title: Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor.
abstract: BACKGROUND
  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).
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: CEA, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), Gif-sur-Yvette, F-91191, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Marquer
authors.forename: Carole
authors.initials: C
authors.lastname: Fruchart-Gaillard
authors.forename: Gilles
authors.initials: G
authors.lastname: Mourier
authors.forename: Olivier
authors.initials: O
authors.lastname: Grandjean
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Girard
authors.forename: Marc
authors.initials: M
authors.lastname: le Maire
authors.forename: Spencer
authors.initials: S
authors.lastname: Brown
authors.forename: Denis
authors.initials: D
authors.lastname: Servent
mesh_terms: D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004546:Elapid Venoms / Q000633:toxicity*; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008024:Ligands; D038761:Photobleaching / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D043643:Receptor, Muscarinic M1 / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility / Q000187:drug effects
chemical_list: D004546:Elapid Venoms; D008024:Ligands; D043643:Receptor, Muscarinic M1; C419876:muscarinic toxin 7