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ANESTHETIC PHARMACOLOGY

RGS Proteins: New Players in the Field of Opioid Signaling and Tolerance Mechanisms

Department of Anesthesia and Perioperative Care, University of California, San Francisco, California

Address correspondence and reprint requests to Pamela Pierce Palmer, MD, PhD, University of California, San Francisco, Department of Anesthesia and Perioperative Care, 513 Parnassus Avenue, Box 0464, Room S-455, San Francisco, CA 94143. Address e-mail to palmerp{at}anesthesia.ucsf.edu.

	Abstract

Top Abstract Introduction Family of RGS Proteins Genomic Linkage of RGS... RGS Proteins Play Essential... RGS Proteins and the... Selectivity of RGS Proteins... References

 
In this article we review recent advances in our understanding of the crucial role of the Regulator of G protein Signaling (RGS) proteins in opioid signaling mechanisms and opioid tolerance development. Opioids exert their physiologic effects via complex G protein-coupled receptor-signaling mechanisms, and RGS proteins are now known to tightly regulate the G protein signaling cycle. RGS proteins contain GTPase-accelerating protein activity within their characteristic RGS domain and various other receptor signaling-related properties of their other functional domains. There have been more than 20 RGS proteins reported in the literature, and multiple RGS proteins have been shown to negatively regulate G protein-mediated opioid signaling, facilitate opioid receptor desensitization and internalization, and affect the rate at which opioid tolerance develops. Using RGS proteins as targets for future drug therapy aimed at modulating opioid effectiveness in both acute and chronic pain settings may be an important advance in the treatment of pain.

	Introduction

Top Abstract Introduction Family of RGS Proteins Genomic Linkage of RGS... RGS Proteins Play Essential... RGS Proteins and the... Selectivity of RGS Proteins... References

 
Opioid analgesia and tolerance development involve complex cellular and molecular mechanisms (1). Decades of scientific research have led to the discovery of endogenous opioid peptides (2), multiple opioid receptors (3) and the opioid-receptor-like (ORL1) receptor, a more recently discovered receptor that shares 60% homology with opioid receptors and has functions in nociceptive pathways (4). Various types of opioid agonists and opioid-like agonists activate classic G protein-coupled receptor signaling pathways via the µ, , opioid, and ORL1 receptors (MOR, DOR, KOR, and ORL1 by IUPHAR nomenclature, respectively), yet the mechanisms of opioid tolerance have proven especially complicated. Numerous studies have demonstrated that multiple mechanisms are involved. Early hypotheses include receptor-G protein uncoupling and loss of membrane surface opioid receptors (internalization and down-regulation) (5,6). Up-regulation of the 3',5'-cyclic adenosine monophosphate (cAMP) pathway (7,8) and activation and inactivation of protein kinases, such as cAMP-dependent protein kinase (9), protein kinase C (10), G protein-coupled receptor kinases (GPRK) (11), and Ca²⁺/calmodulin-dependent kinases (10) have been observed after chronic opioid administration or withdrawal. Involvement of other receptors (such as the N-methyl-d-aspartate receptor), proteins, and anti-opioid peptides is also well established (12,13). Although a large body of evidence supports a role for each of these pathways and molecules in opioid tolerance development, there remain many unanswered questions. New hypotheses are being proposed on the basis of recent studies (14,15), including the concept that rapid morphine tolerance is the consequence of its inability to induce MOR internalization (16). The measurement of relative agonist signaling versus endocytosis is proposed to assess the potential of an opioid agonist for induction of tolerance (16). One of the newest focuses of opioid tolerance research has been on a group of recently discovered proteins, the regulators of G protein signaling (RGS).

	Family of RGS Proteins

Top Abstract Introduction Family of RGS Proteins Genomic Linkage of RGS... RGS Proteins Play Essential... RGS Proteins and the... Selectivity of RGS Proteins... References

 
RGS proteins are a family of cellular proteins containing a homologous RGS domain of approximately 120 amino acids in length. Over 20 different mammalian RGS genes and their cognate proteins have been identified and characterized (17). They have been classified into seven subfamilies based on their sequence similarities and common structural features outside the RGS domain (Table 1). Signaling of a G protein-coupled receptor is initiated by agonist-induced receptor activation, which in turn switches the trimeric Gß complex into separate G and Gß subunits. The G subunit is initially bound to GDP when coupled to the inactive receptor. On receptor activation the G subunit converts to an active GTP-bound state, promoting signal transduction. A primary function of RGS proteins is to negatively regulate G proteins via the GTPase-accelerating protein (GAP) activity of their conserved RGS domain. RGS proteins specifically interact with the G subunits and enhance the intrinsic GTPase activity of G subunits to accelerate GTP hydrolysis and thereby facilitate the switch of G from a GTP-bound active state to a GDP-bound inactive state (18). Therefore, RGS proteins in essence serve to halt the signaling event and return the receptor/G-protein complex to a "ligand-receptive" membrane bound state. RGS proteins fulfill multiple functions through interactions with other proteins by use of their different structural domains (19) (Table 1).

RGS proteins are cell-type and receptor-type specific. It has been recognized that specific members of the RGS family are functionally associated with selective G protein-coupled receptor signal transduction pathways (18). Convincing evidence has shown that RGS proteins play important regulatory roles in G protein-coupled receptors for monoamine and acetylcholine neurotransmitters, many neuropeptides and hormones, by negatively regulating and terminating receptor activation and signaling and by desensitizing and recycling receptors (20). It was not until recently that RGS proteins emerged as key players in opioid signaling and tolerance, an involvement that is hinted at by the close genomic linkage between genes for opioid receptors and various RGS proteins.

	Genomic Linkage of RGS and Opioid Receptor Genes

Top Abstract Introduction Family of RGS Proteins Genomic Linkage of RGS... RGS Proteins Play Essential... RGS Proteins and the... Selectivity of RGS Proteins... References

 
The Human Genome Project and other genome studies have suggested that a close genetic linkage not only indicates coordinated transcription and shared regulation mechanisms for expression but also implies a functional relationship (21). A recent genomic analysis has revealed informative patterns regarding the genomic positions of various RGS genes. Not only are several RGS genes arranged in clusters and often adjacent to G protein-related components, such as G, G subunit genes (GNA, GNG) and GPRK genes, but some RGS genes are also linked to opioid receptor genes. The first of such reports was by Ito et al. (22), who described the genomic coupling of RGS19 (G interacting protein, GAIP) and the ORL1 gene oprl in human chromosome 20. The first exons of RGS19 and oprl are separated by only 83 bp, and this region functions as a bidirectional promoter region for both genes. Subsequently, two more RGS genes were found to be closely linked to and µ opioid receptor genes (oprk and oprm, respectively). RGS20 and oprk are separated by only 0.6 Mb in chromosome 8. RGS17 and oprm are separated by 1.5 Mb in chromosome 6 (23). It is worth noting that RGS19, RGS17, and RGS20, all RGS proteins that are genomically linked to ORL1 and opioid receptors, belong to the same RZ subfamily (Table 1). Studies on the effects of RGS19 and RGS20 on opioid signaling have been reported and will be reviewed in the following sections; no studies, however, have been published on that of RGS17.

The tight genomic coupling between human ORL1 and RGS19 genes strongly suggests a functional relationship, not only at the transcription level where the transcription of the two genes are co-regulated (22) but also possibly at the translation and post-translation levels. There are a number of mammalian genes that, like the human ORL1 and RGS19, are linked (within a distance of about 100–200 bp) head-to-head by a shared bidirectional promoter. Each of these pairs of adjacent genes studied has been found to share a significant functional relationship (21). For example, human dihydrofolate reductase gene and DNA mismatch repair gene MSH3 are linked by an 88-bp bidirectional promoter and transcribed in opposite directions. This physical linkage and transcriptional coregulation bring the DNA repair pathway and the nucleotide metabolism pathway into a cooperative action (24). These findings strongly support the concept that the opioid receptor genes and RGS genes are not only physically linked but are also possibly functionally coupled.

	RGS Proteins Play Essential Role in Opioid Action and Tolerance

Top Abstract Introduction Family of RGS Proteins Genomic Linkage of RGS... RGS Proteins Play Essential... RGS Proteins and the... Selectivity of RGS Proteins... References

 
In addition to the genomic studies, recent functional studies show that RGS proteins, especially members of the RZ, R4, and R7 subfamilies (Table 1), play a crucial role in opioid receptor signaling. They are not only responsible for G protein inactivation, which terminates opioid action (Fig. 1), but are also active components in opioid receptor desensitization, internalization, recycling, and degradation (25). RGS proteins may provide a functional linkage between acute opioid receptor desensitization and chronic opioid tolerance.

View larger version (52K): [in this window] [in a new window]   Figure 1. A schematic of the primary mechanism by which RGS proteins regulate opioid receptor signaling. The binding of a specific opioid agonist to the opioid receptor changes the conformation of the receptor which in turn activates G protein. G subunit switches from a GDP-bound inactive state to a GTP-bound active state, and dissociates from Gß subunits. Activated G protein subunits interact with effectors to transfer signal. RGS protein specifically interacts with GTP-bound G, enhances the intrinsic GTPase activity of G and therefore accelerates the hydrolysis of GTP to GDP, resulting in a rapid return of G subunit to its inactive state and reassociation with Gß units. AC, adenylyl cyclase; GIRK, G protein-coupled inwardly rectifying K+ channels; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PLA2, phospholipase A2; PLCß, phospholipase Cß; VOCC, voltage-operated Ca2+ channels. The essential role of RGS proteins (especially GAIP/RGS19) in opioid receptor internalization and recycling is not illustrated.

The RGS19 member of the RZ subfamily, one of the first RGS proteins discovered, has been found to modulate functions of opioid receptors. It not only accelerates GTPase activity of the Gi subunit, but also facilitates opioid receptor internalization and recycling via clathrin-coated vesicles (25). Hepler et al. (26) reported that RGS19 reduces the inhibitory effect of DOR agonist [leu]enkephalin on cAMP synthesis in NG-108 cells (expressing abundant opioid receptors, predominantly the type). Results from an elegantly designed study using immunofluorescence labeling to investigate DOR trafficking in human embryonic kidney cells have demonstrated that, at resting state, DORs and the inactive trimeric complex Gi/ß are located in noncoated cell membranes, whereas RGS19 proteins are on clathrin-coated pits of the plasma membrane and Golgi apparatus. On addition of the DOR agonist, cyclic [D-penicillamine^2,5] enkephalin, activated DORs and Gi move to the clathrin-coated domain where they bind RGS19. RGS19 enhances the GTPase activity of Gi and hydrolyzes GTP-bound Gi to inactive GDP-bound Gi, the latter moving back to noncoated membrane region where it reassociates with Gß subunits. RGS19 remains on clathrin-coated pits, whereas clathrin-coated vesicles containing DORs bud from the plasma membrane and traffic to endosomes and lysosomes to be recycled or degraded (25). As opioid receptor desensitization and internalization hold the key for opioid signaling and tolerance (1,27), it is not difficult to recognize the crucial role that RGS19 plays in these mechanisms. By intraventricular administration of antisense oligonucleotides to suppress the expression of RGS19 and RGS20 in mouse brain, Garzon et al. (28) were able to demonstrate that a reduction of either RGS19 or RGS20 greatly increases the supraspinal antinociceptive effect of MOR agonists but has no effect on that of DOR agonists. In addition, suppression of RGS19 and RGS20 expression facilitates the development of acute and chronic morphine tolerance (28).

It has been reported that RGS2, RGS4, and RGS8, all members of the R4 subfamily, regulate opioid receptor function, presumably as a result of the GAP activities of their RGS domain and their spatial and temporal coexpression with opioid receptors in certain neuronal cells and tissues. Hepler et al. (26) were the first to report that exogenously added RGS4 successfully reverses DOR agonist [leu]enkephalin-induced inhibition of cAMP synthesis in NG-108 cells. The explanation is that NG-108 cells contain several members of Gi and Go subunits, and opioid receptors inhibit cAMP synthesis by an activation of Gi or Go protein-mediated pathway (inhibition of adenylyl cyclase, AC). RGS4 accelerates the inactivation of subunits of Gi and Go to extinguish the opioid signaling event. In addition, they found that RGS4 is more potent than RGS19 in regulating DOR.

RGS4 regulates MOR and KOR activation as well. Overexpression of RGS4 in human embryonic kidney cells significantly attenuates MOR agonist morphine and [D-Ala²,Phe(N-Me)⁴,Gly-ol⁵]enkephalin (DAMGO)- induced inhibition of AC (29). RGS4 overexpression also accelerates the deactivation of G protein-coupled inwardly rectifying K⁺ channel (GIRK) after termination of MOR signaling (30). Furthermore, Ulens et al. (31) reported that a coexpression of RGS4 and GIRK1/GIRK2 channels in Xenopus oocytes dramatically reduces the basal K⁺ current and accelerates the deactivation of GIRK channels activated by KOR agonist U69593.

Not only can RGS4 negatively regulate opioid receptor signaling, but opioid signaling can also regulate RGS expression and function. It has been demonstrated that RGS4 mRNA is up-regulated by agonist-induced opioid receptor activation in PC12 cells expressing cloned MOR and KOR (32), as well as in rat brain and other neuronal tissues (33). This indicates a negative feedback mechanism in which opioid receptor signaling induces the up-regulation of RGS mRNA and proteins, which in turn rapidly terminates the opioid-induced signal. A decrease (33) or no significant change (34) in RGS4 expression in certain brain regions after opioid agonist administration has been observed, suggesting that the opioid-induced changes in RGS proteins are brain region-specific and depend on methods of opioid administration, as well as the timing of preparation and assay of RGS mRNA and proteins.

Using a cultured Xenopus laevis dermal melanophore cell line stably expressing mouse MORs, Potenza et al. (35) found that overexpression of RGS2 significantly decreases the magnitude of morphine-induced pigment aggregation and shifts the morphine concentration-response curve to the right. In contrast, expression of a mutated Gi1 with impaired binding and interaction with RGS, prolongs morphine-induced pigment aggregation and shifts the morphine concentration-response curve to the left (35). In a glioma cell line C6 stably expressing the rat MORs, it was found that eliminating the effect of endogenous RGS proteins by transfection of an RGS-insensitive Go significantly decreases GTPase activities, greatly enhances the inhibition of AC and the activation of mitogen-activated protein kinase, effects initiated by MOR agonists DAMGO and morphine (36). In contrast, application of RGS8 in. the same system increases MOR agonist-induced GTPase activity (36).

Members of this R4 subfamily, especially RGS4 and RGS2, in addition to regulating acute opioid signaling, are also involved in opioid tolerance mechanisms. Bishop et al. (33) observed that after chronic morphine administration, expression of RGS4 mRNA is significantly up-regulated in dorsal central gray and locus ceruleus, regions known to mediate opioid-induced analgesia as well as physical dependence and withdrawal responses. However, in the red nucleus, a region associated with motor function, expression of RGS4 mRNA is down-regulated. Gold et al. (37) conducted a detailed study on the regulation of RGS expression by chronic morphine in rat locus ceruleus. They found that levels of RGS2 and RGS4 mRNAs are unchanged after 5 days of continuous morphine administration (by a 75-mg morphine pellet implantation) but are increased two- to threefold 6 h after morphine withdrawal (by application of opioid antagonist naltrexone 100 mg/kg). In contrast, protein levels of RGS4 are elevated twofold after chronic morphine but decrease to control levels 6 h after withdrawal. Expression of several other RGS mRNAs and proteins they investigated were not altered. Furthermore, intracellular application of wild-type RGS4 protein, but not a GAP-deficient mutant RGS4 or a boiled RGS4, into locus ceruleus neurons dramatically diminishes morphine-induced electrophysiological current (37). Those findings strongly support that RGS2 and RGS4 are functional modulators in the development of opioid tolerance and actively respond to opioid withdrawal.

Members of the R7 subfamily, particularly RGS9–2, a brain-specific alternative splicing variant of the RGS9 gene, regulate opioid receptors in a unique way: not only through the RGS domain but also through their featured GGL (G protein subunit-like) domain. The GGL domain in R7 subfamily members is homologous in sequence to the G protein subunit and is able to selectively bind and interact with one of the G protein ß subunits, Gß5. RGS9–2 is highly expressed in striatum and at lower levels in periaqueductal gray (PAG) and spinal cord, regions known to have abundant opioid receptor expression (38,39). Similarly, the Gß5 subunit is predominantly expressed in the central nervous system (CNS), especially in thalamus, hypothalamus, PAG, striatum, pons-medulla, cortex, and spinal cord, regions associated with pain transmission, sensation, and modulation (40,41). Overexpression of RGS9–2 by transfection into Xenopus melanophore cells bearing MORs significantly attenuates morphine-induced pigment aggregation (38). Blocking the expression of RGS9–2 or Gß5 with specific antisense oligonucleotides greatly enhances the potency and duration of the antinociceptive effects produced by morphine and DAMGO but has a lesser effect on opioid action. Such treatments also successfully prevent acute morphine tolerance (41–43). When studying chronic morphine tolerance, suppressing RGS9 by antisense oligonucleotide enhances the effects of morphine only for the first 2–3 h. After that short period tolerance still develops, probably as a result of a compensation by other RGS proteins or of the degradation and clearance of oligonucleotides administered (41). But in a cell culture system of mouse hippocampal neuroblastoma cells, suppression of RGS9 expression by antisense oligonucleotide significantly inhibits chronic morphine-induced up-regulation of AC activity and partially reverses the chronic morphine effect on abolishing DAMGO-induced GTPS high-affinity binding (44). Furthermore, a study with RGS9-knockout mice provides convincing evidence for the essential role of RGS9 in opioid action and tolerance (39). Acute morphine administration significantly increases the expression of RGS9–2 mRNA and protein in striatum as well as in PAG and spinal cord, whereas chronic morphine treatment decreases RGS9–2 expression. RGS9-knockout mice show a dramatic increase in morphine analgesia and reward with significantly delayed tolerance. When they become tolerant to morphine, the RGS9-knockout mice exhibit a much stronger response to morphine withdrawal and more severe physical dependence than do wild-type mice. Taken together, these results can be explained by the function of RGS9–2 as a negative regulator for MOR (39). Using antisense technology, RGS6, RGS7, and RGS11 of the same R7 subfamily are also studied, all showing negative regulatory effects on morphine action (43). Interestingly, they display differential effects on MOR and DOR signaling. For example, although inhibition of RGS6 or RGS9–2 greatly increases both the potency and duration of morphine analgesia, it decreases the potency but increases the duration of the analgesic effect of DOR agonists. Inhibition of RGS7 produces an increased response to both MOR and DOR agonists, whereas inhibition of RGS11 reduces the effect of DOR agonist in terms of both potency and duration (43).

As discussed above, RGS proteins of the R7 subfamily regulate opioid action. Similarly, acute and chronic morphine treatments differentially change the neuronal expression of mRNAs and proteins of the R7 subfamily in a region-specific and gene-specific manner (45). Two hours after an intraventricular injection of morphine, RGS7 and RGS9–2 mRNAs are increased in the striatum of the mouse brain, whereas RGS9–2 and RGS11 are decreased in the cortex. During the chronic morphine treatment, mRNA levels of RGS7, RGS9–2, and RGS11 increased in most regions of the brain, especially in the striatum and PAG. The expression of their proteins is also increased after chronic morphine but not after acute morphine (45).

By transfecting an RGS-insensitive Go to eliminate the effect of endogenous RGS proteins on Go-mediated signaling in rat C6 glioma cells, endogenous RGSs are found to suppress MOR agonist-induced, Go-mediated AC supersensitization, a feature of opioid tolerance (46). Although it does not further characterize the identity of these RGS proteins, this study clearly demonstrates the involvement of endogenous RGS proteins in opioid tolerance mechanisms.

Table 2 summarizes the large body of experimental evidence supporting the essential role of RGSs in opioid action. Although there are complications and some of the results are still controversial regarding which RGS proteins are involved, the selectivity of specific RGS proteins toward specific types of opioid receptors, and the qualitative and quantitative changes of RGS expression in acute morphine action and chronic morphine tolerance, these experimental findings have undoubtedly revealed novel mechanisms by which RGS proteins regulate opioid receptor signaling and opioid tolerance development (Table 2).

	RGS Proteins and the ORL1 Receptor

Top Abstract Introduction Family of RGS Proteins Genomic Linkage of RGS... RGS Proteins Play Essential... RGS Proteins and the... Selectivity of RGS Proteins... References

 
The ORL1 receptor, also known as the nociceptin/orphanin FQ receptor, can be considered as a special and atypical member of the opioid receptor family. It possesses very high sequence-homology (>60%) to the classical µ, , and types of opioid receptors. Its endogenous peptide ligand, nociceptin/orphanin FQ, has a marked similarity to opioid peptides, especially to dynorphin. It has been well established that ORL1 plays an important role in opioid signaling and opioid tolerance (47,48). Recent studies in our own laboratory (22,49,50) have suggested that RGS19 is an inseparable part of ORL1 gene expression and also functions as a negative regulator in the ORL1 signal transduction pathway. Human ORL1 and RGS19 are adjacent genes oriented in opposite directions (head to head). Their first exons are separated by an 83-bp region that functions bidirectionally as the core-promoter for a coordinated transcription of the two genes. By using human and mouse multiple tissue cDNA panels, we demonstrated that RGS19 is expressed in all ORL1-positive tissues and cells examined (22,49), which is a prerequisite for RGS19 regulation of ORL1 signaling. Activation of ORL1 by nociceptin/orphanin FQ in NT2 neuronal cells up-regulates RGS19 transcription, which is inhibited by RNA synthesis inhibitor actinomycin D and is blocked by selective ORL1 antagonist [NPhe¹]nociceptin(1–13)NH₂ (50), indicating that the up-regulation is indeed at the transcription level and is ORL1 receptor mediated. In addition, we identified in mice an alternatively spliced short RGS19 mRNA that lacks the exon 2 region and uses an ATG in exon 3 as its translation initiation codon. As a result, the short RGS19 protein does not have the N-terminal 22 amino-acid residues that exist in the long isoform. RGS19 alternative splicing variants are differentially expressed in various tissues (49). Based on these findings, we hypothesize that binding of agonist to ORL1 switches G from a GDP-bound inactive state to a GTP-bound active state and at the same time up-regulates the expression of its negative regulator RGS19. RGS19 alternative splicing may be a mechanism for regulating the function and selectivity of RGS19 isoforms.

	Selectivity of RGS Proteins in Regulating Opioid Receptor Signaling

Top Abstract Introduction Family of RGS Proteins Genomic Linkage of RGS... RGS Proteins Play Essential... RGS Proteins and the... Selectivity of RGS Proteins... References

 
As we mentioned in the preceding sections, RGS proteins display a relative selectivity in regulating opioid receptors in vivo and in vitro. RGS4 is 3–4 times more potent than RGS19 in regulating µ and opioid receptors (26), but RGS19 appears to be more functionally associated with ORL1 (22,49), as well as with opioid receptor internalization and recycling (25). In the melanophore-based assays, only the overexpression of RGS2 (but not RGS1, RGS3, and RGS4) or RGS9–2 (but not RGS9–1, another alternative splicing variant of the RGS9 gene with shorter C-terminus than RGS9–2) has a significant effect on morphine-induced response (35,38). Suppressing the expression of RGS9–2 or RGS11 in mice greatly enhances the antinociceptive effect of MOR agonists but reduces the effect of DOR agonists (43). Another study clearly demonstrating RGS subtype specificity uses RGS9-knockout mice, which exhibit a supersensitivity (10-fold increase as compared to wild-type mice) to the morphine-rewarding effect. This supersensitivity is completely reversed by overexpression of RGS9–2 in the nucleus accumbens but not by overexpression of RGS4 in the same region (39). A recent study demonstrated that blocking the gene expression of RGS19 or RGS20 by antisense oligonucleotides significantly increases the effect of MOR agonists morphine and DAMGO and facilitates the development of morphine tolerance. But blocking the gene expression of these RGSs does not change the effect of DOR agonists (28).

The mechanisms for selectivity of RGS proteins in regulating G protein-mediated receptor signal transduction pathways have just begun to unfold. In general, the selectivity or the preference of an RGS protein for specific receptors is determined by its tissue-specific expression and its specific interaction with the intracellular domains of receptor proteins, G protein subunits, effectors, and other pathway-specific components. In RGS proteins, the distinctive regions outside of the RGS domain are important determinants for selectivity (17,18,51–53). In particular, in the case of RGS4 and RGS19, their N-terminal regions play an essential role in their selectivity toward opioid receptors and ORL1 (49,54). In the case of R7 subfamily RGSs, the specific interaction between the GGL domain and the Gß5 subunit is a significant factor for selective and differential regulation of MORs and DORs (41,43). As for RGS9–2, the tissue-specific expression (RGS9–2 is brain-specific and striatum-enriched, whereas RGS9–1 is retina-specific) and the distinct C-terminus presumably contributes to its selectivity (38). Alternative splicing of RGS mRNAs to produce multiple variants of RGS proteins offers a molecular basis for differential function and selectivity (38,49,55).

Future Directions
It seems apparent that the foundation for the function and selectivity of RGS proteins in regulating opioid signaling lies in their ability to interact with G protein subunits and opioid receptors. The critical role of RGS proteins in receptor desensitization, internalization and recycling may be the basis for their observed effects on opioid tolerance. However, unrecognized mechanisms may exist based on the multifunctional characteristics of RGS proteins.

Although it has not been very long since the discovery of the first RGS protein, and even less time since the first realization of the role of RGS proteins in opioid receptor signaling, we have greatly expanded our knowledge of RGS genes, proteins, and their functions. Still we have just begun to appreciate the critical role that RGS proteins play in opioid tolerance development. RGS proteins have been considered as novel CNS drug targets (56). To promote the development of therapies targeting RGS proteins, we must develop high-affinity and specific antibodies against individual RGS proteins, develop selective RGS activators and inhibitors, and develop transgenic and gene-knockout mice to establish the function of each individual RGS gene. Using these therapies to modulate opioid effectiveness, especially to inhibit the development of opioid tolerance, would be an important advance in the treatment of chronic pain conditions.

	Footnotes

 
Supported, in part, by grants AR45570 and NS042593 from the National Institutes of Health, Bethesda, Maryland, and a John Severinghaus Radiometer Award (to PPP).

Accepted for publication September 23, 2004.

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Top Abstract Introduction Family of RGS Proteins Genomic Linkage of RGS... RGS Proteins Play Essential... RGS Proteins and the... Selectivity of RGS Proteins... References

 

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