Abstract
Fusion proteins between a receptor and a pertussis toxin-insensitive Giα subunit were used to gain insight into the molecular interactions that take place upon μ and δ opioid receptor heterodimerization. When μ opioid receptor-Gi1α fusions were coexpressed with nonfused δ opioid receptors in human embryonic kidney 293 cells, or vice versa, receptor heterodimers were detected by coimmunoprecipitation. In pertussis toxin-treated cells, receptor coexpression decreased the amount of guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) incorporated in the fused Gα protein after the addition of agonists specific for the receptor-Gi1α fusion. In addition, activation of the Gα protein occurred in heterodimers upon addition of an agonist specific for the nonfused receptor. It remained unaffected by an inverse agonist specific for the receptor-Gi1α fusion. These data suggest that signaling through the receptor-Gi1α fusion protein is impaired in heterodimers and support a mechanism in which activation of the Gα subunit is promoted by a direct interaction with the nonfused receptor. Alternatively, receptor coexpression did not modify the ligand binding properties for the high-affinity state of the receptor-Gi1α fusion nor the EC50 values for agonist-induced [35S]GTPγS incorporation in the Gi1α subunit. In addition, no binding competition was observed between δ and μ ligands. Together, the data point to μ-δ opioid receptor heterodimers formed by contact interactions between monomers that retain their structural integrity.
Footnotes
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.106.101220.
-
ABBREVIATIONS: GPCR, G protein-coupled receptor; Tic, 1,2,3,4-tetrahydroisoquinoline; GTPγS, guanosine 5′-O-(3-thio)triphosphate; DOR, δ opioid receptor; MOR, μ opioid receptor; HEK, human embryonic kidney; h, human; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate; PVDF, polyvinylidene difluoride; TBST, Tris-buffered saline/Tween 20; DAMGO, [d-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin; SNC80, (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; TM, transmembrane; Tic-deltorphin, H-Tyr-Tic-Phe-Phe-Val-Val-Gly-NH2; ICI 174,864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; CTAP, d-Phe-Cys-Tyr-d-Trp-Arg-Pen-Thr-NH2.
-
↵1 Current affiliation: Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1.
- Received January 11, 2006.
- Accepted May 2, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|