The cloning, expression and partial characterization of a human delta opioid receptor
Committee ChairYamamura, Henry I.
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PublisherThe University of Arizona.
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AbstractA human delta (δ) opioid receptor (hDOR) was cloned from human brain cDNA libraries by filter replica hybridization screening methods. A single clone (44-11) from a human striatal cDNA library was obtained with a 467 bp cDNA probe produced by PCR amplification using NG 108-15 cells cDNA as the template. The 44-11 clone contained a 0.7 kb fragment corresponding to the 3' end of the mouse δ opioid receptor (mDOR) open reading frame (ORF) including the TGA stop codon. The 0.7 kb segment derived from the 44-11 clone by EcoR I and Not I restriction enzyme digestion, was used to screen a human temporal cortical cDNA library. Three positive 1.0 kb clones were obtained from this library. None of the clones contained the full length δ opioid receptor ORF. Sequence analysis showed that two of these clones were identical to each other and also identical to a portion of the 44-11 clone. However, one of these clones 78-4, was shown to contain a 0.64 kb fragment corresponding to the 5' end of the mDOR ORF including the ATG start codon. Since the 44-11 and 78-4 clones shared identical overlapping regions and a unique Hinc II restriction site, a human cDNA containing a full length of the δ opioid receptor ORF was reconstructed by ligation of these two clones at the Hinc II site. The reassembled human cDNA (78x44) has the full length human δ opioid receptor (hDOR) ORF. It contains 1116 nucleotides encoding a protein of 372 amino acids with 93% amino acid identity to the mDOR and the rat δ opioid receptor (rDOR). The 78x44 cDNA was subcloned into the pcDNA3 expression vector and transiently transfected into COS-7 cells. These cells expressed 1.0 pmole of [³H]naltrindole binding/mg protein. Delta opioid receptor selective ligands exhibited high affinity at the receptors while μ and κ selective ligands showed low affinity at the receptors. These results show that the expressed protein in COS-7 cells transfected with 78x44-pcDNA3 is the human δ opioid receptor.
Degree ProgramPharmacology & Toxicology