Dissecting the conserved NPxxY motif of the M3 muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation (original) (raw)
Related papers
Cellular Physiology and Biochemistry, 2011
Acetylcholine challenge produces M 3 muscarinic acetylcholine receptor activation and accessory/ scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M 3 muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to Gα q/11 after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M 3 R and ARF1 and also for the formation of the ARF/Rho/βγ signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M 3 muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation.
Molecular Pharmacology, 2011
We have used alanine-scanning mutagenesis followed by functional expression and molecular modeling to analyze the roles of the 14 residues, Asn422 to Cys435, C-terminal to transmembrane (TM) helix 7 of the M 1 muscarinic acetylcholine receptor. The results suggest that they form an eighth (H8) helix, associated with the cytoplasmic surface of the cell membrane in the active state of the receptor. We suggest that the amide side chain of Asn422 may act as a cap to the C terminus of TM7, stabilizing its junction with H8, whereas the side chain of Phe429 may restrict the relative movements of H8 and the C terminus of TM7 in the inactive ground state of the receptor. We have identified four residues, Phe425, Arg426, Thr428, and Leu432, which are important for G protein binding and signaling. These may form a docking site for the C-terminal helix of the G protein ␣ subunit, and collaborate with G protein recognition residues elsewhere in the cytoplasmic domain of the receptor to form a coherent surface for G protein binding in the activated state of the receptor.
Cellular Physiology and Biochemistry, 2010
Several motifs found in the third intracellular loop of the M 3 muscarinic receptor are critical for G protein activation and scaffold protein interaction. However, how multiprotein complexes form is not fully understood. A minigene encoding the third intracellular loop of the M 3 muscarinic receptor was constructed to explore whether peptides from this intracellular region could act as inhibitors of the muscarinic multiprotein complex formation and signaling. We found that this construct, when coexpressed with the M 3 receptor, has the ability to act as a competitive antagonist of G protein receptors and receptor-scaffold/accessory proteins. Transient transfection of human embryonic kidney-293 cells with DNA encoding the human M 3 and M 5 receptor subtypes results in a carbachol-dependent increase of inositol phosphate. Co-expression of the M 3 third cytoplasmic loop minigene dramatically reduces both carbachol-mediated G protein activation and inositol phosphate accumulation. Minigene expression also abrogates activation of M 3 and M 5 receptor mitogenactivated protein kinases pathway. Furthermore, minigene expression led to reduced AKT activation. These data, together with results of coimmunoprecipitation of different scaffold and kinase proteins, provide experimental evidence for the role for the third cytoplasmic loop of the human M 3 muscarinic receptor in G-protein activation and multiprotein complex formation.
Muscarinic acetylcholine receptor-interacting proteins (mAChRIPs): targeting the receptorsome
Current drug targets, 2012
Muscarinic acetylcholine receptors comprise a large family of G protein-coupled receptors that are involved in the regulation of many important functions of the central and peripheral nervous system. To achieve such a large range of physiological effects, these receptors interact, in addition to the canonical heterotrimeric G proteins, with a large array of accessory proteins including scaffold molecules, ion channels and enzymes which operate as molecular transducers of muscarinic function. Interestingly, as demonstrated for others G protein-coupled receptors this type of receptors are also able to oligomerize, a fact that has been shown to play a critical role in their subcellular distribution, trafficking, and finetuning modulation of cholinergic signalling. On the other hand, the specificity of these receptor interactions may be largely determined by the occurrence of precise protein-interacting motifs, posttranslational modifications, and the differential tissue distribution and stoichiometry of the receptor-interacting proteins. Thus, the exhaustive cataloguing and documentation of muscarinic acetylcholine receptors interacting proteins and the grasp of their specific function will explain some key physiological differences in muscarinic-mediated cholinergic transmission. Overall, a better comprehension of the muscarinic receptor interactome will have by sure a great impact on the cholinergic pharmacology and thus providing previously unrealized opportunities to achieve greater specificity in muscarinic-related drug discovery and diagnostic.
Muscarinic receptor family interacting proteins: role in receptor function
Journal of neuroscience methods, 2011
a b s t r a c t G protein-coupled receptors constitute one of the most important families of membrane receptors through which cells respond to extracellular stimuli. Receptors of this superfamily likely function as signal transduction complexes. The identification and analysis of their components provide new insights into a better understanding of these receptors' function and regulation. We used tandem-affinity purification and mass spectrometry as a systematic approach to characterize multiprotein complexes in the acetylcholine muscarinic receptor subfamily. To overcome the limitations associated with membrane protein receptor solubilization with detergents, we developed a strategy in which receptors are co-expressed with a cytoplasmic minigene construct, encoding the third intracellular loop and the C-terminal tail tagged to the tandem-affinity-cassette of each receptor subtype. Numerous protein complexes were identified, including many new interactions in various signalling pathways. Systematic identification data set together with protein interactions reported in the literature revealed a high degree of connectivity. These allow the proposal, for the first time, of an outline of the muscarinic interactome as a network of protein complexes and a context for a more reasoned and informed approach to drug discovery and muscarinic receptor subtype specificities.
Journal of Biological Chemistry, 2006
Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cisretinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M 3 muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys 220 residue, Gln 207 , Gly 211 , Arg 213 , Gly 218 , Ile 222 , Phe 224 , Leu 225 , and Pro 228 were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance. . 3 The abbreviations used are: GPCR, G protein-coupled receptor; EGFP, enhanced green fluorescent protein; HA tag, hemagglutinin tag; [ 3 H]NMS, N-[ 3 H]methylscopolamine; i3 loop, the third intracellular loop of G protein-coupled receptors; M3R, M 3 muscarinic receptor; M3R(⌬i3), M 3 muscarinic receptor lacking amino acids Ala 274 -Lys 469 ; M3R⌬(Nterm), M 3 muscarinic receptor lacking amino acids Thr 2 -His 62 ; N-term, the extracellular N-terminal segment of G protein-coupled receptors; o2 loop, the second extracellular loop of G protein-coupled receptors; SC medium, synthetic complete medium; TM I-VII, the seven transmembrane domains of G protein-coupled receptors; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; FLIPR, fluorometric imaging plate reader.
The EMBO Journal, 1990
Communicated by P.Seeburg Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2' release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes.
Structure and activation of muscarinic acetylcholine receptors
Biochemical Society Transactions, 2003
A homology model of the M1 muscarinic acetylcholine receptor, based on the X-ray structure of bovine rhodopsin, has been used to interpret the results of scanning and point mutagenesis studies on the receptor's transmembrane (TM) domain. Potential intramolecular interactions that are important for the stability of the protein fold have been identified. The residues contributing to the binding site for the antagonist, N-methyl scopolamine, and the agonist, acetylcholine, have been mapped. The positively charged headgroups of these ligands probably bind in a charge-stabilized aromatic cage formed by amino acid side chains in TM helices TM3, TM6 and TM7, while residues in TM4 may participate as part of a peripheral docking site. Closure of the cage around the headgroup of acetylcholine may be part of the mechanism for transducing binding energy into receptor activation, probably by disrupting a set of Van der Waals interactions between residues lying beneath the binding site that h...
Regulation of muscarinic acetylcholine receptor signaling
Pharmacology & Therapeutics, 2003
Multiple mechanisms regulate the signaling of the five members of the family of the guanine nucleotide binding protein (G protein)coupled muscarinic acetylcholine (ACh) receptors (mAChRs). Following activation by classical or allosteric agonists, mAChRs can be phosphorylated by a variety of receptor kinases and second messenger-regulated kinases. The phosphorylated mAChR subtypes can interact with b-arrestin and presumably other adaptor proteins as well. As a result, the various mAChR signaling pathways may be differentially altered, leading to short-term or long-term desensitization of a particular signaling pathway, receptor-mediated activation of the mitogenactivated protein kinase pathway downstream of mAChR phosphorylation, as well as long-term potentiation of mAChR-mediated phospholipase C stimulation. Agonist activation of mAChRs may also induce receptor internalization and down-regulation, which proceed in a highly regulated manner, depending on receptor subtype and cell type. In this review, our current understanding of the complex regulatory processes that underlie signaling of mAChR is summarized. D
Overproduction of human M₃ muscarinic acetylcholine receptor: an approach toward structural studies
Biotechnology progress
Human M3 muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral nervous system, is involved in several neurodegenerative and autoimmune diseases. Recently, M3R overexpression has been suggested to play a role in certain forms of cancer, showing promise as a new potential pharmacological target. However, the lack of structural information hampered to develop a new potent selective and potent antagonist. We describe here different strategies for overexpressing functional M3R on the perspective of future biophysical studies. To achieve this goal, four tagged M3R genes were engineered and codon optimized. Different heterologous expression systems, including mammalian cells and viral transfection, were employed to overexpress M3R. Although codon optimization resulted in only twofold to threefold increase of M3R expression, we found that epitope tagging of the synthetic M3R, especially with hemagglutinin and Flag epitope tags, could improve M3R expression levels. On the other hand, viral transfection led to a yield of 27 pmol/mg protein that is the highest level reported so far for this receptor subtype in mammalian cells. Taking together several of the strategies used can help increasing M3R expression, not only to start purification efforts but also for secondary structural analysis trial and functional analyses. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011