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E we show that such peptides (dermorphin and deltorphin), when hugely potent in the human receptors and inactive in frog ORs. The molecular basis for the insensitivity in the frog ORs to these peptides was studied applying chimeras and molecular modeling. Though the molecular mechanism for the insensitivity was not completely resolved as a consequence of its complexity, the delta opioid receptor (DOR) insensitivity to deltorphin was shown to be as a consequence of variation of a single amino acidTrp7.35–which is actually a Leu in mammalian DORs. Notably, Trp7.35 is absolutely conserved in all recognized DOR sequences from lamprey, fish and amphibians. The deltorphin-insensitive phenotype was verified in zebra fish. Our outcomes provide a molecular explanation for the species selectivity of skin-derived opioid peptides and raise an exciting discussion on the potential aspect that the low selectivity of frog ORs may have had within the development of such peptides.EXPERIMENTAL PROCEDURESCell Culture and Transfection Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Corning) supplemented with ten fetal bovine serum, ten U/mL penicillin and 10 g/mL streptomycin (Gibco). The cells had been grown within a humidified incubator in the presence of five CO2 at 37 . Transient transfection (48 h) of wild-type, chimeric, and mutant receptor cDNAs was Fasiglifam Cancer performed in 15-cm tissue culture plates utilizing an optimized calcium phosphate system .Chem Biol. Author manuscript; available in PMC 2016 June 18.Vardy et al.PageConstruction of Chimeric and Mutant ReceptorsAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptHuman opioid receptor cDNAs in the mammalian expression vector pcDNA3.1(+) (Invitrogen) were obtained from the UMR cDNA Resource Center (www.cdna.org). Opioid receptors from Rana pipiens had previously been subcloned in to the exact same vector . Sitedirected mutagenesis was done employing the QuikChange IImutagenesis kit (Agilent) based on the manufacturer’s directions, and all mutations had been confirmed by fulllength automated DNA sequencing (Eton Bioscience, Durham, NC). Generation of inter-species MOR and DOR chimeras was completed working with a two-step overlap PCR method, as previously described , and as diagrammed in Supplementary Fig. 2. The sequences of all primers utilised are offered in Supplementary Table 1. A total of five DOR chimeras (Fig. 1C) and 7 MOR chimeras (Fig. 1B) was made by “humanizing” regions of your frog receptors (i.e., replacing stretches of residues from the frog receptors with their human counterparts). In vitro characterization of OR activation Radioligand Binding Assay–Radioligand binding assays have been performed as previously detailed  utilizing 3H-diprenorphine (Perkin Elmer) for saturation binding assays (10.1 nM), with ten M unlabeled naltrexone (Sigma) to identify nonspecific binding. Functional assay–Inhibition of cAMP production was measured working with the genetically encoded cAMP biosensor, Glosensor-22F (Promega) as previously described . Modeling of DOR-deltorphin interactions Docking of opioid peptides was performed using the hDOR structure recently solved in complex with DIPP-NH2 peptide solved at 2.5 resolution (PDB: 4RWD) . The receptor model was protonated and prepared applying an ICM docking pipeline. Three models had been tested, one with all 23 water molecules retained in the crystal structure, one with out waters, and 1 with only the three water molecules mediating ligand interactions in hDORDIPP-NH2.