Molecular cloning and functional expression of a μ-opioid receptor from rat brain

Opioid drugs act on specific receptors to modulate a wide range of physiological functions. There are at least three types of opioid receptors, μ, δ, and κ. Using a cDNA probe for a mouse δ-opioid receptor in low stringency hybridization, a clone has been isolated from a rat brain cDNA library. This clone contains an open reading frame of 1194 base pairs, with a deduced polypeptide of 398 amino acid residues. The predicted protein exhibits the structural features of guanine nucleotide-binding protein- coupled receptors and displays a high degree of sequence homology with the mouse δ-opioid receptor. When transfected into COS-7 cells, the cDNA conferred a binding site with subnanomolar affinity for [³H]diprenorphine, a high affinity ligand for all three types of opioid receptors. This site also displayed nanomolar affinity for [D-Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAGO), a μ-selective agonist, whereas its affinities for the δ-selective agonist [D-Pen^2,5]-enkephalin and the κ-selective agonist U-50488 were in the micromolar range. Several μ-selective antagonists, including naloxonazine, β-funaltrexamine, and cyprodime, were capable of displacing [³H]diprenorphine binding with nanomolar potency. The pharmacological profile of this binding site thus suggests that it is a μ-type opioid receptor, which we designated MOR-1. In COS-7 cells expressing MOR-1 and stimulated with forskolin, treatment with DAGO decreased the steady state levels of cAMP; this inhibitory effect of DAGO was blocked by naloxonazine. These results suggest that this μ-opioid receptor is functionally coupled to the inhibition of adenylyl cyclase.