title: Dissecting the conserved NPxxY motif of the M3 muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation.
abstract: 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/β γ 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.
authors.affiliation: Centre de Biotecnologia Molecular, Department d'Enginyeria Quémica, Universitat Politècnica de Catalunya, Terrassa, Spain.
authors.forename: Dasiel O
authors.initials: DO
authors.lastname: Borroto-Escuela
authors.forename: Wilber
authors.initials: W
authors.lastname: Romero-Fernandez
authors.forename: Gloria
authors.initials: G
authors.lastname: García-Negredo
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Correia
authors.forename: Pere
authors.initials: P
authors.lastname: Garriga
authors.forename: Kjell
authors.initials: K
authors.lastname: Fuxe
authors.forename: Francisco
authors.initials: F
authors.lastname: Ciruela
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002217:Carbachol / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D009154:Mutation; D010739:Phospholipase D / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D043587:Receptor, Muscarinic M3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; D043587:Receptor, Muscarinic M3; D002217:Carbachol; D054460:rho-Associated Kinases; D010738:Type C Phospholipases; D010739:Phospholipase D; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020823:ADP-Ribosylation Factor 1