A Sincere Thanks

Top Abstract A Sincere Thanks Introduction Differences in µ- and... Differential Regulation of µ-... Differential Regulation of µ-... Regulation of µ- and... Regulation of Opioid Receptor... Regulation of Opioid Receptor... Conclusion References

First of all, I feel deeply honored to receive the Otto Krayer Award and I thank ASPET, the award selection committee, and Zeneca Pharmaceuticals for the privilege of delivering this lecture. I also want to take this opportunity to thank my mentor, Professor Edward Leong Way, for getting me "hooked on morphine" for the past three decades. The enduring total support of my wife and children is an absolute essential component for our work. I also want to thank my long-term collaborator Professor Ping-Yee Law for the many contributions he has made in our laboratory. He was the chief architect for many parts of the work I will present in this talk. Of course, we must not forget that any scientific effort requires the contribution of many individuals and I am very grateful to the many outstanding and dedicated students, postdoctoral fellows, and the entire research staff with whom I was so lucky to have the privilege to work.

During the past three decades our laboratory has focused solely on the pharmacology of opioids, with specific emphasis on the neurochemical mechanism of opioid tolerance. For the presentation today, I'll concentrate on a very limited aspect of our recent work on the regulation of opioid receptor activities.

	Introduction

Top Abstract A Sincere Thanks Introduction Differences in µ- and... Differential Regulation of µ-... Differential Regulation of µ-... Regulation of µ- and... Regulation of Opioid Receptor... Regulation of Opioid Receptor... Conclusion References

The independent reports on the identification of opioid receptors in 1973 by S. Snyder, E. Simon, L. Terenius, and their coworkers (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973) marked the advent of the opioid receptor field. Using radioactive ligand binding techniques, these groups demonstrated the presence of stereoselective binding sites for opioid ligands at the synaptic plasma membrane, with ligand affinities for the binding sites paralleling their pharmacological potencies (Creese and Snyder, 1975). Brain regional localization studies using autoradiography or membranes prepared from individual brain areas indicated that opioid binding is localized to sites known to be associated with the various pharmacological properties of these drugs. Due to the parallels in receptor distribution and the sites of drug actions, it was hypothesized that the change in receptor densities and activity could be responsible for the development of tolerance during chronic exposure to morphine. However, early reports on this subject were equivocal (Klee and Streaty, 1974; Hollt et al., 1975; Rothman et al., 1986; Werling et al., 1989; Brady et al., 1989; Bhargava and Gulati, 1990). There were no data pertaining to receptor activity following chronic drug administration in these early studies.

The presence of a stereoselective binding site for plant alkaloids in neural tissues suggested that endogenous ligands must exist in animal tissues. The family of opioid peptides was established with the first isolation of enkephalin by John Hughes and his coworkers in 1975 (Hughes et al., 1975), the identification of -endorphin by C. H. Li and his colleagues (Li and Chung, 1976), and the subsequent isolation of dynorphin by A. Goldstein and his associates (Goldstein et al., 1979). The dissimilarities among the opioid peptides for the opioid binding sites supported the existence of multiple opioid receptors, as proposed by Martin et al. (1976). Indeed, µ-, -, and -opioid receptors were ultimately characterized using selective radioactive ligands (Chang and Cuatrecasas, 1979a; Chang et al., 1979b), selective ligand alkylation protection studies (Robson and Kosterlitz, 1979; James et al., 1982), and cross-tolerance studies (Schultz et al., 1980; Porreca et al., 1982). These different opioid receptors have distinct brain regional distributions and dissimilar pharmacological properties, with the most dramatic differences being between µ- and -opioid receptors. For example, morphine, a µ-opioid agonist, increases vasopressin release and thereby inhibits diuresis, whereas a -opioid agonist, such as U50,488, decreases vasopressin release, increasing diuresis (Lelander, 1983). Thus, the differential regulation of opioid receptor activities subsequently affects the pharmacological action of a given opioid agonist.

Studies on the cellular regulation of opioid receptor activities are facilitated by the availability of clonal cell lines that express a homogeneous population of -opioid receptors or a mixed population of µ- and -opioid sites. In the early 1980s our laboratory and others demonstrated that chronic exposure of these clonal cell lines, e.g., neuroblastoma × glioma NG108-15 hybrid cells or neuroblastoma N4TG2 cells, to -opioid agonists revealed different types of adaptation processes (Law et al., 1982, 1983b; Chang et al., 1982). These included a decrease in the ability of agonist to regulate adenylyl cyclase activity (receptor desensitization), a decrease in the overall receptor density (receptor down-regulation), and an increase in adenylyl cyclase activity after removal of the agonist (up-regulation of the adenylyl cyclase). The down-regulation of the receptor was found to involve its internalization from the plasma membrane to the lyzosomal compartments in these clonal cells (Law et al., 1983a). Although we could subsequently demonstrate agonist-dependent down-regulation of opioid receptors in the rodent brain (Tao et al., 1987, 1988, 1990), regulation of receptor levels during the chronic administration of an opioid could not account for tolerance to the drug. Thus, the actual molecular events involved in regulating opioid receptor activities could not be elucidated, in part because of the unavailability of receptor reagents that could monitor the effects of covalent modifications of receptor protein or alterations in gene transcription.

These hurdles were overcome by the cloning of -opioid receptors from NG108-15 cells by Evans et al. (1992) and Kieffer et al. (1992). Subsequent cloning of µ- and -opioid receptors was accomplished by others based on the reported sequence of the -opioid receptor (Chen et al., 1993b; Fukuda et al., 1993; Li et al., 1993; Meng et al., 1993; Yasuda et al., 1993). Sequence analysis of these cloned opioid receptors demonstrated unequivocally that they belong to the superfamily of G protein-coupled receptors (GPCR) and the subfamily of rhodopsin receptors. Thus, the µ-, -, and -opioid receptors all have the putative structure of seven transmembrane domains (TMs), an extracellular N-terminus with multiple glycosylation sites, a third intracellular loop (IL-3) with multiple amphiphatic -helixes, and an IL-4 formed by the putative palmitoylation sites at the carboxyl terminus (Evans et al., 1992; Kieffer et al., 1992; Chen et al., 1993b; Fukuda et al., 1993; Li et al., 1993; Meng et al., 1993; Yasuda et al., 1993). On the whole, these receptors are about 60% identical to each other, with the greatest homology found in the TMs (73-76%) and ILs (86-100%). The greatest divergence in amino acid sequence is found in the N-terminus (9-10%), extracellular loops (EL; 14-72%), and the C-termini (14-20%) (Chen et al., 1993a). These opioid receptors are capable of regulating the same second messengers, with activation of the cloned µ-, -, and -opioid receptors causing inhibition of adenylyl cyclase activity (Evans et al., 1992; Kieffer et al., 1992; Chen et al., 1993b; Fukuda et al., 1993; Li et al., 1993; Meng et al., 1993; Yasuda et al., 1993) and N-type (Tallent et al., 1994) and L-type (Piros et al., 1996) Ca²⁺ channels. Activation of these opioid receptors also increases phospholipase C activity, and causes a transient increase in the levels of intracellular Ca²⁺ (Johnson et al., 1994; Spencer et al., 1997), in the activation of inwardly rectifying K⁺ channels (Henry et al., 1995), and the mitogen-activated protein kinases Erk-1 and Erk-2 (Fukuda et al., 1996; Li and Chang, 1996).

	Differences in µ- and -Opioid Receptor Signal Transduction

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Given the similarities among the opioid receptor primary structures and their effectors, what is the reason for the existence of multiple opioid receptors with differing selectivity for various opioid ligands? To examine this issue, we decided to concentrate on the µ- and -opioid receptors because Met-enkephalin, an endogenous ligand, has equal affinity for these two sites, and the selective activation of these receptors results in dramatically different pharmacological responses. Because the two receptors activate the same effectors, the reason for Met-enkephalin having equal affinity for these sites is not readily apparent. When we examined the relative affinities of several ligands for µ- and -opioid receptors stably expressed in Chinese hamster ovary (CHO) cells, the prototypic µ-opioid ligands, such as [D-Ala²,N-MePhe⁴,Gly-ol⁵] (DAMGO) or PL017, displayed high affinity for the µ-opioid receptor, whereas the prototypic -opioid ligands, such as [D-Pen²,D-Pen⁵]-enkephalin (DPDPE) or deltorphin II, displayed high affinity for the -opioid receptor, although DPDPE also has a high affinity for the µ-opioid receptor as well. The ligands clearly having specificity for the expressed -opioid receptor are the agonist deltorphin II and the antagonist Tipp [H-Tyr-Tic[,CH₂NH]Phe-Phe-OH(Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid)], whereas the ligands having specificity for the µ-opioid receptor are the agonist PL017 and the antagonist CTOP (D-Phe-Cys-Try-D-Trp-Orn-Thr-Pen-Thr-NH₂) (Table 1). These findings helped define the differences within the opioid receptor structures involved in ligand recognition. In addition to differences in ligand selectivity, differences were found in the µ- and -opioid receptor activities. Thus, the ratios of the agonist IC₅₀ values, or the antagonist K_e values, versus the K_i values for the -opioid receptor were all 1 (Table 1). This suggested that the inhibition of adenylyl cyclase activity by the -opioid agonist required 10% of the receptor occupancy, implying the presence of spare receptors. On the other hand, because the ratios of IC₅₀, or K_e values, and the K_i for the µ-opioid receptor were 1, it appears the µ-opioid receptor inhibition of adenylyl cyclase in CHO cells requires full receptor occupancy. This difference between receptors was not due to differences in the expression levels of the - and µ-opioid receptor, or the amount of G proteins in the CHO cells. Rather, other components of the signal transduction cascades must be responsible for the differences in µ- and -opioid receptor activities.

View this table: [in this window] [in a new window]   TABLE 1 Relative affinities and potencies of various µ- and -opioid receptor agonists for DOR-1 and MOR-1 stably expressed in CHO cells Relative affinities of various opioid agonists in CHO cells stably expressing DOR-1 or MOR-1 were determined by competition binding studies using [3H]diprenorphine as radioactive ligand. IC50 values of ligands were obtained from computer analysis of competition curves using Sigma Plot (Jandel, San Rafael, CA). Ki values were then calculated using the Cheng-Prusoff equation. Relative potencies were determined by ability of various concentrations of agonists to inhibit 10 µM forskolin-stimulated [3H]cAMP production in CHO cells stably expressing DOR-1 or MOR-1. IC50 values ± S.E. were obtained from computer analysis of dose-response curves using Sigma Plot.

	Differential Regulation of µ- and -Opioid Receptor Signal Cascades at Level of Ligand-Receptor Interaction

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One possible component in the receptor signal cascade that could be responsible for these differences is the ligand-receptor interaction site, because the differential efficiency of G protein activation could be achieved with the formation of different ligand-receptor complexes. To define the conformation of the agonist-receptor complexes, the receptor domains involved in selective ligand binding and the requirements for receptor activation must be defined. Receptor chimera studies followed by mutational analysis has revealed that the TM6 (Fukuda et al., 1995), and the EL-3 (Pepin et al., 1997), are critical for the selective, high-affinity binding of -opioid ligand. As for the µ-opioid receptor, there is some dispute about the domains involved in the selective recognition of DAMGO. Thus, some studies indicated that the first EL-1 (Minami et al., 1995) is involved in DAMGO recognition, whereas µ/ chimeras studies indicated that the EL-3 was critical for DAMGO binding (Xue et al., 1995; Wang et al., 1995). When we conducted studies with the µ/ chimeras and examined receptor affinities and the abilities of the ligands shown on Table 1 to regulate adenylyl cyclase, we found that not only are different domains of the µ- and -opioid receptors involved in the selectivity of the ligands, but also that agonists induce different receptor conformations.

We have constructed 17 µ/ receptor chimeras to investigate the domains involved in µ- and -opioid ligand selectivity and have examined the relative affinities of various agonists and antagonists, both alkaloids and peptides, for these receptor chimeras (Scheme 1). In addition, we examined the ability of agonists to inhibit forskolin-stimulated intracellular [³H]cAMP production and determined K_e values of antagonist to reverse etorphine inhibition. Etorphine was chosen as the agonist in these studies because it displays minimal selectivity for opioid receptor subtypes. The results indicate that the progressive substitution of the -opioid receptor TM domains with the corresponding µ-opioid receptor TM domains resulted in the reduction of DPDPE, deltorphin II, NTB, and TIPP affinities (Table 2). For the most part, the affinities of these ligands for the chimeras were similar to those for the -opioid receptor, provided that TM6 and the EL-3 was present in the chimeras. However, in the µ₁ chimera, where only the sequence of the N-terminus to the beginning of the IL-1 of the -opioid receptor is replaced with the complementary sequence of the µ-opioid receptor, significant reductions in the -opioid agonist affinities were observed (Table 2). Reverse substitution of the same sequence in the µ-opioid receptor with that of the -opioid receptor results in a receptor chimera, ₁µ, capable of binding the -opioid selective ligands with high affinity, the most dramatic of which is for TIPP. Thus, in this case, the ₁µ chimera exhibits nanomolar affinity for TIPP, whereas the wild-type µ-opioid receptor has greater than micromolar affinity for this peptide. Such data suggest the involvement of TM1 in the selectivity of -opioid receptor ligands. If this is the case, then substitution of this -opioid receptor sequence to the µ-opioid receptor should also reduce the affinity for µ-opioid receptor-selective ligands. Indeed, this appears to be case with DAMGO, PL017, oxymorphone, and CTOP affinities being reduced in the ₁µ chimera (Table 2). The only exception to this is naloxone, a ligand that does not distinguish between µ- and -opioid receptors. These data suggest that TM1 of the -opioid receptor is involved in the selective binding of -opioid ligands. If so, what then is the role of EL-3 and TM6 in the binding of -opioid ligands? Because the binding pocket of the receptor is formed by the spatial orientation of various amino acids in different TMs, the binding of receptor-selective ligands could be affected significantly by the interactions among various TMs. The relative interaction of the TM can be studied by examining the ability of the chimera ₅µ to bind -opioid receptor-selective ligands with high affinity. After splicing of only the EL-2 and TM5 of the -opioid receptor into the µ-opioid receptor, the ₅µ chimera exhibited affinities for deltorphin or TIPP in the 10⁸ M range (greater than micromolar) instead of having very low affinity for these ligands (Table 2). Because the other receptor chimera constructs did not reveal the critical involvement of EL-2 or TM5 in -opioid binding, as demonstrated by the relative affinities of these ligands for the chimeras µ_1-4, µ_1-5, _1-4µ, and _1-5µ, one explanation for the high-affinity binding of the -opioid ligands in the ₁µ or ₅µ chimeras is the possible destabilization of µ-opioid receptor structure with the introduction of -opioid receptor TM domains.

View larger version (47K): [in this window] [in a new window]   Scheme 1.   Schematic representation of opioid receptor chimeras constructed from rat MOR-1 and mouse DOR-1.

Similar conclusions can be drawn from comparing the relative affinities of the µ/ chimeras for the µ-opioid receptor-selective ligands. The influence of the TM interactions on the µ-opioid ligand binding is demonstrated by the high-affinity binding of DAMGO, PL017, naloxone, and CTOP to the ₄₅₆µ but not to the µ_1-3 receptor chimeras (Table 2). The difference between these two chimeras is that ₄₅₆µ has the TM7 sequence of the µ-opioid receptor, whereas µ_1-3 does not. However, introduction of the TM6 into the ₄₅₆µ receptor chimera reduces the affinities of these ligands for the receptor. These and other data demonstrate that the relative spatial orientation of the amino acids within the TM affect the affinities of selective ligands. As for nonselective ligands such as etorphine, the substitution of the -opioid receptor sequence into the µ-opioid receptor, or vice versa, does not alter their affinities.

Opioid alkaloids and opioid peptides have overlapping but distinct binding domains. This is demonstrated most dramatically by comparing the relative affinities of naloxone and CTOP for the µ/ receptor chimeras. Thus, the presence of only the TM7 of µ-opioid receptor in the _1-6µ chimera is sufficient for high-affinity binding of naloxone (Table 2), whereas CTOP binds with low affinity to this site (greater than micromolar). Introduction of the -opioid receptor sequence other than the TM1 greatly reduced the affinities of the receptor chimeras for CTOP, but not for naloxone, suggesting that these two opioid antagonists have distinct binding sites within the µ-opioid receptor. In addition, there appear to be differences among the selective agonists' binding sites within the µ-opioid receptor. The introduction of the TM1-5 sequence of the µ-opioid receptor to the -opioid receptor, yielding the µ_1-5 chimera, results in DAMGO and oxymorphone, but not PL017 binding to the chimera with affinities similar to those found with the wild-type µ-opioid receptor (Table 2). In the reverse receptor chimera, _1-5µ, DAMGO and oxymorphone also display high-affinity binding, whereas PL017 exhibits minimal affinity. These data suggest that although PL017 is a µ-opioid receptor agonist, its receptor recognition site is distinct from that of the other two agonists tested.

Differences between agonist binding sites and subsequent receptor conformation is demonstrated further by one of our recent studies on receptor phosphorylation. Agonist-induced receptor phosphorylation has been proposed to be a critical step in the cellular regulation of GPCRs. The phosphorylation of the opioid receptor is reported to involve a G protein-coupled receptor kinase (GRK), but not protein kinase A (PKA; Chen and Yu, 1994; Pei et al., 1995). However, the primary sequence analysis of the opioid receptor predicts consensus sequences for the putative phosphorylation by PKA and protein kinase C (PKC). When we examined the ability of PKA to phosphorylate the µ-opioid receptor, we observed that, in the presence of morphine, PKA phosphorylates the receptor in a morphine concentration- and time-dependent manner. Moreover, this phosphorylation is naloxone reversible and is blocked by pretreating the cells with pertussis toxin (PTX). The in vitro phosphorylation is also blocked by isolating the membrane from cells pretreated with morphine in the presence of forskolin and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Because in vitro phosphorylation is blocked by the PKA-specific inhibitor KT5720, it appears the phosphorylation of the µ-opioid receptor is, in fact, mediated by this enzyme. Surprisingly, PKA-mediated phosphorylation of the receptor in vitro is not observed in the presence of another µ-ligand, DAMGO, or other enkephalins. Rather, only µ-opioid agonists that are alkaloids, or -endorphin, stimulate the in vitro phosphorylation of the receptor. In addition to the putative PKA sites located intracellularly, there are consensus PKA sites on the extracellular portion of the receptor or at the TM. The differences between the ability of morphine and DAMGO to induce in vitro PKA phosphorylation suggest these extracellular or TM sites are accessible to PKA in the presence of opioid alkaloids but not opioid peptides. This difference could be responsible, in part, for the differentiation of the signals induced by the µ-specific opioid alkaloid and peptide ligands.

	Differential Regulation of µ- and -Opioid Receptor at G Protein Level

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Early receptor binding and functional measurements suggested that opioid receptors belong to the super family of GPCR, and the deduced primary sequences of the cloned receptors indicate unequivocally that this is the case. One common feature of these receptors is that their activities are abolished by pretreatment with PTX, suggesting that the G proteins involved in the transduction of the receptor signals are either of the G_i group or the G_o group. Both the -subunit and the dimers of these G proteins could regulate effectors, such as adenylyl cyclase, the Ca²⁺ and K⁺ channels. Studies with other GPCRs suggest that there is specificity with the G_i/G_o proteins in their coupling to the receptor and transduction of the receptor signals. Hence, it is not surprising that in earlier reports opioid receptors appear to exhibit such selectivity. Using G_i/G_o -subunit-selective antibodies and the PTX pretreatment paradigm, it has been reported that -opioid receptors inhibit the adenylyl cyclase in NG108-15 cells via the G_i2 (McKenzie and Milligan, 1990), whereas receptor-mediated inhibition of voltage-dependent Ca²⁺ channels is due to G_o (Hescheler et al., 1987). Studies with the human neuroblastoma SHSY5Y cells indicated that the µ-opioid receptor inhibition of adenylyl cyclase is mediated by G_o proteins (Carter and Medzihradsky, 1993).

We addressed the question of receptor-G protein interaction by directly examining the activation of G_i/G_o by opioid receptor. To this end, we took advantage of the fact that activation of GPCRs leads to the exchange of GDP for GTP and the subsequent dissociation of the heterotrimeric G proteins into -subunits and dimers. This dissociation allows for the ADP-ribosylation of the -subunits of G_i/G_o by cholera toxin (Milligan and McKenzie, 1988). Furthermore, in the presence of the photolabile GTP analog, azidoanilido-[³²P]GTP, receptor agonist promotes the association of this analog, making it possible to identify the G protein that interacts with the receptor. Using these two approaches, we labeled the G protein -subunits coupled to various opioid receptors and optimized the urea/SDS-polyacrylamide gel electrophoresis system to separate G_i/G_o -subunits in a single dimension. By conducting the experiments in NG108-15, NS20Y, and N1E115 cells expressing different levels of -opioid receptor, or in 10 CHO clones that stably express different levels of -opioid receptors, we found that the ability of the -opioid receptor to activate multiple G_i/G_o is independent of receptor density. In the cell lines tested, -opioid receptor activation resulted in the labeling of four different G protein -subunits, G_i2, G_i3, and G_0A, and an unknown protein (Prather et al., 1994a,b). Because the conditions used for these labeling studies favored the binding of azidoanilido-[³²P]GTP to G_i/G_o and not to G_z (Field et al., 1994), activation of G_z and G₁₆, as reported by others (Garzon et al., 1997; Lee et al., 1998) using clonal cell lines stably expressing the opioid receptors, were not detected in our studies.

In addition to being independent of receptor density, the ability of other opioid receptors, µ and , to activate the same four G proteins is observed in CHO cells stably expressing these receptor subtypes (Chakrabarti et al., 1995b; Prather et al., 1995). Furthermore, the potencies of the µ-, -, and -agonists to activate these G_i/G_o proteins is very similar in the CHO cell lines. The notable exception is the G subunit that we could not identify with existing antibodies.

When the type and amount of G protein being activated is examined, the multiple opioid receptors appear to exhibit some preferences (Table 3). Thus, the -opioid receptor displays no selectivity toward the various G_i/G_o, whereas both µ- and -opioid receptors exhibit selectivity toward G_i2 and G₀₂. The maximal levels of G_i2 and G_o2 labeling induced by activation of these two receptors is significantly higher than that observed with G_i3 labeling (Table 3). Because -opioid receptor-induced labeling of G_i3 is equal to that of G_i2 and G_o2, the preferential labeling of G_i2 and G_o2 induced by µ- or -opioid receptors cannot be attributed to differences in G protein levels in the CHO cells. These findings do not suggest, therefore, that µ- and -opioid receptors are associated with G_i2 and G_o2, whereas -opioid receptors are coupled to all G_i/G_o proteins in CHO cells. Other studies suggest that the opioid receptor exhibits changes in the association with G protein upon agonist stimulation (Law and Reisine, 1997). Hence, regulation of opioid receptor activities does not appear to involve their ability to promote the association of GTP onto the G proteins and the subsequent dissociation of heterotrimers. Accordingly, the observed differences in the G proteins involved in the opioid receptor-mediated regulation of adenylyl cyclase and Ca²⁺ channels must lie elsewhere.

View this table: [in this window] [in a new window]   TABLE 3 Relative labeling intensity of Gi/Go by azidoanilido-[-32P]GTP in presence of activated opioid receptors Relative labeling intensity of various Gi/Go -subunits in CHO cells were summarized from data published in Chakrabarti et al., 1995b; Prather et al., 1994b, 1995.

In recent experiments, we observed differences in the efficiency of the receptor regulating the voltage-dependent L-type Ca²⁺ channels and adenylyl cyclase activity. Thus, although we demonstrated µ-opioid receptor-mediated inhibition of L-type channels in GH3 pituitary cells stably expressing the receptor, when a similar level of -opioid receptor is stably expressed in another GH3 clone, regulation of the L-type channels was not observed (Piros et al., 1995). In contrast, in the same two GH3 clonal cell lines both µ- and -opioid agonists inhibit adenylyl cyclase activity. However, when the -opioid receptor is overexpressed in the GH3 cell line that already expresses the µ-opioid receptor, -opioid receptor-mediated inhibition of the L-type Ca²⁺ channels is observed (J. L. Adams, L. Song, E. T. Piros, T. G. Hales, P. Y. Law, and P. L. Prather, unpublished observations). The major difference between the GH3DOR cell line and the GH3 MORDOR cell line is that there is five times more -opioid receptor expressed in the latter. There is also a slight difference in the G proteins that these two opioid receptors activate, with the -opioid receptor inducing azidoanilido-[-³²P]GTP incorporation the most in Go₁, whereas µ-opioid receptor induced GTP incorporation is greatest in G_o2 (J. L. Adams et al., unpublished observations). However, with the GH3 cell lines coexpressing both the µ- and -opioid receptors, the -opioid receptor-mediated inhibition of the L-type Ca²⁺ channels requires a critical concentration of receptor (>0.5 pmol/mg protein) as shown by covalently inactivating the -opioid receptor with SUPERFIT (J. L. Adams et al., unpublished observations). At the same time, the -opioid receptor-mediated inhibition of the adenylyl cyclase activity remains. At this density of µ-opioid receptor, both the L-type channels and adenylyl cyclase activities are regulated by µ-opioid agonists (Piros et al., 1995). Thus, the difference in the requirement of -opioid receptor density to regulate these two effectors suggests different effectors are involved. The difference between the µ- and -opioid receptor regulation of the L-type Ca²⁺ channels also indicates either that different G proteins participate or that there are different efficiencies between these two opioid receptors to activate the same G protein.

There now appears to be little question of differences between the µ- and -opioid receptor activation of the G protein complexes. In addition to the differences in the ratios of the potencies and the affinities of various agonists (Table 1) and the activation of the L-type Ca²⁺ channels, there is a pronounced difference between the µ- and -opioid receptor-G protein complexes. In an earlier study with NG108-15 cells, we reported that after PTX pretreatment the ability of the -opioid agonists to induce receptor internalization and down-regulation remains unaltered, suggesting that G proteins other than the PTX substrates, such as G_i/G_o, are involved in these processes (Law et al., 1985a). When we conduct similar studies with neuroblastoma N₂A cells stably expressing either the µ- or -opioid receptor, a differential response is observed with PTX pretreatment. Similar to the results obtained with the NG108-15 cells, the PTX pretreatment does not diminish the ability of DPDPE to induce down-regulation of the -opioid receptor in N₂A cells. On the other hand, parallel treatment of the N₂A cells expressing the µ-opioid receptor with PTX results in the total elimination of DAMGO-induced receptor internalization or down-regulation (Chakrabarti et al., 1997). In both cases, the ability of the agonist to regulate adenylyl cyclase and intracellular Ca²⁺ level is completely eliminated.

The possibility that the agonist activates other non-PTX substrates is eliminated by mutating the conserved aspartic acid (Asp) in the TM2 to alanine. Previous studies with other GPCR and the opioid receptors (Kong et al., 1993; Surratt et al., 1994) indicates this Asp is critical for agonist activation of the receptor. When the Asp⁹⁵ in the -opioid receptor and the Asp¹¹⁴ in the µ-opioid receptor are mutated to Ala and the mutants stably expressed in the N₂A, the ability of the µ- or -opioid agonist to inhibit adenylyl cyclase activity is not observed. As with the PTX experiments, the Asp⁹⁵ -opioid receptor mutant is down-regulated by agonist pretreatment, whereas the corresponding µ-opioid receptor mutant is not. It was also found that -opioid receptor remains coupled to G proteins even after PTX pretreatment or after mutation of Asp⁹⁵ to Ala (Chakrabarti et al., 1997). Under the same conditions, or the equivalent mutation, the µ-opioid receptor is completely uncoupled from the G proteins and remains in the low-affinity state (Chakrabarti et al., 1997). The existence of the -opioid receptor in the G protein-receptor complex after PTX pretreatment or Asp⁹⁵ mutation is demonstrated by the further reduction of agonist affinity after agonist or PTX treatment, respectively. Thus, these studies not only suggest the need for high-affinity states or the receptor-G protein-coupled states for agonist-induced down-regulation of the receptor, but also that the interactions between the µ-opioid receptor and -opioid receptor and the G proteins are different. This is surprising in view of the homology in the amino acids sequences of the IL in these two receptors. Although the greatest differences between the two receptor sequences are within the carboxyl tail, exchange of this portion of the receptor does not confer -opioid receptor properties to the µ-opioid receptor. That is, the µ/ receptor chimera with only the -opioid receptor carboxyl tail spliced onto the µ-opioid receptor exists in the low-affinity state after PTX pretreatment. Thus, the receptor sequence that regulates the interaction between G proteins and opioid receptors remains to be elucidated.

Molecular dynamic modeling of the GPCR suggests agonist binding causes movement of the TM domains (Luo et al., 1994). Modeling, followed by mutational analysis, indicates that the molecular motion of the TM6 and TM7 is critical for transducing agonist binding signals to the IL-3 or the juxtatransmembrane portion of the carboxyl tail, the two domains known to be responsible for activation of G proteins. Our mutational and receptor chimera studies provided evidence supporting the importance of agonist-induced movements of TM in opioid receptors, as exemplified by comparing relative efficacies of agonists (Table 4). Thus, the efficacies of -opioid agonists are >1, whereas those of the µ-opioid agonists are <1. Although the affinities of the µ-opioid agonists for the µ_1-5 chimera are less than that for the wild-type µ-opioid receptor, the potencies of DAMGO, PL017, and oxymorphone are greater with the wild type and their efficacies in this chimera are >1. The most surprising finding is that whereas PL017 had minimal affinities for the receptor chimera, it is very potent in mediating inhibition of adenylyl cyclase activities. These findings suggest that the µ-opioid receptor sequences in this chimera allow for movement of the TM with minimal agonist-induced activation energy, indicating that the efficiency of opioid receptor activation is influenced by conformational changes in the receptor brought on by agonist binding. This point is substantiated further by studies with the µ_1-5 receptor chimera. With this chimera, the affinities of the -opioid receptor-selective ligands are not altered significantly (Table 2), although the potencies of DPDPE and deltorphin II are greatly reduced and their efficacies are <1. Similar data were obtained with this chimera and µ-opioid receptor-selective agonists (Table 4). This suggests that activation of this chimera is inefficient compared with the wild-type -opioid receptor because high-affinity binding of agonists fails to fully activate this site.

The agonist-induced molecular movement of the TM, or conformational changes that lead to receptor activation, are also found with one of the receptor mutations generated by Taq polymerase. The static receptor model posits that antagonists, unlike agonists, are unable to induce conformational changes in receptors and, therefore, display no efficacy. Overexpression of cloned GPCRs exhibits basal levels of receptor activity even in the absence of agonists, as evidenced by GTP binding studies and the discovery of inverse agonists (Costa and Herz, 1989). To accommodate such observations, the ternary complex model was revised to include isomerization of the receptor R to R* (Cotecchia et al., 1993), with R* representing "activated" receptor. In this case, the agonist binding favors the formation of R*, thus enhancing association with G proteins, whereas antagonist stabilizes the R state of the receptor, reducing its interaction with G proteins. This model suggests, therefore, there is only one receptor conformation capable of activating the system. However, such a model fails to account for results obtained with opioid receptor antagonists. Thus, the structural difference between naloxone, an opioid receptor antagonist, and the corresponding agonist, oxymorphone, is at the N17 position, with oxymorphine having methyl and naloxone an allyl substitution at this position. It has long been thought that the steric hindrance resulting from the bulky group at N17 converts an opioid agonist into the corresponding antagonist. It has also been generally assumed that opioid antagonists compete with agonists for the binding pocket and that the former could not induce a conformational change in the receptor due to the steric hindrance. However, using the µ_1-2 chimera, we found that the "classical" opioid antagonists display agonistic properties (Claude et al., 1996). In this case, opioid antagonists, such as naloxone or naltrexone, do not reverse or block the effect of DPDPE. Instead, these antagonists by themselves inhibited the forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing this receptor chimera, even though their affinities for the receptor remained unchanged. Moreover, the antagonists desensitized and down-regulated the chimera receptor. Thus, in all respects, these antagonists displayed full agonistic properties in this chimera. Following complete sequencing of the receptor chimera, we determined that the Taq polymerase used to generate the receptor fragments introduces a single point mutation at TM4, resulting in the conversion of Ser196 to Leu in the µ_1-2 chimera (Claude et al., 1996). Because this serine is conserved among all three cloned opioid receptors, the mutation of this serine in TM4 to leucine should generate the desired receptor phenotype, i.e., antagonist displaying agonist properties. When Ser196, Ser177, and Ser187 in µ-, -, and -opioid receptors, respectively, are mutated to Leu, naltrexone acts like an agonist in inhibiting adenylyl cyclase activity in CHO cells stably expressing receptor mutants. In addition, this antagonist inhibits the G protein-coupled voltage-dependent inward rectifying potassium channels (GIRK) activities in Xenopus oocytes when GIRK and the receptor mutants were coexpressed in this system. Because there are no observable changes in the mutant receptor affinities for agonists and antagonists, these data suggest that the binding of antagonists to these receptor mutants causes the receptor conformational change required of an agonist. Thus, the conversion of the Ser in the TM4 to a Leu must relieve constraints that prevent G protein activation by antagonists. Because the mutation results in the removal of Ser, an amino acid that has hydrogen bonding capacity, to Leu, an amino acid that has minimal activity in this regard, it appears that the normal constraint is due to the generation of a hydrogen bond. Similar reasoning can be used with respect to the ionic bond formed between the conserved Asp in TM2 with the Asn in TM7. In this case, mutation of the Asp to Asn in TM2 completely abolishes the ability of agonist to regulate G proteins, an effect that is reversed by the mutation of Asn in TM7 to Asp. Studies are underway to determine whether there is such a partner for the conserved Ser in TM4.

The importance of the conserved Ser in TM4 for the constraint within the receptor is demonstrated further with our studies of GIRK regulation by these mutants. In these experiments, we noticed that the efficacies of the antagonists in oocytes expressing both the GIRK and the µ_1-2 chimera paralleled those of the agonists. However, when only the conserved Ser in TM4 is mutated to Leu, the antagonists exhibit only partial agonist properties in oocytes expressing both the GIRK and the mutated - or µ-opioid receptor (Claude et al., 1996). Furthermore, when the conserved Ser in the receptor chimeras ₄₅₆µ or ₄₅µ (Scheme 1) is mutated, the antagonists once again exhibit only partial agonist properties with respect to GIRK channel activity (P. A. Claude, L. J. Erickson-Herbrandson, H. H. Loh, and P. Y. Law, unpublished observations). As seen with the conversion of Ser to Leu in other mutant receptors, the agonist affinities for the receptor, and their potencies and efficacies for regulating both adenylyl cyclase and GIRK channels, are not altered in the ₄₅₆µ or ₄₅µ mutant receptor chimeras. Moreover, the full agonistic properties of the antagonists in the µ₁₂ chimera is due to the incompatibility of the TM1 and TM7 from two different opioid receptors and not to a decrease in the receptor affinity for G proteins. In contrast, the µ_1-2 receptor chimera appears to have higher affinities for G proteins because the stable GTP analogs, GTPS or GppNHp, are less able to decrease agonist binding in these chimeras (P. A. Claude et al., unpublished observations). Thus, the interaction between the TM1 and TM7 of the opioid receptor appears to regulate the efficacies of these ligands. Because the amino acid sequence differences between the TM7 domains of the µ₁₂ chimera and ₄₅₆µ chimera involves only two amino acids, it seems probable that the interaction of these amino acids in TM7 with their partners in TM1 stabilizes the receptor structure, influencing the efficacy of opioids. The identities of these amino acids are currently being deduced.

	Regulation of µ- and -Opioid Receptor Levels by Phosphorylation and Down-Regulation

Top Abstract A Sincere Thanks Introduction Differences in µ- and... Differential Regulation of µ-... Differential Regulation of µ-... Regulation of µ- and... Regulation of Opioid Receptor... Regulation of Opioid Receptor... Conclusion References

Like other GPCRs, the activities of the µ- and -opioid receptors are attenuated after chronic agonist treatment. Studying -opioid receptors in NG108-15 cells, we found that receptor desensitization and down-regulation are associated with attenuation of opioid receptor activities (Chang et al., 1982; Law et al., 1982; Law et al., 1983ab). Reports on other GPCRs, such as the ₂-adrenergic receptor, indicate that agonist-induced receptor phosphorylation is the cellular mechanism responsible for receptor desensitization and down-regulation (Krupnick and Benovic, 1998). The current model suggests that -arrestin binds to the phosphorylated receptor, competing with G protein binding for the attachment site, thereby terminating receptor function. Because the subsequent internalization of the receptor is also arrestin-dependent, it can be stimulated by receptor phosphorylation. The internalized receptor is resensitized by dephosphorylation, after which it is recycled to the membrane. Although other cellular components, such as dynamin, may be involved in the receptor internalization process (Chu et al., 1997), the critical steps for receptor desensitization and internalization are its phosphorylation attachment to -arrestin.

Several groups, including our own, have reported agonist-induced opioid receptor phosphorylation (Arden et al., 1995; Pei et al., 1995; Yu et al., 1997; El Kouhen et al., 1999), with some suggesting that phosphorylation correlates with agonist-induced receptor desensitization. By comparing the ability of agonists to regulate GIRK in oocytes and their ability to phosphorylate receptors in CHO cells, Wang and colleagues (Yu et al., 1997) suggested that DAMGO-induced receptor desensitization is caused by phosphorylation of the µ-opioid site. Using dominant negative mutants of the GRK, and the dominant negative mutant of arrestin, Pei and coworkers (Pei et al., 1995) found that the -opioid agonist-mediated receptor phosphorylation is blocked by the GRK mutant and that desensitization is blocked by the arrestin mutant. The probable involvement of receptor phosphorylation in receptor desensitization is partially supported by data obtained from studying mutated receptors. Thus, Pak et al. (1997) mutated the Thr393 of the µ-opioid receptor into Ala, blunting the DAMGO-induced receptor desensitization. Devi and coworkers (Trapaidze et al., 1996; Cvejic et al., 1996) were unable to block agonist-induced internalization and down-regulation of mutant opioid receptors by mutating Thr352 . Although direct measurements of receptor phosphorylation were made in these studies, the results provide evidence supporting the role of receptor phosphorylation in the regulation of opioid receptor activities.

Others have been unable to establish a casual relationship between receptor phosphorylation and desensitization. Kovoor et al. (1997) reported a very slow rate of DAMGO-induced desensitization of µ-opioid receptor-mediated regulation of GIRK channel activity and found that the rate is not enhanced by overexpression of GRKs. Likewise, when we compared the rate of µ-opioid receptor phosphorylation and the rate of DAMGO-induced receptor desensitization in two different cell lines, we found that agonist-induced receptor phosphorylation occurred within minutes, whereas the reduction in DAMGO-mediated regulation of adenylyl cyclase activities (i.e., desensitization) took hours (El Kouhen et al., 1999). Moreover, the rate of desensitization is not enhanced by inhibition of phosphatase activity using calyculin A, or by promoting phosphorylation either by overexpression of GRKs or stimulation of endogenous protein kinases, such as PKCs. It was also found that overexpression of -arrestin does not increase DAMGO-induced receptor phosphorylation. In other studies with human embryonic kidney cells, HEK293, which express µ- and -opioid receptors, overexpression of GRK-2 and -arrestin potentiate DPDPE-induced -opioid receptor desensitization, but not DAMGO-induced µ-opioid receptor desensitization, even though overexpression of GRK-2 increases both the µ- and -opioid receptor phosphorylation. These results suggest that µ-opioid receptor phosphorylation might not be an obligatory event for desensitization of this receptor.

This conclusion is reinforced by studies involving long-term exposure to morphine. Thus, it is established that chronic administration of morphine completely desensitizes µ-opioid receptors with respect to morphine or DAMGO (Chakrabarti et al., 1995a). However, numerous reports indicate that morphine does not induce receptor phosphorylation (Arden et al., 1995; Zhang et al., 1998), even though one group did find evidence to the contrary (Yu et al., 1997). Moreover, it is generally accepted that morphine cannot induce receptor internalization (Arden et al., 1995; Zhang et al., 1998), a cellular event thought to be closely coincided with receptor phosphorylation. If in fact morphine is unable to induce receptor phosphorylation, then morphine-induced receptor desensitization must involve some other mechanisms.

The ability to induce receptor desensitization in the absence of receptor phosphorylation was demonstrated further by us and Capeyrou et al. (1997) using µ-opioid receptor mutants in which all the putative phosphorylation sites were removed. Replacement of all Ser/Thr within the carboxyl tail, or Ser/Thr within the third intracellular and carboxyl tail (Capeyrou et al., 1997), completely abolished the ability of DAMGO to induce receptor phosphorylation. This suggests that DAMGO-induced receptor phosphorylation is limited to the Ser/Thr in the carboxyl tail. This conclusion is confirmed by analysis of the cyanogen bromide cleavage of the phosphorylated receptor. Interestingly, these receptor mutants could still be desensitized by chronic exposure to agonist. Thus, these experiments suggest that receptor phosphorylation is not a prerequisite for desensitization.

For GPCRs, the receptors in the endosomes are dephosphorylated, resensitized, and recycled, suggesting that the relatively long time needed for desensitization of the µ-opioid receptor is due to recycling. Koch et al. (1998), using monensin, which traps internalized receptor within endosomes, found that the rate of DAMGO-induced desensitization of the MOR1B, a splice variant of the µ-opioid receptor, increases. Similarly, we discovered that the rate of etorphine-induced µ-opioid receptor desensitization in HEK293 cells is increased by the pretreatment with monensin, and that the rate of disappearance of cell surface receptors, as monitored by fluorescence-activated cell-sorting analysis analysis, is also increased by monensin. These data suggest that internalized µ-opioid receptors are being recycled. Because monensin, which blocks the recycling process, increases the rate of desensitization, it appears the ability of agonist to regulate adenylyl cyclase activity depends on the concentration of µ-opioid receptors on the membrane. Pak et al. (1996) proposed that µ-opioid receptor desensitization correlates with the down-regulation of the receptor. In our own experiments, down-regulation of the µ-opioid receptor did not correlate with agonist-induced desensitization. Mutation to Ala of all the Ser/Thr residues in the carboxyl tail of the µ-opioid receptor blocks etorphine-induced receptor down-regulation. However, chronic exposure of the same mutant receptor to etorphine results in a loss of agonist activity. Fluorescence-activated cell-sorting analysis of the receptor cell surface suggests that removal of the putative phosphorylation sites on the carboxyl tail of the µ-opioid receptor does not prevent its internalization. Only under conditions in which internalization of the mutant receptor is blocked by overexpression of dominant negative arrestin, is DAMGO-induced desensitization of the mutant attenuated. However, with HEK293 cells transiently expressing the wild-type µ-opioid receptor, overexpression of dominant negative arrestin does not alter DAMGO-induced receptor desensitization if internalization is inhibited. These results suggest that receptor phosphorylation and the physical removal of the receptors from the cell surface contribute to agonist-induced desensitization of the µ-opioid receptor.

The inability to block etorphine-induced µ-opioid receptor internalization by mutating all of the Ser/Thr residues in the carboxyl tail suggests that phosphorylation of the receptor is not obligatory for this event. However, the same receptor mutation does prevent etorphine-induced receptor down-regulation, indicating that the receptor sorting and redirection of the receptor traffic to the lysosomes for degradation needs a different signal than that required for receptor internalization. Thus, we systematically truncated and mutated the carboxyl tail of the µ-opioid receptor to define receptor domains that might be involved in receptor trafficking. Truncating the receptor after the putative phosphorylation sites revealed that any receptor truncation after the Ser³⁵⁹ does not affect the etorphine-induced down-regulation (Burd et al., 1998). However, truncation after Ser³⁵⁵ blocks etorphine-induced down-regulation, but not internalization, of the receptor. Because the difference between these truncations is four amino acids with the sequence of STIE, this was deleted from the wild type, and the effect on etorphine-induced receptor down-regulation examined. To our surprise, etorphine induces receptor down-regulation of this deletion mutant (Burd et al., 1998). This suggests that either more than one motif is necessary to mediate etorphine-induced receptor down-regulation, or that this region does not play a critical role in this phenomenon. For example, in the rat neurotensin receptor, Thr422 and Tyr424 are critical for agonist-induced internalization of the receptor (Chabry et al., 1995). If, however, these amino acids are modified individually, there is little or no effect on receptor internalization. Thus, it is plausible that more than one motif is involved in the etorphine-induced µ-opioid receptor down-regulation, with one motif being within the STIE, whereas the other is downstream from the Ser359. After studying numerous combinations of mutations, we found that the combination of Ser356 and Ser363 blocks the etorphine-induced down-regulation of the µ-opioid receptor (Burd et al., 1998). Because both Ser356 and Ser363 residues are putative GRK sites, it is likely these mutations block phosphorylation of the receptor. Interestingly, however, direct measurement of ³²P-incorporation into the receptor reveals that the mutation of Ser356 and Ser363 to Ala does not, in fact, attenuate etorphine-induced phosphorylation of the receptor. An explanation for blockade of receptor down-regulation could be that the mutations interfere with the receptor-arrestin interaction, or the formation of other receptor complexes. Our preliminary data indicate that blockade of the etorphine-induced down-regulation by Ser356 and Ser363 mutations is reversed by overexpression of arrestin or GRK-2. Therefore, the cellular processing of µ-opioid receptors requires the formation of multiple protein complexes. The identity and role of individual cellular proteins in these processes are under active investigation in our laboratory.

	Regulation of Opioid Receptor Activities by Associating Proteins

Top Abstract A Sincere Thanks Introduction Differences in µ- and... Differential Regulation of µ-... Differential Regulation of µ-... Regulation of µ- and... Regulation of Opioid Receptor... Regulation of Opioid Receptor... Conclusion References

Various proteins within the membrane microdomain greatly affect opioid receptor signaling. Receptor signaling through scaffold, anchoring, and adaptor proteins is a well-established phenomenon for many membrane receptors, in particular those of the tyrosine kinase family (Pawson and Scott, 1997). The recruitment of other proteins by an adaptor with multiple docking sites allows for the amplification or modulation of signals. An example of this is produced by the Drosophila InaD gene that codes for a protein with five PDZ domains (Tsunoda et al., 1997). The InaD associates through these PDZ domains with a light-activated Ca²⁺ channel (TRP), phospholipase C-, and PKC. The regulation of these effectors by InaD allows for the efficient activation of TRP by phospholipase C- in response to stimulation of rhodopsin and G_q, and inactivation by phosphorylation of TRP by PKC. Thus, by recruitment of cellular proteins containing particular motifs,