I. Introduction

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Although it was long thought that opioid drugs act on specific receptor sites, opioid receptors themselves were not identified until about 25 years ago (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973). Chemists and pharmacologists suspected the existence of multiple opioid receptors (Portoghese, 1965; Gilbert and Martin, 1976; Martin et al., 1976), and radioligand-binding studies provided evidence to divide opioid receptors into three different types (Goldstein, 1987; Pasternak, 1993). Recent molecular cloning techniques have characterized the nucleotide sequence of at least three distinct opioid receptors, namely, the -, -, and µ-opioid receptors. It has been suggested by the International Union of Pharmacology Subcommittee on Opioid Receptors that the designations -, -, and µ-opioid receptors be replaced by the designations OP₁, OP₂, and OP_3, respectively (Dhawan et al., 1996). The OP₁, OP₂, and OP₃ designations, which are based on the order in which these receptors were cloned (Dhawan et al., 1996), have proved to be quite controversial within the research community and will be reconsidered by International Union of Pharmacology in the near future. For this reason, we will use the established -, -, and µ-opioid receptor nomenclature in this review.

The cloned -, -, and µ-opioid receptors are highly homologous, and all three interact with heterotrimeric G proteins (Gilman, 1987; Childers, 1991). The G protein-coupled receptor superfamily, which includes numerous neurotransmitter and hormonal receptors, possesses a common three-dimensional structure that spans the cell membrane seven times, forming three extracellular loops and three intracellular loops. The amino terminus is extracellular, whereas the carboxyl terminus is intracellular (Strosberg, 1991). Studies conducted on the cloned opioid receptors demonstrate that the amino acid sequence of the -, -, and µ-opioid receptors are 65% homologous; hence, it is the other 35% that confer type selectivity (Reisine and Bell, 1993). The domains with the greatest similarity are the transmembrane regions and the intracellular loops, whereas the most divergent regions are the extracellular loops and the amino- and carboxyl-terminals (Fig. 1). Based on results of pharmacological investigations, -, -, and µ-opioid receptors have been further subdivided into receptor subtypes (Satoh and Minami, 1995); however, the molecular basis for subtypes remains to be resolved.

View larger version (66K): [in this window] [in a new window]   Fig. 1.   Human -, -, and µ-opioid receptor amino acid comparison. The TMs are underlined and numbered (modified from Knapp et al., 1995b).

The existence of a fourth opioid receptor, the -opioid receptor, has long been suspected and was initially postulated to explain -endorphin-mediated inhibition of the electrically induced contraction of the rat vas deferens (Wüster et al., 1979; Schulz et al., 1981). These findings were consistent with the presence of a -endorphin-binding receptor in the rat vas deferens that was independent of the - and µ-opioid receptors. Evidence also suggested that the -endorphin-binding site in the rat vas deferens is not a -opioid receptor because a series of benzomorphan compounds, thought to be -selective agonists, were competitive antagonists in the rat vas deferens (Gillan et al., 1981). -Endorphin activity in the rat vas deferens was antagonized by naloxone (Huidobro-Toro et al., 1982), which is consistent with the identification of the -endorphin receptor as an opioid receptor.

-Endorphin-binding sites were also observed in brain tissue (Law et al., 1979; Johnson et al., 1982). These studies suggested the possibility that a portion of the -endorphin binding in the brain was distinct from enkephalin and morphine-binding sites. Further evidence for brain -opioid receptors came from competition-binding studies in rat brain membranes with the universal opioid antagonist [³H]diprenorphine (Chang et al., 1981a). Twenty-seven percent of specific [³H]diprenorphine binding was not competitively excluded from receptors in the presence of sufficient [D-Ala²,D-Leu⁵]enkephalin (DADLE)³ and morphiceptin to block both - and µ-opioid receptors. Conversely, benzomorphan drugs such as cyclazocine were able to totally exclude [³H]diprenorphine binding. These authors named the non-, non-µ-opioid receptor that bound [³H]diprenorphine as benzomorphan-binding sites. -Endorphin also inhibited [³H]diprenorphine binding at the benzomorphan site in rat brain membranes with a K_i value of 10 nM (Chang et al., 1984). In the same study, the potencies of -endorphin, -endorphin fragments, etorphine, DADLE, and Tyr-D-Ala-Gly-NMe-Phe-Met(O)ol in the contraction of the rat vas deferens correlated with the affinities of these agonists at the benzomorphan-binding site in the rat brain.

In contrast to agonists active at other opioid receptors, -endorphin-stimulated antinociception is not directly mediated through pertussis toxin-sensitive G proteins (Tseng and Collins, 1995, 1996). Data also indicate that -opioid receptors are involved in some -opioid receptor-mediated antinociceptive pathways (Suh and Tseng, 1990). Hitherto, the greatest impediment to characterization of -opioid receptor function has been the dearth of selective pharmacological tools. However, a cDNA that may encode the -opioid receptor was cloned from a human genomic library (O'Dowd et al., 1995). If this proves true, expression of this clone in cell lines should allow further characterization of the -opioid receptor. The -opioid receptor was recently reviewed (Narita and Tseng, 1998).

Yet another opioid receptor-like species has been cloned, namely, the opioid receptor-like protein₁ (ORL₁) receptor (Bunzow et al., 1994; Chen et al., 1994; Fukuda et al., 1994; Mollereau et al., 1994; Nishi et al., 1994; Wang et al., 1994a; Wick et al., 1994; Halford et al., 1995; Lachowicz et al., 1995). Like the -, -, and µ-opioid receptors, with which it shares 50 to 60% sequence homology, the cloned ORL₁ receptor is a seven-transmembrane domain (TM)-receptor coupled to G proteins. This naturally occurring receptor is widely distributed in the brain and is responsive to the novel peptide orphanin FQ (Meunier et al., 1995; Reinscheid et al., 1995; also known as nociceptin, Rossi et al., 1997). The cloned receptor mediates the inhibition of forskolin-stimulated cAMP production in a naloxone-insensitive manner (Reinscheid et al., 1995). In contrast to the effects of classic opioid receptors, the ORL₁ receptor appears to mediate hyperalgesia (Meunier et al., 1995; Reinscheid et al., 1995), and this hyperalgesia is insensitive to the opioid antagonist diprenorphine (Rossi et al., 1997). Further investigation has demonstrated that the ORL₁ receptor can also mediate analgesia, although the kinetics of analgesia production differ from those of hyperalgesia and the analgesia is sensitive to the action of opioid antagonists (Rossi et al., 1996, 1997). The ORL₁ receptor is thought to be encoded by the same gene that codes the ₃-opioid receptor; however, antisense knockdown experiments suggest that these receptors are splice variants with differing signaling characteristics (Pasternak and Standifer, 1995; Rossi et al., 1997). For a more extensive review, see Meunier (1997).

Compared with the opioid or opioid-like receptors discussed above, the -opioid receptor is an attractive target for the development of new drugs to control pain. The  opioid receptors have previously been shown to mediate dysphoria (Pfeiffer et al., 1986), ORL₁ receptors mediate hyperalgesia in addition to analgesia (Rossi et al., 1997), and -opioid receptors are still poorly characterized. The -opioid receptor-selective drugs may possess potential clinical benefits compared with the µ-opioid receptor drugs that are currently in use for the relief of pain. These advantages include greater relief of neuropathic pain (Dickenson, 1997), reduced respiratory depression (Cheng et al., 1993), and constipation (Sheldon et al., 1990), as well as a minimal potential for the development of physical dependence (Cowan et al., 1988).

Reflecting the medical importance of opioid receptors, a number of reviews examining the molecular biology of these receptors have appeared (Reisine and Bell, 1993; Reisine et al., 1994; Kieffer, 1995; Knapp et al., 1995b; Minami and Satoh, 1995; Reisine, 1995; Satoh and Minami, 1995; Dhawan et al., 1996; Raynor et al., 1996; Zaki et al., 1996). The present review, while necessarily covering some of the same ground, will endeavor to 1) emphasize the molecular pharmacology of the -opioid receptor and 2) describe the pharmacodynamics of selected agonists that bind to the -opioid receptor. To improve the selectivity of -opioid agonists, there needs to be a corresponding increase in our ability to describe drug activity. One such description is efficacy, which is a measure of the ability of an agonist-bound receptor to stimulate a measurable response in a cell or tissue. This review will suggest that efficacy values are a more meaningful measure of drug activity than the traditional dissociation constants and drug potencies commonly used to describe drug activity.

	II. Role of -Opioid Receptors in Antinociception

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Early studies suggested a prominent role for µ-opioid receptors in opioid drug-mediated analgesia. Morphine, the classic opioid agonist, was recognized as the prototypical µ agonist. The affinity of morphine for the µ-opioid receptor is approximately 50 times higher than that for the -opioid receptor (Emmerson et al., 1994). In initial experiments, opioid drugs capable of eliciting antinociception in vivo were also potent in suppressing electrically stimulated contractions of the guinea pig ileum; however, these drugs did not inhibit electrically evoked contractions in the isolated mouse vas deferens. Because µ-opioid receptors were found to be highly expressed in the guinea pig ileum and -opioid receptors were identified in the mouse vas deferens, it was originally thought that µ receptors were more involved than  receptors in the mediation of analgesia (Heyman et al., 1988). However, with the discovery of compounds with increased selectivity for -opioid receptors, it quickly became clear that these receptors also mediate analgesia. For example, the enkephalin analog Met-kephamide exhibited greater potency than morphine in evoking antinociception and greater in vitro selectivity for the -opioid receptor (Frederickson et al., 1981; Burkhardt et al., 1982). The introduction of cyclic [D-Pen²,D-Pen⁵]enkephalin (where Pen = penicillamine; DPDPE; Mosberg et al., 1983a,b) was also significant because it produced antinociception without the usual gastrointestinal effects or Straub tail phenomenon characteristic of µ-selective agonists (Galligan et al., 1984; Porreca et al., 1984). The additional demonstration that acutely morphine-tolerant mice were not cross-tolerant to the  agonists [D-Ser²,Leu⁵,Thr⁶]enkephalin (DSLET) and [D-Thr²,Leu⁵,Thr⁶]enkephalin (DTLET) was further evidence that -opioid receptors were indeed capable of mediating antinociception (Porreca et al., 1987).

Supporting evidence for -opioid receptor-mediated antinociception was provided by the introduction of pharmacological antagonists with relative selectivity for -opioid receptors, namely, ICI-154,129 [N,N-bisallyl-Tyr-Gly-Gly--(CH₂S)-Phe-Leu-OH; Priestley et al., 1985] and ICI-174,864, [N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, where Aib = -aminoisobutyric acid; Cotton et al., 1984]. Pretreatment with ICI-174,864 antagonized the effects of DPDPE but not morphine or [D-Ala²,MePhe⁴,Gly(ol)⁵]enkephalin (DAMGO) in the mouse tail-flick test, and conversely, the µ receptor blocker -funaltrexamine antagonized the effects of morphine and DAMGO but not DPDPE (Heyman et al., 1987). Additional support for  receptor-mediated antinociception came from experiments using µ-opioid receptor-deficient CXBK mice. These mice show a 10-fold rightward shift in the potency for morphine-induced antinociception and reduced antinociceptive responsiveness to morphine or DAMGO but unaltered responsiveness to DPDPE compared with control mice (Vaught et al., 1988). The CXBK strain was previously demonstrated to have approximately 30% fewer µ₁-opioid-binding sites than C57BL/6BY progenitors (Moskowitz and Goodman, 1985). Recent antinociception studies in recombinant mice, in which expression of the µ-opioid receptor was disrupted, demonstrate that -opioid receptor-selective agonists do not require functional µ-opioid receptors to mediate antinociception (Matthes et al., 1998).

	III. The -Opioid Receptor

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The -opioid receptor was first suggested by the interpretation of studies comparing the effects of morphine and the then newly discovered enkephalins on electrically induced contractions of the guinea pig ileum and mouse vas deferens. The greater potency of morphine in the former bioassay and of enkephalins in the latter suggested that morphine and the enkephalins might act on different populations of opioid receptors (Hughes et al., 1975). The opioid receptor in the mouse vas deferens was assigned the designation "-opioid receptor". Thus, recognition of the -opioid receptor evolved due to differential drug effects in isolated tissues in vitro (Lord et al., 1977), whereas - and µ-opioid receptors were proposed based on differential analgesic drug effects in vivo (Gilbert and Martin, 1976; Martin et al., 1976). Subsequent receptor autoradiographic investigations clearly demonstrated differences in the distribution of opioid receptors within the brain. The pattern of  receptors was distinctly different from that of µ receptors, and the loci of both  and µ receptors were unique from that of the  receptors as well (Sharif and Hughes, 1989; Mansour et al., 1995). As seen for all opioid receptors, the density of -opioid receptors varied widely in different brain regions (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Distribution of the -opioid receptor in the rat brain

In addition to these anatomical differences in receptor localization, the development of , , and µ receptor-selective drugs has provided evidence of a pharmacological difference among opioid receptors. Met-enkephalin and Leu-enkephalin were initially proposed and are still considered the endogenous ligands of the -opioid receptor (Hughes et al., 1975). The introduction of drugs with progressively greater selectivity, such as DADLE (Beddell et al., 1977; Belluzzi et al., 1978), Tyr-D-Ser[-O-C(CH₃)₃]-Gly-Phe-Leu-Thr-O-C(CH₃)₃ (BUBU; Gacel et al., 1988), DPDPE (Mosberg et al., 1983a,b), the deltorphins (Erspamer et al., 1989; Kreil et al., 1989), (±)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-N,N-diethylbenzamide (BW373U86; Chang et al., 1993) and (+)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80; Calderon et al., 1994), further demonstrated that -opioid receptors were capable of mediating antinociception. Recognition of the pharmacological differences among opioid receptors was also supported by drug antagonism studies. It was initially reported that the dose of naloxone required for blocking the  receptor was 10 times greater than that needed to block the µ-opioid receptor (Lord et al., 1977). This finding led to the development of progressively more selective -opioid receptor antagonists, such as naltriben (Portoghese et al., 1988), naltrindole (Takemori and Portoghese, 1992), Tyr-Tic-Phe-Phe-OH (where Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; TIPP; Schiller et al., 1992), and -methyl-2',6'-dimethyltyrosine-L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Liao et al., 1997).

There is pharmacological evidence of distinct subtypes of the -opioid receptor. Initially, inconsistencies in radioligand-binding studies suggested multiple subtypes of  receptors (Vaughn et al., 1990; Negri et al., 1991). Although an alternative explanation was the existence of a single  receptor with multiple affinity states, more definitive evidence arrived with the introduction of -opioid receptor agonists and antagonists with improved subtype selectivity (Portoghese et al., 1992). The antagonist naltriben shifts the potency of DSLET 4-fold but that of DPDPE by only 1.5 times in the tail-flick assay (Sofuoglu et al., 1991). In addition, there is no development of cross-tolerance between DSLET and DPDPE or between DPDPE and [D-Ala²]deltorphin II (Del-II; Mattia et al., 1991). More recently, quantitative autoradiographic studies revealed distinctive patterns of [³H]DPDPE and [³H]DSLET binding in rat brain, in some cases, by as much as a 9:1 ratio of ₂:₁ opioid receptors (Hiller et al., 1996). It is now speculated that the putative ₁ receptor is stimulated by DPDPE and blocked by [Ala²,Leu⁵,Cys⁶]enkephalin, whereas the putative ₂ receptor is stimulated by DSLET and Del-II and blocked by naltrindole-5'-isothiocyanate (Jiang et al., 1991; Vanderah et al., 1994). However, no  subtypes have been cloned, and there remains no definitive molecular evidence for distinct subtypes of the -opioid receptor. We have also observed that DPDPE-mediated analgesia is partly dependent on µ receptors using µ-opioid receptor knockout mice (unpublished results), suggesting the drugs used to establish -opioid receptor subtypes may not be sufficiently selective for this purpose.

One major impediment to the characterization of opioid receptors is the fact that there are multiple opioid receptors, and tissues generally possess more than one type of receptor. This obstacle highly complicates the study of the individual types of receptor but, in recent years, has been addressed by the cloning of the first three opioid receptor types and the expression of each in separate cell lines (Miotto et al., 1995; Table 2).

The first opioid receptor to be cloned was the -opioid receptor. Two groups independently cloned the mouse  receptor by preparing an expression library from mouse neuroblastoma x rat glioma hybrid cells of the NG108-15 cell line and transfecting the library into monkey fibroblast (COS) cells (Evans et al., 1992; Kieffer et al., 1992, 1994). The use of the NG108-15 cell line was a critical step because these cells express -opioid receptors at a greater density than is normally found in brain tissue (Knapp et al., 1995b) and in the absence of other opioid receptors (Kieffer et al., 1992). Both groups used radioligand-binding assays to detect  receptors but used different expression screening procedures. A cDNA sequence encoding a 372-amino acid protein was identified. A later isolation of a mouse -opioid receptor clone from a brain cDNA library (Yasuda et al., 1993) confirmed the sequence identified by these investigators. The rat  receptor was cloned by Fukuda et al. (1993) from a rat cerebellum cDNA library by a hybridization screening method using a mouse -opioid receptor DNA as a probe. The rat receptor also had 372 amino acids with 97% homology to the mouse  receptor. The 3% difference lies in the amino acids of the NH₂- and COOH-terminal sequences and one residue in the second extracellular loop.

The next logical step was to clone the human -opioid receptor because the human receptor is the ultimate target of therapeutic opioid agents. Our laboratory cloned the cDNA for a human  receptor using hybridization screening methods (Knapp et al., 1994). cDNA fragments obtained from human striatum and temporal cortex libraries showed a highly homologous nucleotide sequence to the mouse -opioid receptor, but neither fragment covered the full open reading frame of the receptor protein. Consequently, the sequence fragments were combined by ligation in their overlapping regions. The reassembled open reading frame encoded a 372-residue protein with 93% homology with both the mouse and rat -opioid receptors. Most of the differences in amino acid sequence were in the NH₂- and COOH-terminals, but there were three additional substitutions elsewhere: Met⁸⁰ for Leu in the first cytoplasmic loop, Arg¹⁹⁰ for Gln in the second extracellular loop, and Asp²⁹⁰ for Asn in the third extracellular loop. There were no amino acid differences in any of the TMs. The human -opioid receptor was also cloned concurrently by Simonin et al. (1994) from the SH-SY5Y human neuroblastoma cell line. Regardless of species of origin, these cloned receptors uniformly exhibited greater affinity for Met-enkephalin, -selective agonists (DPDPE, DSLET) and antagonists (naltrindole), than they did for - and µ-selective ligands (Evans et al., 1992; Yasuda et al., 1993).

The use of clonal cells that stably express a recombinant receptor provides a unique system for the study of opioid receptors with several advantages over whole-animal or isolated tissue models (Kenakin, 1996; Mak et al., 1996). First, clonal cells afford a convenient way of inducing a very high level of receptor expression, even to greater levels than might be found naturally. Receptor overexpression in cell systems permits a sufficient density of receptors for clonal characterization (Samama et al., 1993) but may be prone to anomalies in the signaling mechanisms due to supraphysiological receptor densities. These problems can be addressed because recombinant receptors can be stably expressed in a clonal cell line at various densities, including levels comparable to those naturally occurring in tissues. The second advantage of receptor-transfected cells versus tissue is that clonal cells are all identical because they are derived from the same original progenitor cells. Hence, experiments using these cells eliminate experimental variability caused by obtaining tissue samples from different animals or individuals. Finally, clonal cell lines have the added advantage of being transfected to selectively express a given receptor without other receptor types or subtypes that normally coexist in a tissue sample. This eliminates the likelihood of misinterpretation due to the presence of confounding receptors.

	IV. Molecular Biology of -Opioid Receptors

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Gene knockdown is accomplished using short sequences of oligodeoxynucleotide, generally 15 to 25 nucleotides in length, that are complementary to a portion of the mRNA that codes for a particular gene product. Antisense oligodeoxynucleotides (AS oligos) are potentially valuable pharmacological tools, especially in situations where there are no selective antagonists available, and they have been used to inhibit the expression of a specific cannabinoid receptor protein in vivo (Edsall et al., 1996), thus accomplishing essentially the same end as receptor blockade (Albert and Morris, 1994; Weiss et al., 1997).

The pretreatment of experimental animals with AS oligos to the -opioid receptor resulted in reduced antinociceptive response to - but not - or µ-selective receptor agonists (Bilsky et al., 1994; Lai et al., 1994, 1995; Standifer et al., 1994; Tseng et al., 1994). In all of these studies, comparable pretreatment with either sense or mismatch oligodeoxynucleotides was without effect on -opioid receptor-mediated antinociception. In rapid order, selective attenuation of  and µ receptor-mediated antinociception was reported in animals after pretreatment with AS oligos complementary to  (Adams et al., 1994; Chien et al., 1994)- and µ (Rossi et al., 1994; Chen et al., 1995a)-opioid receptor mRNAs, respectively. AS oligos complementary to opioid receptor mRNAs have also been successfully used to implicate , , and µ receptors in the development of opioid tolerance and dependence (Kest et al., 1996), -endorphin-induced antinociception (Tseng and Collins, 1994), and opioid-induced changes in locomotor activity (Mizoguchi et al., 1996) and body temperature (Chen et al., 1995b). Reduced binding of -selective radioligands in cultured NG108-15 cells confirmed the inhibition of -opioid receptor expression by treatment with AS oligos (Standifer et al., 1994).

AS oligo knockdown of opioid receptor expression is reversible. Studies using AS oligos have followed various pretreatment regimens, generally involving multiple injections on a daily basis or sometimes on an alternate-day schedule over 5 days (Table 3). Such pretreatment plans imply the importance of an appropriate time sequence to permit simultaneous degradation of existing receptors and inhibition of the synthesis of new receptors. There is a gradual restoration of sensitivity to -selective agonist-mediated antinociception 5 days after the final AS oligo treatment (Standifer et al., 1994). This is consistent with estimates of 3- to 5-day turnover times for opioid receptors (Ward et al., 1982).

A transgenic µ-opioid receptor knockout mouse has been generated by homologous recombination technology and used to study interactions between - and µ-opioid receptors in the central nervous system (Sora et al., 1997a,b). Although the heterozygous knockout mice exhibit about 54% of wild-type levels of µ receptor expression, the homozygous knockout mice displayed 0% receptor expression. Sora et al. (1997a,b) used hot-plate and tail-flick tests and found that DPDPE induced a weaker than expected antinociceptive effect in µ-knockout mice compared with control animals. The implication of this finding is that the antinociceptive effect of DPDPE, a classic -selective receptor agonist, appears to be dependent on intact µ receptors. On the other hand, G protein activation by Del-II and SNC80 is unimpaired in membranes prepared from the brains of µ-opioid receptor knockout mice, and Del-II-mediated antinociception is not significantly different from that in control mice (unpublished data). These data indicate that the  receptor is functional and mediates antinociception in the absence of the µ-opioid receptor. These findings are similar to those reported by Matthes et al. (1998).

1. Identification of Ligand-Binding Domains. Our current understanding of the regions of the  receptor involved in ligand binding developed from the idea that opioid ligands are bivalent molecules. According to this theory, one portion of the ligand mediates signal transduction while another ligand site determines selectivity toward -, -, or µ-opioid receptors. These regions are referred to as the message and address regions, respectively. The use of this theory to develop - and -selective antagonists has been reviewed previously (Portoghese, 1989; Takemori and Portoghese, 1992). The cloning of the three types of opioid receptors has allowed researchers to identify sites in the -opioid receptor involved in ligand selectivity and binding. The receptor amino acid sequences showed that -, -, or µ-opioid receptors demonstrated extensive sequence homology in the seven TMs and divergent sequence in the intracellular tail and extracellular portions of the receptor. Because many opioid drugs exhibit limited selectivity among opioid receptor types, these findings suggest that the highly homologous TMs form a drug-binding pocket that interacts with the message region of the ligand.

a. THE THIRD EXTRACELLULAR LOOP OF THE -OPIOID RECEPTOR IS CRITICAL TO LIGAND BINDING. Ligand selectivity for  receptors is thought to depend on recognition sites spanning the fifth through seventh TMs. This conclusion was based on findings from binding studies conducted on chimeric receptors constructed from cloned rat - and -opioid receptors (Meng et al., 1995

b. FIRST EXTRACELLULAR LOOP. Other studies have examined the role of the first extracellular loop as a determinant of DAMGO binding to opioid receptors. This was accomplished by replacing the first extracellular loop of the cloned rat -opioid receptor for the same region in the cloned rat µ-opioid receptor and construction of a chimeric /µ/ receptor. This substitution conferred high affinity for [³H]DAMGO to the chimeric receptor (Onogi et al., 1995

c. SECOND EXTRACELLULAR LOOP. Studies using chimeric receptors constructed from cloned rat opioid receptors showed that substitution of the second extracellular loop of the -opioid receptor for that of either the  or µ receptor was insufficient to confer selective binding of the -selective ligands Met-enkephalin, Leu-enkephalin, DPDPE, JOM13, Del-II, or TIPP (Meng et al., 1996

d. TRANSMEMBRANE DOMAINS. The role of residues in the TMs of the -opioid receptor on ligand binding is under active investigation. Asp¹²⁸, a residue in the third TM, was postulated to be involved in ligand binding. A conserved Asp residue in the third TM has previously been shown to affect ligand binding to other G protein-coupled receptors (Befort et al., 1996a

e. N TERMINUS DOMAIN. A subsequent study revealed that both DPDPE and naltrindole bind to the N terminus chimeric (1-78)/(70-372) receptor but not to the reverse chimeric (1-69)/(79-380) receptor; this finding suggests that the N-terminal domain of the -opioid receptor is not critical for binding of -selective ligands (Kong et al., 1994

f. SUMMARY. Findings reviewed above are consistent with the interpretation that the third extracellular loop of the -opioid receptor is a critical region determining the selectivity of  receptor ligands. Data also support a role for the TMs of the -opioid receptor in ligand binding. In contrast, the N-terminal domain and the first and second extracellular loops do not appear to modulate the binding of -selective ligands to the -opioid receptor.

2. -Opioid Receptor Domains Mediating Down-Regulation. Down-regulation of the mouse -opioid receptor was examined with truncation mutants (Cvejic et al., 1996). When the terminal 37 amino acids in the intracellular tail of the receptor were deleted, receptor down-regulation in response to chronic (2-48 h) DADLE treatment was blocked in receptor-transfected Chinese hamster ovary (CHO) cells. Conversely, when the murine -opioid receptor was truncated by 15 amino acids, the receptor did down-regulate on chronic DADLE treatment; however, the receptor levels of the 15-amino acid truncation-mutant were not down-regulated to the same extent as the wild-type. Still, these findings indicated that there are amino acid residues in the cytoplasmic tail of the murine -opioid receptor that regulate receptor down-regulation. When the cytoplasmic tail residue Thr³⁵³ was mutated to an Ala in the mouse  receptor and the mutant receptor expressed in CHO cells, down-regulation was blocked. Although Cvejic et al. (1996) demonstrated that Thr³⁵³ of the mouse -opioid receptor mediates down-regulation, the mechanism of regulation in the human receptor must be different because Thr³⁵³ is already an Ala in the human  receptor sequence (Knapp et al., 1994) and the human receptor down-regulates on chronic agonist exposure (Malatynska et al., 1996).

3. -Opioid Receptor Domains Mediating Signal Transduction Cascades. Studies examining the regions of the -opioid receptor that modulate signal transduction pathways are extremely limited. A role for the carboxyl regions of the cytoplasmic tail in intracellular signaling was precluded by studies in which a 31-amino acid truncation of the tail did not affect DPDPE-mediated inhibition of forskolin-stimulated cAMP production in receptor-transfected CHO cells (Zhu et al., 1997). Merkouris et al. (1996) addressed the question of which -opioid receptor regions mediate interactions with G proteins through the use of synthetic peptides. These investigators examined G protein activation in cell membrane preparations as GTPase activity and [³⁵S]GTPS binding in the presence of peptides (100 mM) homologous to regions of the -opioid receptor. They found that peptides homologous to the third intracellular loop inhibited both GTPase activity and [³⁵S]GTPS binding. Peptides homologous to the second intracellular loop and amino acids 322 through 333 of the cytoplasmic tail did not affect either assay; however, the peptide with homology to the tail slightly enhanced the inhibition of [³⁵S]GTPS binding mediated by one of the third intracellular loop peptides. Because receptor interactions with G proteins are known to modulate the affinity of agonist binding to G protein-coupled receptors, these investigators also examined the effect of the peptides on binding of the -selective agonist [³H]DSLET. Peptides with homologous sequence to the third intracellular loop reduced [³H]DSLET binding, whereas peptides homologous to the second intracellular loop did not. Unexpectedly, the peptide homologous to residues 322 through 333 of the cytoplasmic tail also reduced [³H]DSLET binding. These findings suggest that the cytoplasmic tail may interact with receptor-associated G proteins yet are not vital to signal transduction because a peptide consisting of residues 322 through 333 failed to block either GTPase activity or [³⁵S]GTPS binding.

	V. Opioid Signal Transduction

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Since the initial pharmacological identification of the -opioid receptor, considerable effort has been directed toward understanding the signal transduction pathways that couple this receptor to analgesia and other functional responses. It is well established that most -opioid receptor-mediated events are dependent on the activity of pertussis toxin-sensitive G proteins. It is also well established that  receptor-selective ligands inhibit intracellular cAMP levels and modulate the activity of voltage-gated calcium and potassium channels. More recent studies have addressed -selective ligand-mediated calcium release from intracellular stores and modulation of a variety of protein kinases. In the sections to follow,  receptor-selective ligand-mediated effects on second messenger systems is examined followed by a discussion of the significance of these findings to the physiological role of these drugs.

The role of G proteins in opioid receptor-mediated signaling has been reviewed previously (Childers, 1991; Standifer and Pasternak, 1997). Our present knowledge about the superfamily of seven-helical domain G protein-coupled receptors is based on the work of Lefkowitz and associates on cloned -adrenergic receptors (Ostrowski et al., 1992). Early evidence supporting opioid recepto