Identification of dynorphin a from zebrafish: a comparative study with mammalian dynorphin A.
We report the cloning and molecular characterization of the zfPDYN. The complete open reading frame for this propeptide is comprised in two exons that are localized on chromosome 23. zfPDYN cDNA codes for a polypeptide of 252 amino acids that contains the consensus sequences for four opioid peptides: an Ile-enkephalin, the neo-endorphins, dynorphin A and dynorphin B. Upon comparison between zebrafish (zfDYN A) and mammalian dynorphin A (mDYN A) it has been stated that these two peptides only differ in two amino acids: the Leu(5) is replaced by Met(5) and the Lys(13) by Arg(13). Taking into consideration that mDYN A is able to bind to the three mammalian opioid receptors, we have compared the pharmacological profile of zfDYN A and mDYN A on the zebrafish opioid receptors. By means of radioligand binding techniques, we have established that these two dynorphins bind and activate all of the cloned opioid receptors from zebrafish (delta-, mu- and kappa-like), although with different affinities. zfDYN A and mDYN A displace [(3)H]-diprenorphine binding with K(i) values on the nanomolar range, showing greater affinity for zebrafish opioid receptor (ZFOR) 3 (kappa) receptor. ZFOR1 (delta) and ZFOR4 (delta) present higher affinity for zfDYN A than for mDYN A, while the opposing behavior is observed in ZFOR2 (mu). Functional [(35)S]guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) stimulation experiments indicate that these two peptides fully activate the zebrafish opioid receptors, although the mean effective dose (EC(50)) values obtained for ZFOR2 and ZFOR3 receptors are lower than those seen for ZFOR1 and ZFOR4. A comparative study indicates that mammalian and zebrafish opioid receptors might bind their corresponding dynorphin A in a similar fashion, hence suggesting an important role of the opioid system through the vertebrate evolution.
Gonzalez-Nuñez
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Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Salamanca, Avda Alfonso X El Sabio, s/n 37007 Salamanca, Spain.
Marrón Fernández de Velasco
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Arsequell
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Valencia
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Rodríguez
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D000818:Animals; D001667:Binding, Competitive; D003001:Cloning, Molecular / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004399:Dynorphins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000493:pharmacokinetics; D006801:Humans; D011869:Radioligand Assay; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D013460:Sulfur Isotopes; D015027:Zebrafish / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
D013460:Sulfur Isotopes; D029961:Zebrafish Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004399:Dynorphins