Anton Mestek

Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family

A cDNA was isolated from rat brain by low stringency hybridization with the rat μ opioid receptor cDNA. Sequence analysis of this clone indicated that it contains an open reading frame capable of encoding a 367 amino acid protein. The deduced amino acid sequence of this protein shows high degrees of homology to all three opioid receptors, μ, κ, and δ, suggesting that it is a member of the opioid receptor gene family. RNA blot analysis detected high level expression of the receptor mRNA in the brain. Southern blot analysis suggests that it is a single‐copy gene, and mapping studies localized the gene on mouse chromosome 2. Despite the high sequence homologies between this protein and the other opioid receptors, expression studies of this clone in COS‐7 cells did not show binding to [3H]diprenorphine, a ligand that binds to the other three opioid receptors. Furthermore, co‐expression of this receptor with a G protein‐activated potassium channel in Xenopus oocytes did not show functional coupling upon stimulation with μ, κ and δ agonists. Given the similar degrees of high homology to the μ, κ and δ opioid receptors and the lack of apparent affinity for their ligands, this receptor does not appear to belong to any of the three known classes of opioid receptors. Rather, it represents a novel member of the opioid receptor gene family, not identified from previous pharmacological studies.

Molecular cloning and functional expression of a mu 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, mu, delta, and kappa. Using a cDNA probe for a mouse delta-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 delta-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 mu-selective agonist, whereas its affinities for the delta-selective agonist [D-Pen2,5]-enkephalin and the kappa-selective agonist U-50488 were in the micromolar range. Several mu-selective antagonists, including naloxonazine, beta-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 mu-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 mu-opioid receptor is functionally coupled to the inhibition of adenylyl cyclase.