1 Opioid Receptor

Neuron, Vol. 11, 903-913, November, 1993, Copyright 0 1993 by Cell Press Cloning and Pharmacological Characterization of a Rat ~1Op ioid Receptor Robert C. Thompson, Alfred Mansour, Huda Akil, cortex, and have been shown to bind enkephalin-like and Stanley 1. Watson peptides (Mansour et al., 1988; Wood, 1988). 6 recep- Mental Health Research Institute tors are also thought to modulate several hormonal University of Michigan systems and to mediate analgesia. Ann Arbor, Michigan 48109-0720 The p receptors represent the third member of the opioid receptor family.These receptors bind morphine- like drugs and several endogenous opioid peptides, Summary including P-endorphin (derived from proopiomelanocortin) and several members of both the enkephalin We have isolated a rat cDNA clone that displays 75% and the dynorphin opioid peptide families. These re- amino acid homology with the mouse 6 and rat K opioid ceptors have been localized in many regions of the receptors. The cDNA (designated pRMuR-12) encodes CNS, including the striatum (striatal patches), thalamus, a protein of 398 amino acids comprising, in part, seven nucleus tractus solitarius, and spinal cord (Mansour hydrophobic domains similar to those described for other et al., 1987; McLean et al., 1986; Temple and Zukin, 1987; C protein-linked receptors. Data from binding assays Wood, 1988; Mansour and Watson, 1993). p receptors conducted with COS-1 cells transiently transfected with appear to mediate the opiate phenomena classically a CMV mammalian expression vector containing the full associated with morphine and heroin administration, coding region of pRMuR-12 demonstrated p receptor including analgesia, opiate dependence, cardiovascu- selectivity. In situ hybridization mRNA analysis revealed lar and respiratory depression, and several neuroen- an mRNA distribution in rat brain that corresponds well docrine effects. Pasternak and co-workers (Pasternak to the distribution of binding sites labeled with p-selec- and Snyder, 1975; Wolozin and Pasternak, 1981; Pas- tive ligands. Based upon these observations, we con- ternak and Wood, 1986; Pasternak, 1993) and Rothman clude that pRMuR-12 encodes a p opioid receptor. and co-workers (Rothman et al., 1987) have suggested that p receptors can be subdivided into two classes, Introduction ~1 and ~2. ~1 receptors are thought to have high affinity for both morphine and enkephalins. In contrast, The existence of multiple forms or subtypes of opioid ~2 receptors selectively interact with morphine-like receptors was first suggested by Martin and colleagues compounds and DAMGO ([D-AlaZ,N-MePhe4,Gly-o15]en- (Gilbert and Martin, 1976; Martin et al., 1976), using the kephalin). These studies further suggest that ~1 and chronic spinal dog preparation, and has since been ~2 are functionally distinct: ~1 receptors, unlike ~2 supported by numerous behavioral, pharmacological, receptors, apparently mediate analgesia but not respi- and receptor binding studies (Gillan and Kosterlitz, ratory depression or physical dependence (Pasternak, 1982; Wood, 1982; Goldstein and James, 1984; Herling 1993). and Woods, 1984; James and Goldstein, 1984). Opioid Owing to the importance of opioid receptors in opi- receptors have been divided into at least three main oid pharmacology and physiology, many researchers classes: K, 6, and p. These receptors have unique phar- have attempted to purify these proteins with the aim macological profiles, anatomical distributions, and of ultimately describing the molecular sequence of functions in several species, including man (Wood, these receptors. In one such attempt, Schofield re- 1982; Simon, 1991; Lutz and Pfister, 1992; Mansour and ported the isolation of a protein (opioid-binding cell Watson, 1993). Several pharmacological agents as well adhesion molecule) apparently involved with an opi- as various members of the three endogenous opioid oid receptor (Schofield et al., 1989). This protein has peptide families (prodynorphin, proenkephalin, and been shown to be a member of the immunoglobulin proopiomelanocortin) have been shown to interact superfamily of cell adhesion molecules but lacks trans- with these receptors in distinctive and well-described membrane domains, a feature assumed necessary for manners. K receptors have been shown to exist in signal transduction. Using an expression cloning strat- diverse regions of the CNS; they are particularly en- egy, Xie et al. (1992) detailed the isolation and pharma- riched in the cortex, striatum, and hypothalamus (Man- cological identification of a G protein-coupled pro- sour et al., 1987; Neck et al., 1988; Unterwald et al., tein with opioid receptor binding activity. This protein 1991) and are thought to mediate many of the actions demonstrated several key features of opioid recep- of the dynorphin peptide family in addition to those tors: opioid binding and stereospecificity; however, of benzomorphans. These actions include the modu- the affinity (Ko) for pharmacologically relevant agents lation of drinking, water balance, food intake, gut mo- was low and not selective. The biochemical purifica- tility, temperature control, and various endocrine func- tion of the p receptor proteins has been reported by tions (Morley and Levine, 1983; Leander et al., 1985; several groups (Simon et al., 1975; Bidlack and Abood, lyengar et al., 1986; Manzanares et al., 1990). 6 recep- 1980; Ruegg et al., 1980; Simonds et al., -1980; Cho et tors are also found throughout the CNS, particularly al., 1981, 1986; Howells et al., 1982; Chow and Zukin, in forebrain areas such as the striatum and cerebral 1983; Demoliou-Mason and Barnard, 1984; Maneckjee Neuron 904 et al., 1985; Ueda et al., 1987). Many of these reports hZAPll cDNA librarywas plated and screened with the suggest that this protein is approximately 40-60 kd. radiolabeled DNA insert from p5A-1. Twelve positive Additionally, this receptor can be functionally cou- clones were obtained. Restriction mapping and South- pled to G proteins (Koski and Klee, 1981; Barchfeld ern blot analysis were performed on rescued plasmid and Medzihradsky, 1984; Hsia et al., 1984; Puttfarchen DNA. One clone, designated pRMuR-12, demonstrated et al., 1988; Attali et al., 1989). However, the low abun- a strong hybridization signal on Southern blots and dance of the protein and its apparently high hydro- contained a cDNA insert of approximately2.0 kb. Vari- phobicity have proved to be major stumbling blocks ous restriction enzyme digestion fragment of pRMuR- for the sequencing of sufficiently long peptide frag- 12 were subcloned into Bluescript and completely se- ments necessary for the molecular cloning of this re- quenced in both orientations. DNA sequence analysis ceptor. revealed that pRMuR-12 contained a full open reading Kieffer et al. (1992) and Evans et al. (1992) reported frame with 74% homology with the mouse 8 and rat the isolation and pharmacological characterization of K opioid receptor proteins. the first high affinity opioid receptor, the 6 opioid receptor, using an expression cloning strategy. This DNA and Protein Structure Analysis receptor was shown to contain seven hydrophobic An EcoRl-Hindlll fragment isolated form pRMuR-12 domains characteristic of membrane receptors known was shown to contain the entire protein coding region to transduce extracellular signals (e.g., ligand binding) of this cDNA and was used for all subsequent experi- via G proteins to the intracellular environment. The ments. The complete DNA sequence contained an cloned receptors exhibited high affinity and selectiv- open reading frame encoding a protein of 398 amino ity for 8 opioid ligands, and Evans et al. (1992) showed acids comprising, in part, seven hydrophobic domains. that activation of their receptor with selective 8 ago- GenBank data base searches using both the amino nists could decrease CAMP levels in transfected COS acid and the nucleic acid sequences revealed that the cells. The mouse S receptors were found to be highly pRMuR-12 sequence was novel and highly homolo- homologous to several members of the G protein- gous to the mouse 6 opioid receptor (74% similar and coupled receptor family, particularlythesomatostatin 59.5% identical at the amino acid level) and the so- receptors. The cloning of the mouse 6 receptors facili- matostatin receptors (60.5% similar and 39.5% identi- tated the cloning of other members of the opioid re- cal at the amino acid level). As can be seen in Figure ceptor family. Recently, Yasuda and co-workers de- 1, the regions of greatest homology were found in scribed the isolation of a mouse K receptor cDNA the hydrophobic domains, particularly domains I, II, that had been isolated as a member of the related III, VI, and VII. The regions of greatest divergence somatostatin receptor family (Yasuda et al., 1993). In- included the amino and carboxyl termini of the pro- dependently, our laboratory, using DNA fragments tein, the region surrounding and including hydropho- homologous to the mouse 6 opioid receptor and low bic domain IV, and the region between hydrophobic stringency cDNA library screening, isolated and phar- domains V and VI. In addition to the seven hydropho- macologically characterized a rat K receptor cDNA bic domains classically associated with G protein- (Meng et al., 1993). The rat and mouse K receptors coupled receptors, the open reading frame contains contain seven transmembrane domains and are nega- several other features of this receptor family, includ- tivelycoupled toadenylatecyclase via G proteins. This

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