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-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.
Footnotes
- Received April 24, 2014.
- Accepted May 29, 2014.
This research was supported by a Collaborative Health Research Project grant from the Natural Sciences and Engineering Research Council of Canada and the Canadian Institutes of Health Research [Grants RPCS-385882-2010 (to M.G. and L.G.) and 311997 (to G.P.)] and the Fonds de Recherche Québec–Santé [Junior 2 salary support (to L.G.) and a Master scholarship (to P.B.)]. M.G. holds the Tier 2 Canada Research Chair in Nanopharmacology and Atomic Force Microscopy.
This work was previously presented in part at the following workshop: Bourassa P, Grandbois M, Gendron L (2013) Surface plasmon resonance as a tool to assess functional selectivity at the mu opioid receptor. 43rd Annual Meeting for the Society for Neuroscience; 2013 Nov 9–13; San Diego, CA.
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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