A single nucleotide polymorphism (SNP) in the human -opioid receptor gene (OPRM1 A118G) has been widely studied for its association in a variety of drug addiction and pain sensitivity phenotypes; however, the extent of these adaptations and the mechanisms underlying these associations remain elusive. To clarify the functional mechanisms linking the OPRM1 A118G SNP to addiction and analgesia phenotypes, we derived a mouse model possessing the equivalent nucleotide/amino acid substitution in the Oprm1 gene. Mice harboring this SNP (A112G) demonstrated several phenotypic similarities to humans carrying the A118G SNP, including reduced mRNA expression and morphine-mediated antinociception. We found additional phenotypes associated with this SNP including significant reductions of receptor protein levels, morphine-mediated hyperactivity, and the development of locomotor sensitization in mice harboring the G112 allele. In addition, we found sex-specific reductions in the rewarding properties of morphine and the aversive components of naloxone-precipitated morphine withdrawal. Further cross-species analysis will allow us to investigate mechanisms and adaptations present in humans carrying this SNP.analgesia ͉ morphine ͉ -opioid receptor ͉ sex differences ͉ SNP M OPR (-opioid receptors) are integrally involved in the modulation of several pathways including pain, stress, and drug reward. Genetic mutations of the MOPR alter endogenous and exogenous opioidergic function, thus influencing behavior. A single nucleotide polymorphism (SNP) in exon 1 of the -opioid receptor gene (OPRM1), in which an adenine-toguanine substitution (A118G) exchanges an asparagine for an aspartic acid at a putative N-glycosylation site (N40D), is common in persons of European (15Ϫ30%) and Asian ancestry (49-60%), with lower prevalence in African American and Hispanic populations (1-3). The A118G SNP has been associated with an altered vulnerability to opioid addiction (4-6), a decreased response to opioid-induced analgesia (7,8), and an enhanced response to therapies for alcohol (9, 10) and nicotine addiction (7, 11). However, some association studies report divergent effects (12, 13), and sex-specific associations (14-16), underscoring the need to understand the functional significance of this SNP.Examination of the A118G variant in heterologous expression systems has yielded inconsistent results. Initial in vitro studies indicated that expression of the human G118 MOPR variant in AV-12 cells increases the binding affinity of ␤-endorphin to 3-fold higher than that of the human A118 MOPR and results in higher potency for activation of G protein-coupled potassium channels (17), suggesting a gain of function of the receptor. However, other studies report no differences in agonist binding, functional coupling, or desensitization (18). Using an allelic expression assay, Zhang and colleagues (19) found a 1.5-fold reduction in allele-specific mRNA expression in postmortem brain tissue and also a 10-fold reduction in protein levels in CHO cells expressi...

Kappa opioid receptor (KOR) agonists produce analgesic and anti-pruritic effects, but their clinical application was limited by dysphoria and hallucinations. Nalfurafine, a clinically used KOR agonist, does not cause dysphoria or hallucinations at therapeutic doses in humans. We found that in CD-1 mice nalfurafine produced analgesic and anti-scratch effects dose-dependently, like the prototypic KOR agonist U50,488H. In contrast, unlike U50,488H, nalfurafine caused no aversion, anhedonia, or sedation or and a low level of motor incoordination at the effective analgesia and anti-scratch doses. Thus, we established a mouse model that recapitulated important aspects of the clinical observations. We then employed a phosphoproteomics approach to investigate mechanisms underlying differential KOR-mediated effects. A large-scale mass spectrometry (MS)-based analysis on brains revealed that nalfurafine perturbed phosphoproteomes differently from U50,488H in a brain-region specific manner after 30-min treatment. In particular, U50,488H and nalfurafine imparted phosphorylation changes to proteins found in different cellular components or signaling pathways in different brain regions. Notably, we observed that U50,488H, but not nalfurafine, activated the mammalian target of rapamycin (mTOR) pathway in the striatum and cortex. Inhibition of the mTOR pathway by rapamycin abolished U50,488H-induced aversion, without affecting analgesic, anti-scratch, and sedative effects and motor incoordination. The results indicate that the mTOR pathway is involved in KOR agonist-induced aversion. This is the first demonstration that phosphoproteomics can be applied to agonist-specific signaling of G protein-coupled receptors (GPCRs) in mouse brains to unravel pharmacologically important pathways. Furthermore, this is one of the first two reports that the mTOR pathway mediates aversion caused by KOR activation.

Salvinorin A, acrylamido]morphinan hydrochloride), and 3FLB (diethyl 2,4-di-[3-fluorophenyl]-3,7-dimethyl-3,7-diazabicyclo[3.3.1]nonane-9-one-1,5-dicarboxylate) are structurally distinctly different from U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate], the prototypic selective agonist. Here, we investigated their in vitro pharmacological activities on receptors expressed in Chinese hamster ovary cells and in vivo antiscratch and antinociceptive activities in mice. All three compounds showed high selectivity for the opioid receptor (KOR) over the opioid receptor (MOR) and ␦ opioid receptor (DOR) and nociceptin or orphanin FQ receptors. In the guanosine 5Ј-O-(3-[35 S]thio)triphosphate ([ 35 S]GTP␥S) binding assay, all three were full agonists on the KOR. The rank order of affinity and potency for the KOR was TRK-820 Ͼ Ͼ U50,488H ϳ salvinorin A Ͼ Ͼ 3FLB. TRK-820 acted as a partial agonist on MOR and DOR, whereas salvinorin A and 3FLB showed no activities on these receptors. Salvinorin A, TRK-820, and 3FLB caused internalization of the human KOR in a dosedependent manner. Interestingly, although salvinorin A and U50,488H had similar potencies in stimulating [ 35 S]GTP␥S binding, salvinorin A was about 40-fold less potent than U50,488H in promoting internalization. Following 4-h incubation, all three compounds induced down-regulation of the human KOR, with salvinorin A causing a lower extent of down-regulation. Although TRK-820 was potent and efficacious against compound 48/80-induced scratching, salvinorin A showed low and inconsistent effects, and 3FLB was inactive. In addition, salvinorin A and 3FLB were not active in the acetic acid abdominal constriction test. The discrepancy between in vitro and in vivo results may be due to in vivo metabolism of salvinorin A and 3FLB and possibly to their effects on other pharmacological targets.At least three types of opioid receptors, , ␦, and , mediate pharmacological effects of opioid drugs and physiological actions of endogenous peptides (for review, see Chang, 1984;Mansour et al., 1988). Opioid receptors are coupled to G i /G o proteins to affect several different effectors, including inhibition of adenylyl cyclase, enhancement of K ϩ conductance, decrease in Ca 2ϩ conductance, and activation of p42/p44 mitogen-activated protein kinases (for review, see Law et al., 2000). In addition, opioid receptors are shown to act through Gz to inhibit adenylyl cyclase and G 16 to activate phospholipase C (Lai et al., 1995;Lee et al., 1998), and opioid receptors stimulate Na , ␦, and opioid receptors of several species have been cloned (for review, see Kieffer, 1995;Knapp et al., 1995). In addition, a receptor with high sequence similarity to the opioid receptors, termed the ORL1 receptor, was cloned and found to be coupled to G i /G o proteins (for review, see Kieffer, 1995;Knapp et al., 1995). Subsequently, the endogenous ligand for the ORL1 receptor was identified and named noThis work was supported by National I...

No abstract

The sodium-hydrogen exchanger regulatory factors (NHERF-1 and NHERF-2) are a family of adaptor proteins characterized by the presence of two tandem PDZ protein interaction domains and a C-terminal domain that binds the cytoskeleton proteins ezrin, radixin, moesin, and merlin. The NHERF proteins are highly expressed in the kidney, small intestine, and other organs, where they associate with a number of transporters and ion channels, signaling proteins, and transcription factors. Recent evidence has revealed important associations between the NHERF proteins and several G protein-coupled receptors such as the beta2-adrenergic receptor, the kappa-opioid receptor, and the parathyroid hormone receptor, as well as growth factor tyrosine kinase receptors such as the platelet-derived growth factor receptor and the epidermal growth factor receptor. This review summarizes the emerging data on the biochemical mechanisms, physiologic outcomes, and potential clinical implications of the assembly and disassembly of receptor/NHERF complexes.