta-amyloid precursor protein secretion

Full and functionally selective M1 muscarinic agonists (carbachol and AF102B, respectively) activate secretion of the soluble form of amyloid precursor protein (APPs) in PC12 cells expressing the m1 muscarinic receptor (PC12M1 cells). This activation is further augmented by neurotrophins such as nerve growth factor and basic fibroblast growth factor. Muscarinic stimulation activates two transduction pathways that lead to APPs secretion: protein kinase C (PKC)-dependent and mitogen-activated protein kinase (MAPK)-dependent pathways. These pathways operate in parallel and converge with transduction pathways of neurotrophins, resulting in enhancement of APPs secretion when both muscarinic agonist and neurotrophins stimulate PC12M1 cells. These conclusions are supported by the following findings: (a) Only partial blockade of APPs secretion is observed when PKC, p21ras, or MAPK is fully inhibited by their respective specific inhibitors, GF109203X, S-trans, trans-farnesylthiosalicylic acid, and PD98059. (b) K252a, which blocks PKC and phorbol 12-myristate 13-acetate-induced APPs secretion, enhances both muscarinic-stimulated MAPK activation and APPs secretion. (c) Activation of MAPK in PC12M1 cells by muscarinic agonists is Ras-dependent but PKC-independent and is enhanced synergistically by neurotrophins. These results suggest that muscarinic stimulation of APPs secretion is mediated by at least two independent pathways that converge and enhance the signal for APPs secretion at the convergence point.     

Localization of the m2 muscarinic acetylcholine receptor protein and mRNA in cortical neurons of the normal and cholinergically deafferented rhesus monkey

The m2 muscarinic acetylcholine receptor in the cerebral cortex has traditionally been thought of as an autoreceptor located on cholinergic fibers that originate from neurons in the nucleus basalis of Meynert. We now provide evidence for widespread localization of the m2 receptor in noncholinergic neurons and fibers of the cerebral cortex. The cellular and subcellular distribution of the m2 receptor protein and mRNA were examined in normal monkeys and in monkeys in which the cortical cholinergic afferents were selectively lesioned by injection of the specific immunotoxin, anti-p75NTR-saporin into the nucleus basalis. Both in normal and immunolesioned monkeys, the m2 mRNA and protein were localized in pyramidal and nonpyramidal neurons. In pyramidal neurons, membrane-associated receptor immunoreactivity was found exclusively in dendritic spines receiving asymmetric synapses, indicating that the m2 receptor may modulate excitatory neurotransmission at these sites. In nonpyramidal neurons, the m2 immunoreactivity was present along the cytoplasmic surface of membranes in cell bodies, dendrites and axons. Both in pyramidal and nonpyramidal neurons of normal and lesioned monkeys, the m2 receptor was located peri- and extra-synaptically, suggesting that it may be contacted by acetylcholine via volume transmission. The localization of the m2 receptor in cortical neurons and the sparing of m2 immunoreactivity in lesioned monkeys indicates that the m2 receptor is synthesized largely within the cortex and/or is localized to noncholinergic terminals of either intrinsic or extrinsic origin. These findings open the possibility that the loss of the m2 receptor in Alzheimer's disease may in part be due to degenerative changes in m2 positive neurons of the cortex rather than entirely due to the loss of autoreceptors.     

Binding-site modeling of the muscarinic m1 receptor: a combination of homology-based and indirect approaches

A model of the muscarinic m1 receptor has been constructed on the basis of the putative three-dimensional structural similarity between bacteriorhodopsin and G-protein coupled receptors. The homology-based m1 receptor model takes into account hydrophobicity and conserved amino acids and information from site-directed mutagenesis studies and from hydropathy plots. The resulting model was used in conjunction with an indirect model which describes a proposed active agonist conformation of acetylcholine and a number of related compounds. A receptor-excluded volume was constructed by superimposing these muscarinic agonists and calculating their combined van der Waals volume. The resulting m1 receptor excluded volume was used to define the agonist binding site, which consists of nine amino acids and which binds agonists primarily through interaction with Asp105 (ionic interaction). Thr192 and Asn382 (hydrogen bonds). The model is flexible since the conformation of the nine amino acids may change in response to the agonist structure. The combination of indirect and homology-based approaches is particularly attractive since it utilizes more experimental data than a purely homology-based model and since a binding-site model might be more realistic and general in terms of applicability than indirect models. Docking of the ligands was performed by optimizing attractive interactions and minimizing repulsive interactions. In addition to the agonists used to define the binding site, structurally different agonists are also accommodated by the binding-site model. Furthermore, the m1 receptor binding-site model is able to reproduce experimentally determined stereoselectivities.     

Activation of the genetically defined m1 muscarinic receptor potentiates N-methyl-D-aspartate (NMDA) receptor currents in hippocampal pyramidal cells

Evidence suggests that cholinergic input to the hippocampus plays an important role in learning and memory and that degeneration of cholinergic terminals in the hippocampus may contribute to the memory loss associated with Alzheimer's disease. One of the more prominent effects of cholinergic agonists on hippocampal physiology is the potentiation of N-methyl-D-aspartate (NMDA)-receptor currents by muscarinic agonists. Here, we employ traditional pharmacological reagents as well as m1-toxin, an m1 antagonist with unprecedented selectivity, to demonstrate that this potentiation of NMDA-receptor currents in hippocampal CA1 pyramidal cells is mediated by the genetically defined m1 muscarinic receptor. Furthermore, we demonstrate the colocalization of the m1 muscarinic receptor and the NR1a NMDA receptor subunit at the electron microscopic level, indicating a spatial relationship that would allow for physiological interactions between these two receptors. This work demonstrates that the m1-muscarinic receptor gene product modulates excitatory synaptic transmission, and it has important implications in the study of learning and memory as well as the design of drugs to treat neurodegenerative diseases such as Alzheimer's.     

Combined treatment with a 5HT1A receptor agonist and a muscarinic acetylcholine receptor antagonist disrupts water maze navigation behavior

In the present study, the effects of PACAP27, PACAP38 and VIP in a concentration range from 10(-13) to 10(-6) M were studied in vitro on the spontaneous and directed mobility of lymphocytes from rat spleen and thymus. The results show that VIP and both PACAPs inhibit significantly and in a similar way the mobility of lymphocytes from thymus and spleen, and the maximal effects were observed at 10(-9) M and 10(-8) M. The three neuropeptides significantly increased cAMP concentrations. Moreover, incubation with increasing PMA concentrations showed a progressive enhancement of chemotaxis of lymphocytes, which was partially prevented by VIP, and both PACAPs. Incubation with forskolin caused decrease in the chemotaxis of thymocytes and splenocytes, and the presence of VIP or PACAP peptides was not synergistic in the inhibitory effect on lymphocyte chemotaxis, suggesting that the three neuropeptides and forskolin mediate their actions by the same intracellular pathway. This study showed the ability of the VIP receptor antagonist (N-Ac-Tyr1,D-Phe2)-GRF(1-29)-NH2 to partially reverse the inhibitory effect of both PACAPs and VIP on chemotaxis, suggesting that PACAP receptors are identical or very similar to VIP receptors in both thymocytes and splenocytes. These data suggest that PACAP27 and PACAP38 can be included as two novel immunoregulatory peptides that can modulate cell mobility on central and peripheral lymphoid organs.     

Pharmacological characterization of human m1 muscarinic acetylcholine receptors with double mutations at the junction of TM VI and the third extracellular domain

A mutant human m5 receptor containing the mutations of Ser465 to Tyr and Thr466 to Pro showed constitutive activity. By replacing the equivalent Ser388 with Tyr and Thr389 with Pro, we created a mutant human m1 (Hm1) receptor with comparable double mutations. The mutant receptor, Hm1(Ser388Tyr, Thr389Pro), was stably expressed in A9 L cells and displayed enhanced responses to classical muscarinic agonists with significantly increased potencies. Choline, a normal component of growth media, showed an efficacy comparable to acetylcholine and carbachol at Hm1(Ser388Tyr, Thr389Pro) receptors. Methylcarbachol, a selective nicotinic agonist, exhibited partial agonist activity at human m1 wild-type receptors and full agonist activity at Hm1(Ser388Tyr, Thr389Pro) receptors. l-Hyoscyamine inhibited the activities of choline and methylcarbachol. Muscarinic antagonists displayed small reductions in binding affinities, although muscarinic agonists showed greatly increased binding affinities for Hm1(Ser388Tyr, Thr389Pro) receptors. All agonists, including choline and methylcarbachol, showed multiple affinity states at Hm1(Ser388Tyr, Thr389Pro) receptors in the absence of GppNHp. The high affinity binding sites for acetylcholine, arecoline and choline were shifted in the presence of GppNHp. These results suggest that Hm1(Ser388Tyr, Thr389Pro) is conformationally favorable for agonist binding and receptor activation.     

Arrestin-independent internalization of the m1, m3, and m4 subtypes of muscarinic cholinergic receptors

To understand what processes contribute to the agonist-induced internalization of subtypes of muscarinic acetylcholine receptors, we analyzed the role of arrestins. Whereas the m2 mAChR has been shown to undergo augmented internalization when arrestins 2 and 3 are overexpressed (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682-23689), the agonist-induced internalization of m1, m3, and m4 mAChRs was unchanged when arrestins 2 or 3 were overexpressed in transiently transfected HEK-tsA201 cells. Furthermore, when a dominant-negative arrestin was used to interrupt endogenous arrestin function, there was no change in the internalization of the m1, m3, and m4 mAChR whereas the internalization of the beta2 adrenergic receptor was completely blocked. Wild-type and GTPase-deficient dominant-negative dynamin were used to determine which endocytic machinery played a role in the endocytosis of the subtypes of mAChRs. Interestingly, when dynamin function was blocked by overexpression of the GTPase-deficient dynamin, agonist- induced internalization of the the m1, m3, and m4 mAChRs was suppressed. These results suggested that the internalization of the m1, m3, and m4 mAChRs occurs via an arrestin-independent but dynamin-dependent pathway. To ascertain whether domains that confer arrestin sensitivity and dynamin insensitivity could be functionally exchanged between subtypes of mAChRs, chimeric m2/m3 receptors were analyzed for their properties of agonist-induced internalization. The results demonstrated that the third intracellular loop of the m2 mAChR conferred arrestin sensitivity and dynamin insensitivity to the arrestin-insensitive, dynamin-sensitive m3 mAChR while the analogous domain of the m3 mAChR conferred arrestin resistance and dynamin sensitivity to the previously arrestin-sensitive, dynamin-insensitive m2 mAChR.     

Sequestration of muscarinic acetylcholine receptor m2 subtypes. Facilitation by G protein-coupled receptor kinase (GRK2) and attenuation by a dominant-negative mutant of GRK2

Sequestration of m2 receptors (muscarinic acetylcholine receptor m2 subtypes), which was assessed as loss of N-[3H]methylscopolamine ([3H]NMS) binding activity from the cell surface, was examined in COS 7 and BHK-21 cells that had been transfected with expression vectors encoding the m2 receptor and, independently, vectors encoding a G protein-coupled receptor kinase (GRK2) (beta-adrenergic receptor kinase 1) or a GRK2 dominant-negative mutant (DN-GRK2). The sequestration of m2 receptors became apparent when the cells were treated with 10(-5) M or higher concentrations of carbamylcholine. In this case, approximately 40% or 20-25% of the [3H]NMS binding sites on COS 7 or BHK-21 cells, respectively, were sequestered with a half-life of 15-25 min. In cells in which GRK2 was also expressed, the sequestration became apparent in the presence of 10(-7) M carbamylcholine. Approximately 40% of the [3H]NMS binding sites on both COS 7 and BHK-21 cells were sequestered in the presence of 10(-6) M or higher concentrations of carbamylcholine. When DN-GRK2 was expressed in COS 7 cells, the proportion of [3H]NMS binding sites sequestered in the presence of 10(-5) M or higher concentrations of carbamylcholine was reduced to 20-30%. These results indicate that the phosphorylation of m2 receptors by GRK2 facilitates their sequestration. These results are in contrast with the absence of a correlation between sequestration and the phosphorylation of beta-adrenergic receptors by the GRK2 and suggests that the consequences of phosphorylation by GRK2 are different for different receptors.