Site Mutation in the Rat μ‐Opioid Receptor Demonstrates the Involvement of Calcium/Calmodulin‐Dependent Protein Kinase II in Agonist‐Mediated Desensitization

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166

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,...

Section: N40d N152d R265h and S268p Polymorphism In The -Opioid Rementioning

confidence: 71%

Section: N40d N152d R265h and S268p Polymorphism In The -Opioid Rementioning

confidence: 89%

Section: N40d N152d R265h and S268p Polymorphism In The -Opioid Rementioning

confidence: 99%

See 1 more Smart Citation

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

Befort

Filliol

Décaillot

et al. 2001

234

166

“…Whether phosphorylation of Ser 363 and Thr 358 involved a GRK remains to be demonstrated. Some reports suggested that agonist-induced phosphorylation of the opioid receptors could be mediated by Ca 2+ /calmodulin-dependent protein kinase II (26), or by mitogen-activated protein kinase (27), but the amino acid motif surrounding Thr 358 or Ser 363 does not correspond to that of either kinase. Thus, it is tempting to suggest that a yet unknown Ser/Thr kinase is responsible for the DPDPEinduced phosphorylation of DOR.…”

Section: Discussionmentioning

confidence: 99%

Hierarchical Phosphorylation of δ-Opioid Receptor Regulates Agonist-induced Receptor Desensitization and Internalization

Kouhen

Wang

Solberg

et al. 2000

Treatment of HEK293 cells expressing the δ-opioid receptor with agonist [D-Pen 2,5 ]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr 358 and Ser 363 , were phosphorylated, with Ser 363 being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser 363 as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr 358 and Ser 363 , with Thr 358 being involved in the receptor internalization.Prolonged exposure to opioid drugs often produces tolerance, dependence, and addiction, and the molecular mechanisms underlying these phenomena are still poorly understood. Opioid receptors belong to the G protein-coupled receptor (GPCR)1 superfamily (1, 2). Therefore, one component of opioid tolerance is likely to be mediated by a phosphorylationdependent desensitization of the receptor. Phosphorylation of the prototypic β 2 -adrenergic receptor by protein kinases, including the G protein-coupled receptor kinases (GRKs), promotes the association with the receptor of inhibitory proteins called arrestins (3,4). This association uncouples the receptor from the G proteins and promotes targeting of activated receptors to clathrin-coated pits for subsequent internalization, thus blunting receptor signaling.Agonist-induced phosphorylation of opioid receptors has been demonstrated in different systems (5-10). Concrete demonstration of the phosphorylation of δ-opioid receptor (DOR) was first reported by Pei and colleagues (6). Although the sites of agonist-dependent phosphorylation have not yet been identified, truncation of the carboxyl tail (C-tail) of DOR showed that major phosphorylation sites are localized within this domain (11,12 Phosphorylation of DOR appears to be the mechanism for agonist-induced receptor desensitization (6,13,14). 5 ]enkephalin (DPDPE)-induced phosphorylation of DOR seems to involve one or more GRKs (6), and Ala substitution of the last four carboxylterminal Ser and Thr of the receptor impaired the GRK-and arrestin-mediated receptor desensitization (13). In Xenopus oocytes, co-expression of GRK3 and β-arrestin 2 resulted in an increased rate of agonist-induced homologous desensitization of DOR (13). However, truncation of the COOH-terminal 31 amino acids of DOR did not affect the agonist-induced desensitization of the receptor in CHO cells (15), unlike what Zhao and coll...

“…For example, phosphorylation by G protein receptor kinases following agonist treatment has been proposed to play a role in opioid receptor desensitization and tolerance (2). Other serine/ threonine kinases such as protein kinase C and calcium/calmodulin-dependent kinase II in some systems may also regulate opioid receptors (3).…”

mentioning

confidence: 99%

Tyrosine Phosphorylation of the κ-Opioid Receptor Regulates Agonist Efficacy

Appleyard¹,

McLaughlin²,

Chavkin³

2000

To explore the role of highly conserved tyrosine residues in the putative cytoplasmic domains of the seven-transmembrane G protein-coupled opioid receptors, we expressed the rat -opioid receptor (KOR) in Xenopus oocytes and then activated the intrinsic insulin receptor tyrosine kinase. KOR activation by the agonist U69593 produced a strong increase in potassium current through coexpressed G protein-gated inwardly rectifying potassium channels (K IR 3). Brief pretreatment with insulin caused a 60% potentiation of the KOR-activated response. The insulin-induced increase in -opioid response was blocked by the tyrosine kinase inhibitor genistein. In contrast, insulin had no effect on the basal activity of K IR 3, suggesting that KOR is the target of the tyrosine kinase cascade. Mutation of tyrosine residues to phenylalanines in either the first or second intracellular loop of KOR to produce KOR(Y87F) and KOR(Y157F) had no effect on either the potency or maximal effect of U69593. However, neither KOR(Y87F)-nor KOR(Y157F)-mediated responses were potentiated by insulin treatment. Insulin pretreatment shifted the dose-response curve for U69593 activation of KOR by increasing the maximal response without changing the EC 50 value for U69593. These results suggest that insulin increases the efficacy of KOR activation by phosphorylating two tyrosine residues in the first and second intracellular loops of the receptor. Thus, tyrosine phosphorylation may provide an important mechanism for modulation of G protein-coupled receptor signaling.Opioid receptors are widely expressed throughout the nervous system, and opioid drugs affect pain perception, learning and memory, epilepsy, and food intake, among other diverse functions (1). A common mechanism for post-translational regulation of protein function is by phosphorylation, and phosphorylation of opioid receptors is likely to regulate opioid actions. For example, phosphorylation by G protein receptor kinases following agonist treatment has been proposed to play a role in opioid receptor desensitization and tolerance (2). Other serine/ threonine kinases such as protein kinase C and calcium/calmodulin-dependent kinase II in some systems may also regulate opioid receptors (3).To date, few studies have reported any effects of tyrosine kinases on acute opioid receptor function. Tyrosine residues in the putative cytoplasmic domains of G protein-coupled receptors are extremely common, and regulation of opioid receptor signaling by tyrosine kinase cascades would provide a powerful mechanism of cellular coordination. Indeed, a portion of the agonist-induced internalization of the -opioid receptor is dependent on tyrosine kinase activation (4 -6). Opioid receptor activation has been shown to increase tyrosine kinase activity (7,8), and the agonist-induced internalization mediated by tyrosine kinase could potentially involve a process that is directly activated by opioid receptors. These findings are consistent with data on the ␤ 2 -adrenergic receptor in which tyrosine phosphorylation of...