Cloning, heterologous expression and pharmacological characterization of a kappa opioid receptor from the brain of the rough-skinned newt, Taricha granulosa

Abstract

A full-length cDNA that encodes a kappa ( ) opioid receptor has been isolated from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa. The deduced protein contains 385 amino acids and possesses features commonly attributed to G protein-coupled receptors, such as seven putative transmembrane domains. The newt receptor has 75% sequence identity to opioid receptors cloned from mammals, and 66% sequence identity to the opioid receptor reported for the zebrafish, with the greatest divergence in the extracellular N-terminus, the second and third extracellular loops and the intracellular C-terminus. Membranes isolated from COS-7 cells expressing the newt receptor possessed a single, high-affinity (K d =1·5 nM) binding site for the -selective agonist U69593. In competition binding assays, the expressed newt receptor displayed high affinity for the -selective agonists GR89696, dynorphin A(1-13), U69593, U50488 and BRL52537, as well as the -selective antagonist nor-binaltorphimine and the non-selective antagonist naloxone. Rank order potencies and affinity constants were similar in competition binding studies that used either whole brain homogenates or membranes isolated from COS-7 cells expressing the newt receptor. The expressed receptor displayed essentially no affinity for the -selective agonist DPDPE ([d-penicillamine, d-penicillamine]enkephalin), but showed moderate affinity for the µ-selective agonist DAMGO ([d-Ala-MePhe, Gly-ol]enkephalin) and moderately high affinity for nociceptin (orphanin FQ), the endogenous ligand for the opioid receptor-like (ORL)1 receptor. These findings support the conclusions that a gene for the opioid receptor is expressed in amphibians and that the pharmacology of the newt receptor closely matches mammalian opioid receptors. However, the functional dichotomy between the classic opioid receptors ( , , µ) and ORL1 appears less strict in amphibians than in mammals.

Suppression of sex behavior by kappa opiates and stress steroids occurs via independent neuroendocrine pathways

“…This notion was supported by significant evidence: both j-opioid agonists and CORT suppress clasping behavior (Deviche and Moore, 1987), CORT suppresses clasping by acting at a membrane receptor mCR (Orchinik et al, 1991) and rapidly up-regulating endogenous cannabinoid signaling (Coddington et al, 2007), mCR has j-like properties (Bradford et al, 2005), and specific j-opioid agonists bind and compete with CORT for the mCR with high affinity (Evans et al, 2000). Consistent with past research, this study found that the j-opioid agonist U50488 significantly suppressed clasping in a dose-dependent manner.…”

“…U50488 binds to both the mCR and it's own native jOR in in vitro preparations of Taricha brains; binding to the mCR with the same affinity as CORT (K i = 250 nM; (Evans et al, 2000)), and binding its own receptor (jOR) with a higher affinity (K i = 3.4 nM; (Bradford et al, 2005). Furthermore, the mCR has a single CORT binding site that has very high specificity and affinity for the CORT and j-opiate ligands, and very low affinity for mineralocorticoids or classical intracellular glucocorticoid receptor antagonists (Orchinik et al, 1991;Evans et al, 2000).…”

Evolution of vertebrate opioid receptors

“…The genes arose by duplications in the common ancestor of tetrapods and bony fishes (13). Opioid receptor sequences have been reported for a few nonmammalian tetrapods (14)(15)(16)(17)(18)) and a few teleost fishes (19)(20)(21)(22)(23)(24), and a partial sequence has been reported for a hagfish (19). Functional studies in amphibians and bony fishes have shown that the opioid system is involved in nociception also in these species (25,26).…”

Bioinformatics and Evolution of Vertebrate Nociceptin and Opioid Receptors

“…The first full-length clone sequenced from a nonmammalian species was ccMOR from the brain of the white suckerfish, Catostomus commersoni (Darlison et al, 1997). Other nonmammalian species with full-length opioid receptors cloned are the zebrafish, Danio rerio, by Rodriguez and colleagues (Alvarez et al, 2006;Barrallo, González-Sarmiento, Alvar, & Rodriguez, 2000;Barrallo, González-Sarmiento, Porteros, Gracia-Isídoro, & Rodríguez, 1998;Porteros, Garcia-Isodoro, Barrallo, González-Sarmiento, & Rodriguez, 1999) and the rough-skinned newt, Taricha granulosa, in the laboratory of Moore (Bradford, Walthers, Searcy, & Moore, 2005;Bradford, Walthers, Stanley, Baugh, & Moore, 2006;. Using the same sets of primers that Evans group used in the first phylogenetic study of opioid receptors (Li et al, 1996b), our group was able to clone all four opioid receptor types expressed in R. pipiens brain tissue: rpMOR, rpDOR, rpKOR, and rpORL (Stevens, Brasel, & Mohan, 2007).…”