A Single Nucleotide Polymorphic Mutation in the Human μ-Opioid Receptor Severely Impairs Receptor Signaling
2001; Elsevier BV; Volume: 276; Issue: 5 Linguagem: Inglês
10.1074/jbc.m006352200
ISSN1083-351X
AutoresKatia Befort, Dominique Filliol, Fabien M. Décaillot, Claire Gavériaux‐Ruff, Margret R. Hoehe, Brigitte L. Kieffer,
Tópico(s)Pain Management and Opioid Use
ResumoLarge scale sequencing of the human μ-opioid receptor (hMOR) gene has revealed polymorphic mutations that occur within the coding region. We have investigated whether the mutations N40D in the extracellular N-terminal region, N152D in the third transmembrane domain, and R265H and S268P in the third intracellular loop alter functional properties of the receptor expressed in mammalian cells. The N152D receptor was produced at low densities. Binding affinities of structurally diverse opioids (morphine, diprenorphine, DAMGO and CTOP) and the main endogenous opioid peptides (β-endorphin, [Met]enkephalin, and dynorphin A) were not markedly changed in mutant receptors (<3-fold). Receptor signaling was strongly impaired in the S268P mutant, with a reduction of efficacy and potency of several agonists (DAMGO, β-endorphin, and morphine) in two distinct functional assays. Signaling at N40D and R265H mutants was highly similar to wild type, and none of the mutations induced detectable constitutive activity. DAMGO-induced down-regulation of receptor-binding sites, following 20 h of treatment, was identical in wild-type and mutant receptors. Our data show that natural sequence variations in hMOR gene have little influence on ligand binding or receptor down-regulation but could otherwise modify receptor density and signaling. Importantly, the S268P mutation represents a loss-of-function mutation for the human μ-opioid receptor, which may have an incidence on opioid-regulated behaviors or drug addiction in vivo. Large scale sequencing of the human μ-opioid receptor (hMOR) gene has revealed polymorphic mutations that occur within the coding region. We have investigated whether the mutations N40D in the extracellular N-terminal region, N152D in the third transmembrane domain, and R265H and S268P in the third intracellular loop alter functional properties of the receptor expressed in mammalian cells. The N152D receptor was produced at low densities. Binding affinities of structurally diverse opioids (morphine, diprenorphine, DAMGO and CTOP) and the main endogenous opioid peptides (β-endorphin, [Met]enkephalin, and dynorphin A) were not markedly changed in mutant receptors (<3-fold). Receptor signaling was strongly impaired in the S268P mutant, with a reduction of efficacy and potency of several agonists (DAMGO, β-endorphin, and morphine) in two distinct functional assays. Signaling at N40D and R265H mutants was highly similar to wild type, and none of the mutations induced detectable constitutive activity. DAMGO-induced down-regulation of receptor-binding sites, following 20 h of treatment, was identical in wild-type and mutant receptors. Our data show that natural sequence variations in hMOR gene have little influence on ligand binding or receptor down-regulation but could otherwise modify receptor density and signaling. Importantly, the S268P mutation represents a loss-of-function mutation for the human μ-opioid receptor, which may have an incidence on opioid-regulated behaviors or drug addiction in vivo. μ-opioid receptor d-Phe-Cys-Tyr-d-Trp-Orn-Pen-Thr-NH2 [d-Ala2,MePhe4,Gly-ol5] enkephalin cloned human μ-opioid receptor G protein-coupled receptors guanosine 5′-O-(thio)triphosphate phosphate buffer saline secreted alkaline phosphatase wild type The opioid system controls pain perception and mood and is generally implicated in a wide variety of behaviors that are essential in facing threatening situations (1Akil H. Watson S.J. Young E. Lewis M.E. Khachaturian H. Walker J.J. Annu. 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In support of this, gene targeting experiments have shown the absence of morphine-induced analgesia (6Matthes H.W.D. Maldonado R. Simonin F. Valverde O. Slowe S. Kitchen I. Befort K. Dierich A. LeMeur M. Dollé P. Tzavara E. Hanoune J. Roques B.P. Kieffer B.L. Nature. 1996; 383: 819-823Crossref PubMed Scopus (1390) Google Scholar, 7Sora I. Takahashi N. Funada M. Ujike H. Revay R.S. Donovan D.M. Miner L.L. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1544-1549Crossref PubMed Scopus (463) Google Scholar, 8Tian M. Broxmeyer H.E. Fan Y. Lai Z. Zhang S. Aronica S. Cooper S. Bigsby R.M. Steinmetz R. Engle S.J. Mestek A. Pollock J.D. Lehman M.N. Jansen H.T. Ying M. Stambrook P.J. Tischfield J.A. Yu L. J. Exp. Med. 1997; 185: 1517-1522Crossref PubMed Scopus (155) Google Scholar, 9Schuller A.G.P. King M. Zhang J. Bolan E. Pan Y.-X. Morgan D.J. Chang A. Czick M.E. Unterwald E.M. Pasternak G.W. Pintar J.E. Nat. 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Neurosci. 1998; 18: 7285-7295Crossref PubMed Google Scholar), or constipation (14Roy S. Liu H.-C. Loh H.H. Mol. Brain Res. 1998; 56: 281-283Crossref PubMed Scopus (98) Google Scholar) in MOR-deficient mice, demonstrating unambiguously that the μ-opioid receptor is a main molecular target for morphine action in vivo. The finding of genetic mutations altering the expression or functional activity of MOR is therefore important to understand inter-individual variable responses to the major opioid drugs, both in the clinical management of pain or heroin addiction.In addition to the direct mediation of opiate-induced euphoria, tolerance, and dependence, μ-opioid receptors have been shown to regulate the effects of other substances with high addictive potential such as cocaine or alcohol (15Kreek M. Mol. Psychiatry. 1996; 1: 232-254PubMed Google Scholar). As an example, in humans, the μ-receptor antagonists naloxone and naltrexone have been shown not only to reverse heroin overdose but also to alter alcohol consumption (16Reid L. GA H. Alcohol. 1984; 1: 33-37Crossref PubMed Scopus (169) Google Scholar, 17Herz A. Psychopharmacology. 1997; 129: 99-111Crossref PubMed Scopus (596) Google Scholar). Studies using animal models also point at a possible role of the MOR gene in ethanol use. Recently we have reported that MOR-deficient mice do not self-administer alcohol (18Roberts A. Mcdonald J.S. Heyser C.J. Kieffer B.L. Matthes H.W.D. Koob G.F. Gold L.H. J. Pharmacol. Exp. Ther. 2000; 293: 1002-1008PubMed Google Scholar), suggesting that μ receptors are essential in mediating the reinforcing properties of this substance. Also a quantitative trait loci (QTL) analysis in mice showed that oral morphine preference is largely mediated by a single locus in chromosome 10 which harbors the MOR gene (19Berrettini W.H. Ferraro T.N. Alexander R.C. Buchberg A.M. Vogel W.H. Nat. Genet. 1994; 7: 54-58Crossref PubMed Scopus (168) Google Scholar). Thus, among the various genetic components of the opioid system, the μ-opioid receptor gene is an evident candidate in the search for genes potentially involved in the susceptibility to drug abuse.The cloning of the human opioid receptor gene (20Mestek A. Hurley J.H. Bye L.S. Campbell A.D. Chen Y. Tian M. Liu J. Schulman H. Yu L. J. Neurosci. 1995; 15: 501-527Crossref Google Scholar, 21Wang J.B. Johnson P.S. Persico A.M. Hawkins A.L. Griffin C.A. Uhl G.R. FEBS Lett. 1994; 338: 217-222Crossref PubMed Scopus (288) Google Scholar) has prompted studies of DNA sequence variability within the hMOR gene (22Berrettini W.H. Hoehe M.R. Ferraro T.N. DeMaria P.A. Gottheil E. Addiction Biol. 1997; 3: 303-308Crossref Scopus (82) Google Scholar, 23Bergen A.W. Kokoszka J. Peterson R. Long J.C. Virkkunen M. Linnoila M. Goldman D. Mol. Psychiatry. 1997; 2: 490-494Crossref PubMed Scopus (263) Google Scholar, 24Bond C. Laforge K.S. Tian M. Melia D. Zhang S. Borg L. Gong J. Schluger J. Strong J.A. Leal S.M. Tischfield J.A. Kreek M.J. Yu L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9608-9613Crossref PubMed Scopus (974) Google Scholar). We have recently conducted a comprehensive polymorphic study of the MOR gene in 250 patients using large scale multiplex DNA sequencing. We have identified 43 variants within 7-kilobase pair regulatory, exonic, and intronic sequences (25Hoehe M.R. Kopke K. Wendel B. Rohde K. Flachmeier C. Kidd K.K. Berrettini W.H. Church G.M. Hum. Mol. Genet. 2000; 9: 2895-2908Crossref PubMed Google Scholar). We have found six mutations in the coding region. Five of these mutations modify the encoded protein sequence, and these include the previously identified A6V and N40D mutations in the N-terminal region of the receptor (22Berrettini W.H. Hoehe M.R. Ferraro T.N. DeMaria P.A. Gottheil E. Addiction Biol. 1997; 3: 303-308Crossref Scopus (82) Google Scholar, 23Bergen A.W. Kokoszka J. Peterson R. Long J.C. Virkkunen M. Linnoila M. Goldman D. Mol. Psychiatry. 1997; 2: 490-494Crossref PubMed Scopus (263) Google Scholar, 24Bond C. Laforge K.S. Tian M. Melia D. Zhang S. Borg L. Gong J. Schluger J. Strong J.A. Leal S.M. Tischfield J.A. Kreek M.J. Yu L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9608-9613Crossref PubMed Scopus (974) Google Scholar), as well as three yet unreported mutations. One of these novel mutations is located in the third transmembrane domain of the receptor and changes an asparagine residue into an aspartic acid residue (N152D). The two other mutations are found in the third intracellular loop of the receptor and replace an arginine and a serine by a histidine (R265H) and a proline (S268P) residue, respectively.The three latter mutations occur in regions that may be critical for receptor function, and this has prompted us to determine whether these natural MOR variants indeed exhibit an altered pharmacological activity profile. We have constructed the three novel hMOR variants by site-directed mutagenesis. We also have generated the N40D mutant, some properties of which have been described earlier (24Bond C. Laforge K.S. Tian M. Melia D. Zhang S. Borg L. Gong J. Schluger J. Strong J.A. Leal S.M. Tischfield J.A. Kreek M.J. Yu L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9608-9613Crossref PubMed Scopus (974) Google Scholar), to extend our knowledge on the functional properties of this frequent polymorphic variant. We have expressed the four mutant receptors in mammalian cells, and we determined binding affinities for four prototypic opioid ligands (morphine, DAMGO, diprenorphine, and CTOP) and the main opioid peptides ([Met]enkephalin, β-endorphin, and dynorphin A). We also have investigated agonist-induced functional responses of the receptor using the [35S]GTPγS binding assay, as well as a reporter gene assay. Finally, we have examined receptor down-regulation following chronic exposure to the potent μ-agonist DAMGO. Our results show no obvious modification in receptor binding or down-regulation. However, the data indicate decreased heterologous expression for the N152D mutant receptor and a remarkable decrease of receptor signaling for the S268P variant.DISCUSSIONThe detection of genetic variation affecting the opioid system is an interesting approach to understand the origin of inter-individual differences in response to opiates, in opioid-associated pathophysiology or in diseases of substance dependence. Vulnerability to heroin abuse is thought to result from polygenic influences. It was shown associated with a polymorphism in the D4 dopamine receptor gene (39Kotler M. Cohen H. Segman R. Gritsenko I. Nemanov L. Lerer B. Kramer I. Zer-zion M. Kletz I. Ebstein R. Mol. Psychiatry. 1997; 2: 251-254Crossref PubMed Scopus (183) Google Scholar) or with a mutation in the δ-opioid receptor gene, in one of two studies (40Mayer P. Rochlitz H. Rauch E. Rommelspacher H. Hasse H.E. Schmidt S. Höllt V. Neuroreport. 1997; 8: 2547-2550Crossref PubMed Scopus (95) Google Scholar, 41Franke P. Nothen M. Wang T. Neidt H. Knapp M. Lichtermann D. Weiffenbach O. Mayer P. Höllt V. Propping P. Maier W. Am. J. Med. Genet. 1999; 88: 462-464Crossref PubMed Scopus (59) Google Scholar). The μ-opioid receptor has been the focus of several polymorphism studies because this receptor type is the major target for opiate drugs and also is a main modulator of reward pathways in the brain. A number of single nucleotide polymorphisms have now been described for the μ-opioid receptor gene. Two mutations that modify the N-terminal sequence of the encoded protein (A6V and N40D) have been found repeatedly. N40D, the most frequent mutation, occurs at an allelic frequency of 10–20% depending on the study (23Bergen A.W. Kokoszka J. Peterson R. Long J.C. Virkkunen M. Linnoila M. Goldman D. Mol. Psychiatry. 1997; 2: 490-494Crossref PubMed Scopus (263) Google Scholar, 24Bond C. Laforge K.S. Tian M. Melia D. Zhang S. Borg L. Gong J. Schluger J. Strong J.A. Leal S.M. Tischfield J.A. Kreek M.J. Yu L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9608-9613Crossref PubMed Scopus (974) Google Scholar), whereas the A6V mutation seems less frequent (22Berrettini W.H. Hoehe M.R. Ferraro T.N. DeMaria P.A. Gottheil E. Addiction Biol. 1997; 3: 303-308Crossref Scopus (82) Google Scholar, 23Bergen A.W. Kokoszka J. Peterson R. Long J.C. Virkkunen M. Linnoila M. Goldman D. Mol. Psychiatry. 1997; 2: 490-494Crossref PubMed Scopus (263) Google Scholar, 24Bond C. Laforge K.S. Tian M. Melia D. Zhang S. Borg L. Gong J. Schluger J. Strong J.A. Leal S.M. Tischfield J.A. Kreek M.J. Yu L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9608-9613Crossref PubMed Scopus (974) Google Scholar). Our recent large scale sequence analysis of the μ-opioid receptor gene in 250 patients has not only confirmed the occurrence of the A6V and N40D mutations but also has led to the identification of novel polymorphic variants (25Hoehe M.R. Kopke K. Wendel B. Rohde K. Flachmeier C. Kidd K.K. Berrettini W.H. Church G.M. Hum. Mol. Genet. 2000; 9: 2895-2908Crossref PubMed Google Scholar). The present study shows that some of these natural mutations, which alter the protein sequence of the encoded receptor, may have functional consequences on receptor activity.The N40D Mutant, Subtle Changes in Receptor FunctionalityThe N40D is the most frequent mutation, and several association studies have been conducted. The analysis of alcohol dependence has led to variable results, depending on the populations under study. Although no association was found in two studies (23Bergen A.W. Kokoszka J. Peterson R. Long J.C. Virkkunen M. Linnoila M. Goldman D. Mol. Psychiatry. 1997; 2: 490-494Crossref PubMed Scopus (263) Google Scholar, 42Sander T. Gscheidel N. Wendel B. Samochowiec J. Smolka M. Rommelspacher H. Schmidt L.G. Hoehe M.R. Alcohol Clin. Exp. Res. 1998; 22: 2108-2110PubMed Google Scholar), an increased risk factor for the WT allele or genotype was described in another study (43Town T. Abdullah L. Crawford F. Schinka J. Ordorica P.I. Francis E. Hughes P. Duara R. Mullan M. Am. J. Med. Genet. 1999; 88: 458-461Crossref PubMed Scopus (103) Google Scholar), suggesting a possible protective effect of the mutation. In a group of alcoholics under acute withdrawal, increased dopaminergic sensitivity was found for the variant genotype compared with the WT genotype (44Smolka M. Sander T. Schmidt L.G. Samochowiec J. Rommelspacher H. Gscheidel N. Wendel B. Hoehe M.R. Psychoneuroendocrinology. 1999; 24: 629-638Crossref PubMed Scopus (53) Google Scholar). Recently, a higher frequency of the mutant allele was reported in idiopathic absence epilepsy patients (45Sander T. Berlin W. Gscheidel N. Wendel B. Janz D. Hoehe M.R. Epilepsy Res. 2000; 39: 57-61Crossref PubMed Scopus (46) Google Scholar), indicating that the N40D μ-opioid receptor variant may not only contribute to the etiology of alcoholism but may be implicated in neuronal excitability. A first step toward understanding the molecular mechanisms underlying associations between this mutation and complex behaviors is to determine whether the change in protein sequence alters receptor function.The N40D mutation is located in the N-terminal region of the receptor and results in the loss of a putative glycosylation site. One possible consequence is an alteration of the glycosylation status of the receptor that could potentially modify receptor expression (46Liles W.C. Nathanson N.M. J. Neurochem. 1986; 46: 85-95Google Scholar). However, this was not observed; our data show that receptor densities do not significantly differ from WT when the N40D receptor is transiently expressed in COS cells. This is in accordance with previous studies that have shown that the lack of N-terminal tail (hence the lack of glycosylation sites) does not prevent expression of the receptor in heterologous host cells (47Wang J.B. Imai Y. Eppler C.M. Gregor P. Spivak C.E. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10230-10234Crossref PubMed Scopus (404) Google Scholar, 48Surratt C.K. Johnson P.S. Moriwaki A. Seidleck B.K. Blaschak C.J. Wang J.B. Uhl G.R. J. Biol. Chem. 1994; 269: 20548-20553Abstract Full Text PDF PubMed Google Scholar, 49Wang W.W. Shahrestanifar M. Jin J. Howells R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12436-12440Crossref PubMed Scopus (85) Google Scholar, 50Shahrestanifar M. Wang W.W. Howells R.D. J. Biol. Chem. 1996; 271: 5505-5512Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 51Chaturverdi K. Shahrestanifar M. Howells R.D. Mol. Brain Res. 2000; 76: 64-72Crossref PubMed Scopus (55) Google Scholar).Modifications of pharmacological properties were hardly detectable for the N40D receptor. First, binding affinities of a large set of opioid ligands were highly similar for N40D and WT receptors. Second, maximal activation of the N40D mutant by the agonist DAMGO was slightly enhanced, but no significant change in potency was observed. Third, down-regulation tended to be more prominent for the N40D receptor, but again the difference was not statistically different from WT. Collectively, the data demonstrate that the N40D polymorphism does not impair receptor activity. On the contrary, we may speculate that, although our expression conditions may not have been optimal to reveal marked modifications of N40D functionality, these subtle differences altogether could contribute in enhancing N40D receptor responsivity under specific circumstances. This would be in agreement with the association studies mentioned earlier (24Bond C. Laforge K.S. Tian M. Melia D. Zhang S. Borg L. Gong J. Schluger J. Strong J.A. Leal S.M. Tischfield J.A. Kreek M.J. Yu L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9608-9613Crossref PubMed Scopus (974) Google Scholar, 43Town T. Abdullah L. Crawford F. Schinka J. Ordorica P.I. Francis E. Hughes P. Duara R. Mullan M. Am. J. Med. Genet. 1999; 88: 458-461Crossref PubMed Scopus (103) Google Scholar). These authors have suggested that a potentially enhanced N40D receptor function could translate into a hyperactivity of the endogenous opioid system that would contribute to increase the activity of the hypothalamic-pituitary-adrenal axis and diminish vulnerability to opioid dependence (24Bond C. Laforge K.S. Tian M. Melia D. Zhang S. Borg L. Gong J. Schluger J. Strong J.A. Leal S.M. Tischfield J.A. Kreek M.J. Yu L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9608-9613Crossref PubMed Scopus (974) Google Scholar) or alcoholism (43Town T. Abdullah L. Crawford F. Schinka J. Ordorica P.I. Francis E. Hughes P. Duara R. Mullan M. Am. J. Med. Genet. 1999; 88: 458-461Crossref PubMed Scopus (103) Google Scholar).N152D, Decreased Receptor ExpressionThe N152D polymorphism was not described previously and represents a rare mutation (2/250 individuals). In our study this mutant was expressed at lower receptor density compared with wild-type receptor. Another mutation in the transmembrane domain III of the δ-opioid receptor was previously shown to alter receptor expression (26Befort K. Tabbara L. Bausch S. Chavkin C. Evans C. Kieffer B.L. Mol. Pharmacol. 1996; 49: 216-223PubMed Google Scholar), suggesting that structural integrity of transmembrane domain III, believed to be the most buried transmembrane domain within the helical bundle, is important for receptor stability. Otherwise, no obvious affinity change was observed for all the opioid ligands that we have tested. Therefore, together the data suggest that the general conformation of the N152D receptor is maintained. Whether expression of this receptor variant is modifiedin vivo remains to be determined.S268P, a Loss-of-Function MutationIn this paper we have studied two mutations that occur in the third intracellular domain (i3), a key region for GPR activity. The amphipathic structures of the membrane-proximal regions of i3 have been shown to be critical for productive interaction of GPRs with the α-subunit of G proteins and therefore receptor signaling (see Ref. 52Strader C.D. Fong T.M. Tota M.R. Unterwood D. Dixon R.A.F. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar). This loop is highly conserved across opioid receptor subtypes (53Kieffer B.L. Cell. Mol. Neurobiol. 1995; 15: 615-635Crossref PubMed Scopus (349) Google Scholar), is involved in their coupling to Go/Gi subunits (54Merkouris M. Dragatsis I. Megaritis G. Konidakis G. Zioudrou C. Milligan G. Georgoussi Z. Mol. Pharmacol. 1996; 50: 985-993PubMed Google Scholar), and may interact with other proteins, as was reported for calmodulin and the μ-receptor (55Wang D. Sadée W. Quillan J.M. J. Biol. Chem. 1999; 274: 22081-22088Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). This receptor domain also harbors putative phosphorylation sites that could be involved in the regulation of receptor activity (34Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). The amino acid residue Ser-268 itself represents a putative phosphorylation site for Ca2+/calmodulin-dependent protein kinase II (56Koch T. Kroslak T. Mayer P. Raulf E. Höllt V. J. Neurochem. 1997; 69: 1767-1770Crossref PubMed Scopus (96) Google Scholar). We therefore expected that mutations within this region would alter receptor function.The R265H mutation has some influence on hMOR activity, which leads to decreased signaling. This modification, as well as changes in opioid binding, remains minor presumably because of the conservative nature of the mutation. Otherwise receptor down-regulation was unchanged. It is therefore unlikely that the R265H polymorphism (rare: 1/250) would drastically alter MOR activity, at least for receptor responses that we have investigated.In contrast, the S268P mutation strongly impairs receptor signaling, independently from the agonist or the functional assays that are used. Site-directed mutagenesis of this serine residue into alanine was described previously in the rat μ-opioid receptor (56Koch T. Kroslak T. Mayer P. Raulf E. Höllt V. J. Neurochem. 1997; 69: 1767-1770Crossref PubMed Scopus (96) Google Scholar). The authors found no impairment of agonist-induced inhibition of adenylyl cyclase when a double mutant receptor (S261A/S266A) was expressed in HEK cells, as well as no impairment of agonist-evoked increase in inward K+ currents using the Xenopus oocyte expression system. Very recently, the same authors investigated the human S268P mutant and reported a strong impairment of signaling (57Koch T. Kroslak T. Averbeck M. Mayer P. Schröder H. Raulf E. Höllt V. Mol. Pharmacol. 2000; 58: 328-334Crossref PubMed Scopus (62) Google Scholar), as we do in this study. A likely explanation for the distinct consequences of the alanine or proline mutations is that the alanine mutation leaves the general structure of the loop intact, and therefore functional coupling to the G protein is maintained. On the contrary, the proline residue of the polymorphic mutant disrupts the tertiary structure of this functionally critical loop, thus severely compromising signal transduction. Down-regulation of hMOR S268P was otherwise unaltered, suggesting that the absence of this putative phosphorylation site has no obvious consequences on long term agonist-induced regulation. We cannot exclude, however, that there may be an impairment of rapid agonist-induced desensitization, as was shown for the S261A/S266A mutant in the rat (56Koch T. Kroslak T. Mayer P. Raulf E. Höllt V. J. Neurochem. 1997; 69: 1767-1770Crossref PubMed Scopus (96) Google Scholar) or recently suggested for the human S268P mutant (57Koch T. Kroslak T. Averbeck M. Mayer P. Schröder H. Raulf E. Höllt V. Mol. Pharmacol. 2000; 58: 328-334Crossref PubMed Scopus (62) Google Scholar).Many polymorphic variants have been identified among GPR genes, and some of these mutations account for inherited disorders and diseases (for a review see Ref. 58Spiegel A.M. Annu. Rev. Physiol. 1995; 58: 143-170Crossref Scopus (187) Google Scholar). Most mutations are loss-of-function mutations, and here we provide an additional example for such a mutation (S268P). Like the R265H mutation described here, the S268P mutation was found in one individual only (1/250). The individual is heterozygous for the mutation, suggesting that he may produce an intact version of the receptor from the WT allele. Although rare and monoallelic, this polymorphic mutation is of considerable interest because the consequence of amino acid replacement strongly impairs receptor signaling. Consequently, the level of fully functional receptors in this individual is most probably close to half of that from most individuals, as observed in heterozygous MOR-deficient mice with one allele inactivated (6Matthes H.W.D. Maldonado R. Simonin F. Valverde O. Slowe S. Kitchen I. Befort K. Dierich A. LeMeur M. Dollé P. Tzavara E. Hanoune J. Roques B.P. Kieffer B.L. Nature. 1996; 383: 819-823Crossref PubMed Scopus (1390) Google Scholar). Therefore, future studies will aim at enlarging the DNA sampling to find more examples of the S268P polymorphism. In a preliminary study, we have investigated the possible presence of the mutation in other members of the patient's family, and DNA sequencing has revealed the existence of the same S268P mutation in one of the siblings (one allele also, not shown). An expanded study of this particular mutation should indicate whether individuals homozygous for the mutation do exist. Finally, the clinical examination of patients expressing the S268P polymorphic receptor from one or the two alleles may provide interesting insights into the functional consequences of decreased μ-opioid receptor function in humans.AknowledgementsWe thank J. L. Mandel and W. Berrettini for helpful comments. We are grateful to F. Pattus and P. Chambon for continuing encouragement. We also thank Valérie Favier for participation in the work and Lei Yu for the hMOR cDNA. We are grateful to Philippe Walker, Manon Valiquette, and Thierry Groblewski (AstraZeneca, Montreal, Canada) for their contributions. The opioid system controls pain perception and mood and is generally implicated in a wide variety of behaviors that are essential in facing threatening situations (1Akil H. Watson S.J. Young E. Lewis M.E. Khachaturian H. Walker J.J. Annu. Rev. 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In support of this, gene targeting experiments have shown the absence of morphine-induced analgesia (6Matthes H.W.D. Maldonado R. Simonin F. Valverde O. Slowe S. Kitchen I. Befort K. Dierich A. LeMeur M. Dollé P. Tzavara E. Hanoune J. Roques B.P. Kieffer B.L. Nature. 1996; 383: 819-823Crossref PubMed Scopus (1390) Google Scholar, 7Sora I. Takahashi N. Funada M. Ujike H. Revay R.S. Donovan D.M. Miner L.L. Uhl G.R. Proc.
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