PT - JOURNAL ARTICLE AU - Philippe Bourassa AU - Hanieh Bagheri Tudashki AU - Graciela Pineyro AU - Michel Grandbois AU - Louis Gendron TI - Label-Free Monitoring of <em>μ</em>-Opioid Receptor–Mediated Signaling AID - 10.1124/mol.114.093450 DP - 2014 Aug 01 TA - Molecular Pharmacology PG - 138--149 VI - 86 IP - 2 4099 - http://molpharm.aspetjournals.org/content/86/2/138.short 4100 - http://molpharm.aspetjournals.org/content/86/2/138.full SO - Mol Pharmacol2014 Aug 01; 86 AB - 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-NH2), 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-NH2) 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.