Edinburgh Napier University

  The molecular evolution of the opioid receptor family has been studied by isolating cDNAs that encode six distinct opioid receptor-like proteins from a lower vertebrate, the teleost fish Catostomus commersoni. One of these, which has been obtained in full-length form, encodes a 383-amino acid protein that exhibits greatest sequence similarity to mammalian μ-opioid receptors; the corresponding gene is expressed predominantly in brain and pituitary. Transfection of the teleost cDNA into HEK 293 cells resulted in the appearance of a receptor having high affinity for the μ-selective agonist [d-Ala2, MePhe4-Gly-ol5]enkephalin (DAMGO) (Kd = 0.63 ± 0.15 nM) and for the nonselective antagonist naloxone (Kd = 3.1 ± 1.3 nM). The receptor had negligible affinity for U50488 and [d-Pen2, d-Pen5]enkephalin (DPDPE), which are κ- and δ-opioid receptor selective agonists, respectively. Stimulation of transfected cells with 1 μM DAMGO lowered forskolin-induced cAMP levels, an effect that could be reversed by naloxone. Experiments in Xenopus oocytes have demonstrated that the fish opioid receptor can, in an agonist-dependent fashion, activate a coexpressed mouse G-protein-gated inward-rectifying potassium channel (GIRK1). The identification of six distinct fish opioid receptor-like proteins suggests that additional mammalian opioid receptors remain to be identified at the molecular level. Furthermore, our data indicate that the μ-opioid receptor arose very early in evolution, perhaps before the appearance of vertebrates, and that the pharmacological and functional properties of this receptor have been conserved over a period of ≈400 million years implying that it fulfills an important physiological role.