Cloning and bioinformatics of amphibian mu, delta, kappa, and nociceptin opioid receptors expressed in brain tissue: evidence for opioid receptor divergence in mammals

Abstract

Opioid agonists produce analgesia in humans and other mammals by binding to three distinct types of G protein-coupled receptors; mu (MOR), delta (DOR), and kappa (KOR) opioid receptors. A fourth member of the opioid receptor family is the nociceptin or orphanin FQ receptor (ORL), however the role of the ORL receptor in analgesia is less clear. In the Northern grass frog, Rana pipiens, systemic and central administration of morphine and selective MOR, DOR, and KOR agonists produced dose-dependent antinociceptive effects blocked by the general opioid antagonist, naltrexone. The present study reports on the sequence, expression, and bioinformatics of four opioid receptor cDNAs cloned from Rana pipiens; rpMOR, rpDOR, rpKOR, and rpORL. These were the first opioid receptors cloned from a species of Class Amphibia, are selectively expressed in brain tissue, and show 70-84% identity to their homologous mammalian opioid receptors. Comparisons within species showed that MOR, DOR, and KOR proteins are significantly less divergent in earlier-evolved vertebrates compared to humans and other mammals. Among the four types of opioid receptors, MOR proteins show the least sequence variation among the six vertebrate species. Additionally, phylogenetic analysis supports the hypothesis that the family of opioid receptor proteins are coded by four genes that arose from two gene duplications of a single ancestral opioid receptor gene.

Fig. 1

Tissue expression of rpMOR, rpDOR, rpKOR, and rpORL in amphibian brain, heart, GI, liver, and muscle tissues (lanes 2–6). Lane 1 is the standardized bp ladder. For control, β-actin expression was also assayed in each of the tissues. Numbers on the left margin are the predicted size for each of the receptor amplicons, based on gene-specific primers used in the PCR reactions.

Fig. 2

Phylogenetic analysis of MOR, DOR KOR and ORL sequences in six vertebrates. MEGA software was used to generate a radial phylogenetic tree using the neighbor-joining method, rooted with the available matching sequences of rhodopsin (RHO). Protein sequences from Rana pipiens (rp) were provided by conceptual translation of the cloned cDNA sequences deposited in GenBank. Abbreviations and access codes for MOR, DOR, KOR, and ORL sequences from other vertebrates were: dr: Dano rerio (zebrafish; AAK01143, AAP86771, AAG60607, AAN46747), tg: Taricha granulosa (newt; AAV28689, AAV28690, AAU15126, AAU26067), m: Mus musculus (mouse; P42866, P32300, P33534, P35377), r: Rattus norvegicus (rat; P33535, P33533, P34975, P35370), h: Homo sapiens (human; P35372, P41143, P41145, AAH38433). The arrow shows the bifurcation of MOR + DOR sequences from KOR + ORL. Values on plot are mean percent identity (%ID). Asterisk (*) indicates that these two values were significantly different by t-test. Branch length is equal to the proportional difference among the sequences (scale bar = 0.05 or 5% difference in amino acid sequence). Inset box: The molecular evolution of vertebrate opioid receptors. For simplicity, the genes are referred to by the same acronym as the opioid receptor proteins they encode. ‘S’ denotes slow and ‘F’ fast rate of adaptive evolution. See text for further details.