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 (κ) 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 κ receptor has 75% sequence identity to κ opioid receptors cloned from mammals, and 66% sequence identity to the κ 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 κ receptor possessed a single, high-affinity (Kd =1.5 nM) binding site for the κ-selective agonist U69593. In competition binding assays, the expressed newt κ receptor displayed high affinity for the κ-selective agonists GR89696, dynorphin A(1–13), U69593, U50488 and BRL52537, as well as the κ-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 κ receptor. The expressed receptor displayed essentially no affinity for the δ-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 κ opioid receptor is expressed in amphibians and that the pharmacology of the newt κ receptor closely matches mammalian κ opioid receptors. However, the functional dichotomy between the classic opioid receptors (κ, δ, μ) and ORL1 appears less strict in amphibians than in mammals.