Endogenous heptapeptide Met-enkephalin-Gly-Tyr binds differentially to duplicate delta opioid receptors from zebrafish

Met-enkephalin-Gly-Tyr (MEGY) is an endogenous peptide that binds to opioid sites in zebrafish and in rat brain homogenates. The aim of this work is to characterize the binding profile of this opioid ligand on two duplicate delta receptors from zebrafish, ZFOR1 and ZFOR4. Our results show that, while ZFOR1 presents one single binding site for [3H]-MEGY (KD = 4.0 ± 0.4 nM), the experimental data from ZFOR4 fit better to the two-site binding model (KD1 = 0.8 ± 0.2 nM and KD2 = 30.2 ± 10.2 nM). Two other MEGY synthetic analogues, (D-Ala2)-MEGY and (D-Ala2, Val5)-MEGY were also prepared and tested, together with the original peptide MEGY and other opioid ligands, in competition binding assays. While these peptides presented Ki values on the nanomolar range when using [3H]-MEGY as radioligand, these parameters were two orders higher in competition binding assays with the antagonist [3H]-diprenorphine. Functional [35S]GTPγS stimulation analysis has revealed that these two receptors can be activated by several opioid agonists. Our results prove that although the MEGY peptide acts as an agonist on ZFOR1 and ZFOR4, there are subtle pharmacological differences between these two delta opioid receptors from zebrafish.