A Single Nucleotide Polymorphic Mutation in the Human μ-Opioid Receptor Severely Impairs Receptor Signaling

Abstract

Large scale sequencing of the human -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 (␤-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, ␤-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 -opioid receptor, which may have an incidence on opioid-regulated behaviors or drug addiction in vivo.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 (1, 2). Opioid receptors also mediate the strong analgesic and addictive actions of opiate drugs. Pharmacological studies indicate that the prototypic opiate morphine, the main clinically useful opiates such as fentanyl or methadone, and the closely related drug of abuse heroin preferably act by activating the -opioid receptor (MOR) 1 rather than ␦-or -opioid receptors (3-5). In support of this, gene targeting experiments have shown the absence of morphine-induced analgesia (6 -10), reward and physical dependence (6), immunosuppression (11, 12), respiratory depression (13), or constipation (14) in MOR-deficient mice, demonstrating unambiguously that the -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, -opioid receptors have been shown to regulate the effects of other substances with high addictive potential such as cocaine or alcohol (15). As an example, in humans, the -receptor antagonists naloxone and naltrexone have been shown not only to reverse heroin overdose but also to alter alcohol consumption (16,...

Genetic variability of the μ-opioid receptor influences intrathecal fentanyl analgesia requirements in laboring women ☆

“…While an increased binding affinity for β-endorphins was reported by Bond et al [4], this was not the case in their study for smaller endogenous opioid peptides or μ-preferring opioid agonists, or for the opioid antagonist, naloxone, nor was this finding replicated by others [2,3] or by the same group recently [26]. In fact, taken together, in vitro studies tend to suggest that the 304A/ G polymorphism is more likely to affect μOR function via alterations in expression, transduction systems or receptor trafficking rather than via altered receptor binding affinity [32].…”

“…A number of single nucleotide polymorphisms (SNPs) have been described for OPRM1 [2,47,48]. An adenine to guanine substitution in gene OPRM1 1 that results in an asparagine residue replacing an aspartate 2 has been reported to occur at a minor allelic frequency of f(−) 0.10-0.30 [4,20,28].…”

Variation at the mu-opioid receptor gene (OPRM1) influences attachment behavior in infant primates

“…Similar to the rhOPRM1 C77G, a polymorphism encoding an amino acid substitution in the N-terminal arm of OPRM1 has independently arisen in humans (OPRM1 A118G) (14). Although its consequences at the molecular level remain to be fully elucidated (14,16,17,31,32), recent in vivo data support the notion that the OPRM1 118G is indeed a gain-of-function variant, because it is associated with increased pain threshold, increased sensitivity to euphorogenic effects of addictive drugs, increased cortisol response after challenge with the opioid antagonist naltrexone, and higher rates of therapeutic response to the opioid antagonist naltrexone in alcoholism (19,20,(33)(34)(35). Findings in rhesus macaques suggest that rhOPRM1 77G has a gain-of-function role as well (15,18), indicating the human and rhesus OPRM1 variants are functionally similar.…”

Altered Levels of Basal Cortisol in Healthy Subjects with a 118G Allele in Exon 1 of the Mu Opioid Receptor Gene

“…This A118G polymorphism (rs1799971) results in an asparagine to aspartic acid substitution at amino-acid position 40 and leads to increased receptor-binding affinity for the endogenous opioid beta-endorphin and increased potency of ion channel activation following beta-endorphin binding (Bond et al, 1998). Two other studies, one in transiently-expressing COS cells (Befort et al, 2001), the other in stable HEK293 cell lines (Beyer et al, 2004), did not replicate these findings. In our prior study (Bond et al, 1998), the variant and prototype receptors were stablytransfected AV-12 cells, which confer N-glycosylation.…”