Human M2 Muscarinic Acetylcholine Receptor Research Articles

Developing anticholinergic drugs for the treatment of asthma with improved efficacy

Anticholinergics are used in treating asthma, a chronic inflammation of the airways. These drugs block human M1 and M2 muscarinic acetylcholine receptors, inhibiting bronchoconstriction. However, studies have reported complications of anticholinergic usage, such as exacerbated eosinophil production and worsened urinary retention. Modification of known anticholinergics using bioisosteric replacements to increase efficacy could potentially minimize these complications. The present study focuses on identifying viable analogs of anticholinergics to improve binding energy to the receptors compared to current treatment options. Glycopyrrolate (G), ipratropium (IB), and tiotropium bromide (TB) were chosen as parent drugs of interest, due to the presence of common functional groups within the molecules, specifically esters and alcohols. Docking score analysis via AutoDock Vina was used to evaluate the binding energy between drug analogs and the muscarinic acetylcholine receptors. The final results suggest that G-A3, IB-A3, and TB-A1 are the most viable analogs, as binding energy was improved when compared to the parent drug. G-A4, IB-A4, IB-A5, TB-A3, and TB-A4 are also potential candidates, although there were slight regressions in binding energy to both muscarinic receptors for these analogs. By researching the effects of bioisosteric replacements of current anticholinergics, it is evident that there is a potential to provide asthmatics with more effective treatment options.

Chlorpyrifos and parathion regulate oxidative stress differentially through the expression of paraoxonase 2 in human neuroblastoma cell

Organophosphate (OP) compounds are frequently linked to both chronic and acute forms of nervous system disorders. Chlorpyrifos (CPF) and parathion (PA) are two of the most widely used OP insecticides throughout the world. These compounds are acetylcholinesterase inhibitors and cause a cholinergic crisis. However, there are other non-cholinergic effects of the OP compounds as well. The role of Paraoxonase 1 (PON1) in the metabolism of OP compounds is well established owing to its significant organophosphatase activity. Since PON2 has no paraoxonase activity and the level of its expression is 20–40 fold higher in the brain, in this article the role of PON2 in response to CPF and PA exposure concerning both cholinergic and non-cholinergic effects are explored. The effect of these OPs on cell viability, reactive oxygen species (ROS), PON2 gene expression, and function was studied. Glutathione level, esterase activity, and paraoxonase activity were also measured in CPF- and PA-treated IMR-32 cells. At these levels, both CPF and PA showed different impacts on IMR-32 cells. PA at higher concentrations (50–200 µM) proved to be less toxic than CPF. Interestingly, induction of ROS was also lower in the case of PA-treated cells as compared to the CPF. However, PON2 protein expression was increased with the increasing concentration of PA and decreased with the increasing concentration of CPF. To explore the possible mechanism of the differential regulation of PON2 gene expression by CPF and PA, we investigated the possible binding and signaling through the human M2 muscarinic acetylcholine receptor (M2AChR). Since M2AChRs are similar to G-protein coupled receptors and function through cAMP signalling, we measured the cAMP level after CPF and PA treatment. CPF- and PA-treated IMR-32 cells can be used as a model to study the mechanism by which PON2 acts as a ROS scavenger in response to xenobiotics stimulation in the brain.

Vibrational spectroscopy analysis of ligand efficacy in human M2 muscarinic acetylcholine receptor (M2R)

The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M2 muscarinic acetylcholine receptor (M2R). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M2R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M2R and paves the path to rationally design ligands with varied efficacy towards the target GPCR.

BRET- and fluorescence anisotropy-based assays for real-time monitoring of ligand binding to M2 muscarinic acetylcholine receptors

BRET and fluorescence anisotropy (FA) are two fluorescence-based techniques used for the characterization of ligand binding to G protein-coupled receptors (GPCRs) and both allow monitoring of ligand binding in real time. In this study, we present the first direct comparison of BRET-based and FA-based binding assays using the human M2 muscarinic acetylcholine receptor (M2R) and two TAMRA (5-carboxytetramethylrhodamine)-labeled fluorescent ligands as a model system. The determined fluorescent ligand affinities from both assays were in good agreement with results obtained from radioligand competition binding experiments. The assays yielded real-time kinetic binding data revealing differences in the mechanism of binding for the investigated fluorescent probes. Furthermore, the investigation of various unlabeled M2R ligands yielded pharmacological profiles in accordance with earlier reported data. Taken together, this study showed that BRET- and FA-based binding assays represent valuable alternatives to radioactivity-based methods for screening purposes and for a precise characterization of binding kinetics supporting the exploration of binding mechanisms.

The optimization of a novel selective antagonist for human M2 muscarinic acetylcholine receptor

Muscarinic acetylcholine receptors (mAChRs) comprise five distinct subtypes denoted M1 to M5. The antagonism of M2 subtype could increase the release of acetylcholine from vesicles into the synaptic cleft and improve postsynaptic functions in the hippocampus via M1 receptor activation, displaying therapeutic potentials for Alzheimer’s disease. However, drug development for M2 antagonists is still challenged among different receptor subtypes. In this study, by optimizing a scaffold from virtual screening, we synthesized two focused libraries and generated up to 50 derivatives. By measuring potency and binding selectivity, we discovered a novel M2 antagonist, ligand 47, featuring submicromolar IC50, high M2/M4 selectivity (~30-fold) and suitable lipophilicity (cLogP = 4.55). Further study with these compounds also illustrates the structure–activity relationship of this novel scaffold. Our study could not only provide novel lead structure, which was easy to synthesize, but also offer valuable information for further development of selective M2 ligands.

Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

Spectral investigations, DFT computations and molecular docking studies of 1,7,8,9-tetrachloro-10,10-dimethoxy-4-{3-[4-(2-methylphenyl)piperazin-1-yl]propyl}-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione

The optimized molecular structure, vibrational frequencies, corresponding vibrational assignments of the title compound have been investigated experimentally and theoretically. The HOMO and LUMO analysis is used to determine the charge transfer within the molecule. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analysed using NBO analysis. The hyperpolarisability calculation reveals the present material has a reasonably good propensity for nonlinear optical activity. Due to the different potential biological activity of the title compound, molecular docking study is also reported and the compound might exhibit inhibitory activity against human M2 muscarinic acetylcholine receptor.

Proteoliposome-based Selection of a Recombinant Antibody Fragment Against the Human M2 Muscarinic Acetylcholine Receptor

The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins.

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M1 muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M1 mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M1 mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M1 mAChRs, we show that once internalized, M1 mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M1 mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.

Toward activated homology models of the human M1 muscarinic acetylcholine receptor

Structure-based virtual screening offers a good opportunity for the discovery of selective M1 muscarinic acetylcholine receptor (mAChR) agonists for the treatment of Alzheimer's disease. However, no 3-D structure of an M1 mAChR is yet available and the homology models that have been previously reported are only able to identify antagonists in virtual screening experiments. In this study, we generated a homology model of the human M1 mAChR, based on the crystal structure of an M3 mAChR as the template. This initial model was modified, using the agonist-bound crystal structure of a β2-adrenergic receptor as a guide, to give two possible activated structures. The T192 side chain was adjusted in both structures and one of the structures also had the whole of transmembrane (TM) 5 rotated and tilted toward the inner channel of the transmembrane region. The binding sites of all three structures were then refined by induced-fit docking (IFD) with acetylcholine. Virtual screening experiments showed that all three refined models could efficiently differentiate agonists from decoy molecules, with the TM5-modified models also giving good agonist/antagonist selectivity. The whole range of agonists and antagonists was observed to bind within the orthosteric site of the structure obtained by IFD refinement alone, implying that it has inactive state character. In contrast, the two TM5-modified structures were unable to accommodate the antagonists, supporting the proposition that they possess activated state character.

Activation and allosteric modulation of a muscarinic acetylcholine receptor

Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously binding the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into activation mechanism and allosteric modulation of muscarinic receptors.

Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

N-Terminal Fusion Tags for Effective Production of G-Protein-Coupled Receptors in Bacterial Cell-Free Systems

G-protein-coupled receptors (GPCR) constitute one of the biggest families of membrane proteins. In spite of the fact that they are highly relevant to pharmacy, they have remained poorly explored. One of the main bottlenecks encountered in structural-functional studies of GPCRs is the difficulty to produce sufficient amounts of the proteins. Cell-free systems based on bacterial extracts fromE. colicells attract much attention as an effective tool for recombinant production of membrane proteins. GPCR production in bacterial cell-free expression systems is often inefficient because of the problems associated with the low efficiency of the translation initiation process. This problem could be resolved if GPCRs were expressed in the form of hybrid proteins with N-terminal polypeptide fusion tags. In the present work, three new N-terminal fusion tags are proposed for cell-free production of the human β2-adrenergic receptor, human M1 muscarinic acetylcholine receptor, and human somatostatin receptor type 5. It is demonstrated that the application of an N-terminal fragment (6 a.a.) of bacteriorhodopsin fromExiguobacterium sibiricum(ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein fromB. subtilisallows to increase the CF synthesis of the target GPCRs by 5-38 times, resulting in yields of 0.6-3.8 mg from 1 ml of the reaction mixture, which is sufficient for structural-functional studies.

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

The parasympathetic limb of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G protein coupled receptors (GPCRs) that mediate the response to acetylcholine released from parasympathetic nerves.1–5 Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiologic control of cardiovascular function through activation of G protein-coupled inwardly-rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of antagonist-bound M2 receptor, the first human acetylcholine receptor to be characterized structurally. The antagonist QNB binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all 5 muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The M2 receptor structure provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor1

The idea that GPCRs (G-protein-coupled receptors) may exist as homo- or hetero-oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM1 (human M1 muscarinic acetylcholine receptor subtype). We analysed the hM1 oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native-PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO-M1 cells (Chinese-hamster ovary cells expressing muscarinic M1 receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M1 receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M1 receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor. Our results suggest that MT7 binds to a dimeric form of hM1 receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre-existing muscarinic receptor homodimers.

Single nucleotide polymorphisms of the human M1 muscarinic acetylcholine receptor gene.

The gene encoding the human muscarinic receptor, type 1 (CHRM1), was genotyped from 245 samples of the Coriell Collection (Coriell Institute for Medical Research, Camden, NJ). Fifteen single nucleotide polymorphisms (SNPs) were discovered, 9 of which are located in the coding region of the receptor. Of these, 8 represent synonymous SNPs, indicating that CHRM1 is highly conserved in humans. Only a single allele was found to contain a nonsynonymous SNP, which encodes an amino acid change of Cys to Arg at position 417. This may have functional consequences because a C417S point mutation in rat M1 was previously shown to affect receptor binding and coupling. Furthermore, 0 of 4 SNPs within CHRM1 previously deduced from sequencing of the human genome were found in this study despite a prediction that a majority of such inferred SNPs are accurate. The consensus sequence of CHRM1 obtained in our study differs from the deposited reference sequence (AC NM_000738) in 2 adjacent nucleotides, leading to a V173M change, suggesting a sequencing error in the reference sequence. The extraordinary sequence conservation of the CHRM1 gene-coding region was unexpected as M1-knockout mice show only minimal functional impairments.

Interaction of human chagasic IgG with human colon muscarinic acetylcholine receptor: molecular and functional evidence

BACKGROUND AND AIMSGastrointestinal disorders is one of the clinical manifestations of chronic Chagas' disease. The pathogenesis seems to be associated with autonomic dysfunction. Here, we consider the muscarinic cholinoceptor mediated...

Residues specifically involved in down-regulation but not internalization of the m1 muscarinic acetylcholine receptor.

Human m1 muscarinic acetylcholine receptor mutants were screened to determine receptor domains and cellular pathways relevant to down-regulation. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for receptor activation or internalization had been previously demonstrated in HEK293 cells, were selected for this study. To assess receptor down-regulation, the m1 receptor mutants were transfected into Chinese hamster ovary cells. Because receptor internalization is expected to precede down-regulation, mutants displaying intact internalization were selected to permit interpretation of mutational effects on down-regulation alone. Four mutations were identified that specifically impaired down-regulation without altering receptor internalization: V127A, I211A, E360A, and K362A. The results define new receptor domains in the second intracellular loop and the junctions of the third intracellular loop that are involved in down-regulation. These same four mutants were also defective in signaling via the phospholipase C and the adenylyl cyclase pathways and in G protein activation, as measured by [35S]GTP gamma S binding. However, the level of second messenger stimulation correlated poorly with the extent of down-regulation. In summary, several mutations of the m1 receptor selectively affect down-regulation, demonstrating that internalization and down-regulation represent distinct events driven by different cellular mechanisms.

Challenges and opportunities for developing muscarinic receptor subtype-based therapeutic agents

Drug Development ResearchVolume 40, Issue 2 p. 101-103 IntroductionFree Access Challenges and opportunities for developing muscarinic receptor subtype-based therapeutic agents James C. Blosser, Corresponding Author James C. Blosser Astra Arcus U.S.A., Rochester, New YorkAstra Arcus U.S.A., Rochester, New YorkSearch for more papers by this authorJohn E. Macor, John E. Macor Astra Arcus U.S.A., Rochester, New YorkSearch for more papers by this authorWilliam S. Messer Jr., William S. Messer Jr. Department of Medicinal and Biological Chemistry, University of Toledo, Toledo, OhioSearch for more papers by this author James C. Blosser, Corresponding Author James C. Blosser Astra Arcus U.S.A., Rochester, New YorkAstra Arcus U.S.A., Rochester, New YorkSearch for more papers by this authorJohn E. Macor, John E. Macor Astra Arcus U.S.A., Rochester, New YorkSearch for more papers by this authorWilliam S. Messer Jr., William S. Messer Jr. Department of Medicinal and Biological Chemistry, University of Toledo, Toledo, OhioSearch for more papers by this author First published: 06 December 1998 https://doi.org/10.1002/(SICI)1098-2299(199702)40:2<101::AID-DDR1>3.0.CO;2-PCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References Allard WJ, Sigal IS, Dixon RA (1987): Sequence of the gene encoding the human M1 muscarinic acetylcholine receptor. Nucleic Acids Res 15: 10604. Bluml KE, Mutschler, Wess J (1994a): Functional role in ligand binding and receptor activation of an asparagine residue present in the sixth transmembrane domain of all muscarinic receptors. J Biol Chem 269: 18870– 18876. Bluml KE, Mutschler, Wess J (1994b): Insertion mutagenesis as a tool to predict the secondary structure of a muscarinic receptor domain determining specificity of G-protein coupling. Proc Nat Acad Sci USA: 7980– 7984. Bonner TI, Buckley NJ, Young AC, Brann MR (1987): Identification of a family of muscarinic acetylcholine receptor genes. Science 237: 527– 532. Bonner TI, Young AC, Brann MR, Buckley NJ (1988): Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes. Neuron 1: 403– 410. Dale HH (1914): The action of certain esters and ethers of choline, and their relation to muscarine. J Exp Ther 6: 147– 190. 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Phosphorylation of Human m1 Muscarinic Acetylcholine Receptors by G Protein-coupled Receptor Kinase 2 and Protein Kinase C

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 alpha, beta, and gamma types, PKC). The m1 receptor was phosphorylated by either GRK2 or PKC in an agonist-dependent or independent manner, respectively. G protein beta gamma 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 phoshorylation 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.