title: Label-free monitoring of μ-opioid receptor-mediated signaling.
abstract: In this study, we used a combination of traditional signaling investigation approaches, bioluminescence resonance energy transfer (BRET) biosensors, and the label-free approach surface plasmon resonance (SPR) spectroscopy to monitor the signaling cascades of the μ-opioid receptor (MOP). In human embryonic kidney cells stably expressing a Flag-tagged version of human MOP, we compared the signals triggered by the noninternalizing and internalizing MOP agonists morphine and DAMGO (Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol), respectively. We studied three major and well described components of MOP signaling: receptor internalization, G protein coupling, and activation of extracellular signal-regulated kinase ERK1/ERK2. Our results show that morphine and DAMGO display different profiles of receptor internalization and a similar ability to trigger the phosphorylation of ERK1/ERK2. Our SPR analyses revealed that morphine and DAMGO evoke similar SPR signatures and that Gαi, cAMP-dependent pathways, and ERK1/ERK2 have key roles in morphine- and DAMGO-mediated signaling. Most interestingly, we found that the so-called MOP neutral antagonists CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2)), naloxone, and naltrexone behave like partial agonists. Even more intriguing, BRET experiments indicate that CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) induces similar conformational changes as naltrexone at the Gαi-βγ interface, whereas it appears as an inverse agonist based on its SPR response thus indicating distinct signaling mechanisms for the two ligands. Taken together, our results support the usefulness of label-free methods such as SPR to study whole-cell responses and signaling cascades triggered by G protein-coupled receptors and complement the conventional approaches by revealing cellular responses that would have been otherwise undetectable.
authors.affiliation: Départements de Physiologie et Biophysique (P.B., L.G.), Pharmacologie (H.B.T., M.G.), and Psychiatrie (G.P.), Centre de Recherche du CHU Ste-Justine (H.B.T, G.P.), Université de Montréal, Montreal, Quebec, Canada; Institut de Pharmacologie de Sherbrooke (M.G., L.G.), Centre de Recherche du CHU Sherbrooke (P.B., M.G., L.G.), Université de Sherbrooke (P.B., M.G., L.G.), Sherbrooke, Quebec, Canada; and Quebec Pain Research Network, Quebec City, Quebec, Canada (L.G.).
authors.forename: Philippe
authors.initials: P
authors.lastname: Bourassa
authors.affiliation: Départements de Physiologie et Biophysique (P.B., L.G.), Pharmacologie (H.B.T., M.G.), and Psychiatrie (G.P.), Centre de Recherche du CHU Ste-Justine (H.B.T, G.P.), Université de Montréal, Montreal, Quebec, Canada; Institut de Pharmacologie de Sherbrooke (M.G., L.G.), Centre de Recherche du CHU Sherbrooke (P.B., M.G., L.G.), Université de Sherbrooke (P.B., M.G., L.G.), Sherbrooke, Quebec, Canada; and Quebec Pain Research Network, Quebec City, Quebec, Canada (L.G.).
authors.forename: Hanieh Bagheri
authors.initials: HB
authors.lastname: Tudashki
authors.affiliation: Départements de Physiologie et Biophysique (P.B., L.G.), Pharmacologie (H.B.T., M.G.), and Psychiatrie (G.P.), Centre de Recherche du CHU Ste-Justine (H.B.T, G.P.), Université de Montréal, Montreal, Quebec, Canada; Institut de Pharmacologie de Sherbrooke (M.G., L.G.), Centre de Recherche du CHU Sherbrooke (P.B., M.G., L.G.), Université de Sherbrooke (P.B., M.G., L.G.), Sherbrooke, Quebec, Canada; and Quebec Pain Research Network, Quebec City, Quebec, Canada (L.G.).
authors.forename: Graciela
authors.initials: G
authors.lastname: Pineyro
authors.affiliation: Départements de Physiologie et Biophysique (P.B., L.G.), Pharmacologie (H.B.T., M.G.), and Psychiatrie (G.P.), Centre de Recherche du CHU Ste-Justine (H.B.T, G.P.), Université de Montréal, Montreal, Quebec, Canada; Institut de Pharmacologie de Sherbrooke (M.G., L.G.), Centre de Recherche du CHU Sherbrooke (P.B., M.G., L.G.), Université de Sherbrooke (P.B., M.G., L.G.), Sherbrooke, Quebec, Canada; and Quebec Pain Research Network, Quebec City, Quebec, Canada (L.G.) Louis.Gendron@USherbrooke.ca Michel.Grandbois@USherbrooke.ca.
authors.forename: Michel
authors.initials: M
authors.lastname: Grandbois
authors.affiliation: Départements de Physiologie et Biophysique (P.B., L.G.), Pharmacologie (H.B.T., M.G.), and Psychiatrie (G.P.), Centre de Recherche du CHU Ste-Justine (H.B.T, G.P.), Université de Montréal, Montreal, Quebec, Canada; Institut de Pharmacologie de Sherbrooke (M.G., L.G.), Centre de Recherche du CHU Sherbrooke (P.B., M.G., L.G.), Université de Sherbrooke (P.B., M.G., L.G.), Sherbrooke, Quebec, Canada; and Quebec Pain Research Network, Quebec City, Quebec, Canada (L.G.) Louis.Gendron@USherbrooke.ca Michel.Grandbois@USherbrooke.ca.
authors.forename: Louis
authors.initials: L
authors.lastname: Gendron
mesh_terms: D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000235:genetics; D010766:Phosphorylation / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D043562:Receptors, G-Protein-Coupled; D017450:Receptors, Opioid, mu; D019204:GTP-Binding Proteins