Pharmacological characterization of a nociceptin receptor from zebrafish (Danio rerio).
The nociceptin receptor (NOP) and its endogenous ligand, nociceptin/orphanin FQ (OFQ), are involved in a wide range of biological functions, such as pain, anxiety, learning, and memory. The zebrafish has been proposed as a candidate to study the in vivo effects of several drugs of abuse and to discover new pharmacological targets. We report the cloning, expression, and pharmacological characterization of a NOP receptor from zebrafish (drNOP). The full-length cDNA codes a protein of 363 residues, which shows high sequence similarity to other NOPs. Phylogenetic analysis indicates that NOPs are broadly conserved during vertebrate evolution, and that they stand for the most divergent clade of the opioid/OFQ receptor family. Expression studies have revealed that drNOP mRNA is highly expressed in the central nervous system, and low expression levels are also found in peripheral tissues such as gills, muscle, and liver. Pharmacological analysis indicates that drNOP displays specific and saturable binding for [Leucyl-3,4,5-(3)H]nociceptin, with a K(d)=0.20 ± 0.02 nM and a B(max)=1703 ± 81 fmol/mg protein. [(3)H]Nociceptin binding is displaced by several opioid ligands such as dynorphin A (DYN A), naloxone, bremazocine, or the κ-selective antagonist nor-binaltorphimine. [(35)S]GTPγS stimulation studies showed that drNOP receptor is functional, as nociceptin is able to fully activate the receptor and DYN A behaves as a partial agonist (50% stimulation). Our results indicate that drNOP receptor displays mixed characteristics of both NOP and κ opioid receptors. Hence, drNOP, which has retained more of the likely ancestral features, bridges the gap between nociceptin and opiate pharmacology.
Department of Biochemistry and Molecular Biology, University of Salamanca, 37007 Salamanca, Spain.
Angel A
AA
Rivas-Boyero
M Javier
MJ
Herrero-Turrión
Veronica
V
Gonzalez-Nunez
Fátima Macho
FM
Sánchez-Simón
Katherine
K
Barreto-Valer
Raquel E
RE
Rodríguez
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D005880:Gills / Q000737:chemistry; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D023421:Models, Animal; D008969:Molecular Sequence Data; D018847:Opioid Peptides / Q000378:metabolism / Q000494:pharmacology*; D010802:Phylogeny; D011485:Protein Binding / Q000187:drug effects* / Q000502:physiology; D011957:Receptors, Opioid / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017473:Receptors, Opioid, kappa / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism; D000094942:Nociceptin Receptor; D000097629:Nociceptin
D018076:DNA, Complementary; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D018847:Opioid Peptides; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D011994:Recombinant Proteins; D000094942:Nociceptin Receptor; D000097629:Nociceptin