The bovine mu-opioid receptor: cloning of cDNA and pharmacological characterization of the receptor expressed in mammalian cells

The cDNA coding for the bovine mu-opioid receptor has been cloned and seque nced. Conserved sequences from murine delta-receptor cDNA were used as prim ers in polymerase chain reaction (PCR) to amplify cDNA, prepared by reverse transcription of bovine brain mRNA. This cDNA was used to probe a bovine b rain library. The partial sequence obtained was extended to provide the ful l length clone by PCR. The cDNA has an open reading frame of 1203 base pair s (bp) with a 3'-untranslated region of 1900 bp and a 5'-untranslated regio n of 265 bp. The protein contains 401 amino acids and has 94% amino acid id entity with the human and 91% with the rat mu-opioid receptor. It has the p utative seven transmembrane domains, characteristic of G protein-coupled re ceptors and contains 5 potential N-linked glycosylation sites near the N-te rminus. Several potential phosphorylation sites and a putative palmitoylati on site are also present. The receptor was stably expressed in HEK293 cells . The binding profile was found to be that of a typical mu receptor, i.e., mu agonists and antagonists, but not delta and kappa ligands, bound with hi gh affinity. Functional assays, namely, opioid stimulation of [S-35]GTP gam ma S binding and inhibition of forskolin-activated adenylyl cyclase, were a lso found to be highly specific for mu-opioid agonists. The receptor was do wnregulated by chronic exposure to mu agonists but not delta or kappa agoni sts. Evidence is presented indicating that the cloned receptor is the same as the bovine mu receptor previously purified to homogeneity in our laborat ory. No evidence was found for genes for multiple mu-type opioid receptors. (C) 1999 Elsevier Science B.V. All rights reserved.