| Abstract No.: | C-B3064 |
| Country: | Canada |
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| Title: | IN VIVO AND IN VITRO CHARACTERIZATION OF THE MU-DELTA OPIOID RECEPTOR HETEROMER: A NOVEL OPIOID RECEPTOR SUBTYPE |
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| Authors/Affiliations: | 1 Noufissa Kabli*; 1 Brian F. O'Dowd; 1 Susan R. George;
1 Department of Pharmacology & Toxicology, University of Toronto, ON, Canada |
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| Content: | Opioid receptors (OR) are G-Protein Coupled Receptors that modulate key biological functions including analgesia and reward. Although only three OR types have been cloned -mu, delta, and kappa-, pharmacological studies predict a greater number of subtypes, which may result from direct receptor-receptor interactions. Indeed, we have shown that co-expressed mu- and delta-ORs associate physically into hetero-oligomeric complexes with a unique pharmacological and novel signaling profile. Deltorphin II was identified as a selective agonist for the mu-delta heteromer, and activated a novel pertussis toxin(PTX)-resistant Gz-coupled signaling cascade. To date, our understanding of the pharmacological profile, and localization and function of the mu-delta heteromer in physiological tissues remains rudimentary. OBJECTIVES: Thus, we sought to determine whether mu-, delta-, and mu-delta-selective ligands differentially activate PTX-sensitive and resistant G proteins in the brain, to demonstrate the physical interaction between the mu-delta heteromer and Gz, and to characterize the agonist internalization profile of the mu-delta heteromer in heterologous cells. METHODS & RESULTS: Using the 35S-GTPgS incorporation assay, we demonstrate that the muOR agonist DAMGO, deltaOR agonist DPDPE, and mu/deltaOR agonist deltorphin II activated receptor-coupled G proteins equally in membrane preparations from rodent hippocampus and striatum. Quantification of 35S-GTPgS incorporation into specific immunoprecipitated Ga proteins revealed that deltorphin II and DPDPE induced robust activation of the PTX-resistant Gz in the hippocampus, which was significantly different from basal and DAMGO levels. In the striatum, deltorphin II induced robust activation of Gz, which differed significantly from basal, DAMGO-, and DPDPE-stimulated levels, while all agonists activated Gi3 modestly. Using Bioluminescence Resonance Energy Transfer, we demonstrate that the interaction between mOR and Gz was more conformationally favourable in the presence of a physical interaction between mu- and delta-OR. Using cell surface receptor binding of a radiolabeled muOR agonist in intact cells, we demonstrate that the mu-delta heteromer has a novel agonist internalization profile. Delta OR agonists induced internalization of surface muOR in cells co-expressing mu- and delta-OR, an effect abolished by the deltaOR antagonist naltrindole. As expected deltaOR agonists did not induce internalization of surface muOR in cells singly expressing muOR. Thus, deltaOR agonists may induce internalization of muOR in complexes with deltaOR. Interestingly, deltaOR agonist-mediated internalization of surface muOR in cells co-expressing mu- and delta-OR, was preserved after PTX treatment suggesting that internalization was independent of PTX-sensitive G proteins. CONCLUSIONS: Taken together, these findings further our understanding of the mechanism of opioid action in distinct brain regions and demonstrate that the mu-delta heteromer may be expressed in hippocampus and striatum. Furthermore, the mu-delta heteromer interacts preferentially with Gz, is expressed on the cell surface, and is regulated by internalization. Characterizing the mu-delta heteromer will shed light onto the neuronal circuits it modulates as this receptor may be a promising target for the treatment of neurological disorders. (Funded by CIHR, NIH, OMHF, CAMH, & a CRC to S.R.G) |
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