Opioid drugs play important roles in the clinical management of pain, as well as in the development and treatment of drug abuse. The mu opioid receptor is the primary site of action for the most commonly used opioids, including morphine, heroin, fentanyl, and methadone. By sequencing DNA from 113 former heroin addicts in methadone maintenance and 39 individuals with no history of drug or alcohol abuse or dependence, we have identified five different single-nucleotide polymorphisms (SNPs) in the coding region of the mu opioid receptor gene. The most prevalent SNP is a nucleotide substitution at position 118 (A118G), predicting an amino acid change at a putative N-glycosylation site. This SNP displays an allelic frequency of approximately 10% in our study population. Significant differences in allele distribution were observed among ethnic groups studied. The variant receptor resulting from the A118G SNP did not show altered binding affinities for most opioid peptides and alkaloids tested. However, the A118G variant receptor binds ␤-endorphin, an endogenous opioid that activates the mu opioid receptor, approximately three times more tightly than the most common allelic form of the receptor. Furthermore, ␤-endorphin is approximately three times more potent at the A118G variant receptor than at the most common allelic form in agonist-induced activation of G protein-coupled potassium channels. These results show that SNPs in the mu opioid receptor gene can alter binding and signal transduction in the resulting receptor and may have implications for normal physiology, therapeutics, and vulnerability to develop or protection from diverse diseases including the addictive diseases.The mu opioid receptor is the primary site of action of several of the endogenous opioid peptides including ␤-endorphin, Met-enkephalin-Arg-Phe, and the recently identified endomorphins (1). This receptor is also the major target for clinically important opioid analgesic agents including morphine, methadone, fentanyl, and related drugs (2, 3). Activation of this receptor has diverse physiological effects (4, 5). Furthermore, it is the major molecular site of action for heroin (6, 7). Rapid activation of the mu opioid receptor, such as that which occurs in the setting of drug abuse, results in a euphoric effect, thus conferring the reinforcing or rewarding effects of the drug, contributing to the development of addiction. Clinical observations have suggested that individuals have varied sensitivity to opioids, suggesting potential variability in the receptor protein and gene. Naturally occurring polymorphisms are well known to exist in human genes; some have been shown to produce profound effects on the function of the corresponding proteins. Molecular cloning of the mu opioid receptor (8-11) has made it possible to determine potential sequence polymorphism, as shown by two recent studies (12, 13). The mu opioid receptor is a member of the G protein-coupled receptor family (8,14). There are a number of well documented cases where natural...

We thank Drs. Lily Jan and Henry Lester for their generous gifts of cDNAs encoding the G-protein-activated K' channel, Dr. Leonard Adam for technical advice and Drs.

A cDNA was isolated from rat brain by low stringency hybridization with the rat p 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, p, K, and S, 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 ['I-Ildiprenorphine, 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 p, K and 6 agonists. Given the similar degrees of high homology to the ,u, K and 6 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.Key work Opioid receptor; Molecular cloning; Tissue distribution; Chromosome mapping IIltroductionExogenous opioid alkaloids and endogenous opioid peptides play a central role in mediating analgesia and many other physiological activities [l]. Opioids achieve their functions by interacting with three different types of opioid receptors, ,u, K and 6 [2]. It has been suggested by pharmacological studies that different subtypes may exist within each of the major types [3]. A member from each of the three opioid receptor classes has been cloned [48]. Sequence comparison of these three receptors reveals highly conserved sequence similarities among them, reflecting the overlapping functions of these receptors [9]. In an effort to isolate other members of the opioid receptor gene family, we used the p receptor cDNA to screen a rat brain cDNA library under low stringency conditions. We report here the isolation of a cDNA clone with a high degree of sequence homology to the other three opioid receptors. Experimental Library screening1.4 kb Hind111 cDNA fragment containing the complete protein coding region of the rat p opioid receptor [4] was used to screen a rat brain cDNA library under low stringency. Hybridization and the final wash were performed at 55'C using previously described conditions [4]. Sequence analysis of 24 isolated clones showed that four identical cDNA clones were similar to the three opioid receptors, ~1, K and 6. One of four isolates was used for subsequent sequence analysis using doublestranded DNA and Sequenase version 2 (IJSB). Hydropathy analyses were performed and putative post-translational modification si...