Allelic Variation S268P of the Human μ-Opioid Receptor Affects Both Desensitization and G Protein Coupling

Opioid analgesics are highly effective in controlling pain elicited by noxious stimuli, but their repeated use causes tolerance and physical dependence and can lead to addiction (1, 2). Individual predisposition toward abuse likely involves genetic factors in addition to behavioral ones (3-7). As the main molecular target of opioid analgesics, the opioid receptor (MOR) 1 represents an obvious candidate in the search for genetic variations that affect the response of an individual to opioid analgesics. Genotyping of the human MOR gene has revealed several single nucleotide polymorphisms (SNPs) that exist in the general population (7-9). One SNP mapping to the N-terminal region of the seven-transmembrane structure of MOR has already been shown to alter the binding affinity for ␤-endorphin (8), but the clinical significance of this genetic variation remains uncertain (10). We hypothesize that by analyzing the effect of additional polymorphisms on the complex signaling behavior of MOR, we might gain further clues as to the molecular basis of opioid drug response. Opioid receptors are functionally dynamic, seven-transmembrane-spanning proteins known to transmit signals via the GTP-binding proteins, G i /G o . However, they cannot be understood adequately in terms of acute G protein activation alone. Activation of MOR also results in long term changes associated with receptor phosphorylation, internalization, and alterations in gene expression (1). Recently, a direct interaction between the ubiquitous calcium-sensitive regulatory protein calmodulin (CaM) and the third intracellular (i3) loop of MOR has been demonstrated (11). The importance of the i3 loop to G protein coupling (12) and CaM binding (11,13), the presence of multiple phosphorylation consensus sequences (14), and recent observations that this region regulates spontaneous basal G protein coupling (11, 13, 15) all strengthen the rationale for examining i3 loop polymorphisms. A range of functional consequences could emanate from alterations in the i3 loop. At least three allelic, nonsynonymous SNPs that alter the MOR-i3 loop have so far been identified in single individuals, leading to the human variants 9). The allele frequency of these three sporadic variants is unknown. In this study we have genotyped exon 3 of the hMOR gene in 252 subjects (Coriell collection) to obtain more information on allele frequency and discover any further MOR variants. Examination of S268P-MOR had already revealed differences in G protein coupling efficiency and in desensitization kinetics, differences that could stem from structural changes induced by proline insertion and loss of the CaM-kinase II phosphorylation site at Ser 268 (14). Moreover, Befort et al. (16) have proposed that the S268P substitution significantly impaired agoniststimulated G protein coupling of MOR, a finding that we have re-examined in the present study.Recently, a significant level of basal G protein coupling has been demonstrated for MOR (11,13,15). Basal signaling is observable even at relatively low...

supporting

confidence: 47%

Section: Discussionmentioning

confidence: 66%

mentioning

confidence: 99%

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Single Nucleotide Polymorphisms in the Human μ Opioid Receptor Gene Alter Basal G Protein Coupling and Calmodulin Binding

Wang¹,

Quillan²,

Winans³

et al. 2001

“…Studies of desensitization of MOP activation of GIRK channels in Xenopus laevis oocytes have also reported significant reductions in opioid agonist responses with prolonged incubations (8,24,64,65). In studies where MOP desensitization is promoted by coexpressed GRK and ␤-arrestin, the desensitization to high efficacy agonists such as DAMGO proceeds with a similar time course to that in the LC.…”

Section: Table II Relative Efficacy For Opioid Agonist Inhibition Of mentioning

confidence: 87%

Opioid Agonists Have Different Efficacy Profiles for G Protein Activation, Rapid Desensitization, and Endocytosis of Mu-opioid Receptors

Borgland

Connor

Osborne

et al. 2003

144

179

The differential ability of various -opioid receptor (MOP) agonists to induce rapid receptor desensitization and endocytosis of MOP could arise simply from differences in their efficacy to activate G proteins or, alternatively, be due to differential capacity for activation of other signaling processes. We used AtT20 cells stably expressing a low density of FLAG-tagged MOP to compare the efficacies of a range of agonists to 1) activate G proteins using inhibition of calcium channel currents (I Ca ) as a reporter before and after inactivation of a fraction of receptors by ␤-chlornaltrexamine, 2) produce rapid, homologous desensitization of I Ca inhibition, and 3) internalize receptors. Relative efficacies determined for G protein coupling were [Tyr-D-Ala-Gly-MePhe-Glyol]enkephalin (DAMGO) (1) > methadone (0.98) > morphine (0.58) > pentazocine (0.15). The same rank order of efficacies for rapid desensitization of MOP was observed, but greater concentrations of agonist were required than for G protein activation. By contrast, relative efficacies for promoting endocytosis of MOP were DAMGO (1) > methadone (0.59) > > morphine (0.07) > pentazocine (0.03). These results indicate that the efficacy of opioids to produce activation of G proteins and rapid desensitization is distinct from their capacity to internalize -opioid receptors but that, contrary to some previous reports, morphine can produce rapid, homologous desensitization of MOP.Tolerance to the analgesic and other effects of opioid drugs, such as morphine, undermines their use in long term treatment (1). Although analgesic tolerance is likely to be a complex phenomenon, an understanding of how -opioid (MOP) 1 receptors are regulated by opioid agonists will lead to important insights into this phenomenon. MOPs are similar to many other G protein-coupled receptors (GPCR) in that they undergo desensitization within several minutes of stimulation by agonists (2-4), and continued agonist exposure results in removal of receptors from the cell surface (5-7). MOP phosphorylation is increased as a consequence of agonist exposure, leading to a commonly held assumption that receptor phosphorylation is essential for MOP desensitization and internalization (5, 8 -10). The mechanism that may be responsible for uncoupling MOP from G protein activation and then promoting receptor sequestration is as follows: phosphorylation of the agonist occupied MOP by a G protein receptor kinase (GRK), subsequent binding of ␤-arrestins to the phosphorylated receptor, and removal of MOP from the cell surface via a clathrin-dependent process (11-15). The enhanced analgesic potency of morphine and diminished analgesic tolerance in ␤-arrestin2 knockout mice suggest that the capacity of opioid agonists to cause receptor desensitization and endocytosis is one of the key cellular mechanisms underlying tolerance (16). Phosphorylation of MOP by protein kinase C (PKC) or calmodulin-dependent kinase II can also reduce the capacity of MOP to activate G proteins (8,17), also contributing to opioid tole...

“…This region is thought to be involved with G protein binding along with the third intracellular loop and the C-terminal tail (26,27). It is possible that regulation of the phosphorylation state of these residues alters the affinity of KOR for the GTP-bound state of the G protein, and so more receptors are associated with G proteins and available to activate the downstream effectors.…”

Section: Discussionmentioning

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...