The most common single nucleotide polymorphism in the coding region of the human mu opioid receptor gene is the A118G variant, an adenine to guanine transition at nucleotide position 118 of the coding sequence of the gene. This polymorphism codes for an asparagine to aspartic acid substitution at amino acid 40 in the amino-terminus, thereby removing a potential extracellular glycosylation site. Using in vitro cellular expression assays, this variant has been reported to change binding of the endogenous agonist beta-endorphin and signaling of the receptor following binding of beta-endorphin. Three clinical studies report that A118G genotype affects opioid antagonist-mediated increases in cortisol levels. These studies demonstrate a functional role of this variant in responses to endogenous and exogenous opioids. To further characterize function, we expressed the prototype and variant receptors in two types of cells (human 293 embryonic kidney cells and Syrian hamster adenovirus-12-induced tumor cells). Stable expression of variant and prototype receptors was characterized by differences in levels of cell surface binding capacity (B max ), forskolin-induced cAMP accumulation, as well as agonist-induced accumulation of cAMP (EC 50 ) for several agonists, but not for beta-endorphin. In contrast, transiently expressed variant receptors showed only a minor difference in cell surface binding capacity compared to the prototype, and no differences in cAMP EC 50 values. Opioid receptors modulate many endogenous physiological and neurobiological systems. They are also essential drug targets in the treatment of pain and pivotally involved in addiction to drugs of abuse, including opiates, cocaine and alcohol. Mu, kappa, and delta opioid receptors (encoded by the OPRM1, OPRK1, and OPRD1 genes, respectively), are G-protein coupled receptors (GPCRs), which couple to inhibitory (G i /G o ) heterotrimeric G-proteins. Several polymorphic variants of the human OPRM1 gene have been described (e.g. Bergen et al. 1997;Berrettini et al. 1997;Bond et al. 1998;Hoehe et al. 2000), including variants that alter amino acid sequence of the receptor. Some of these naturally occurring sequence alterations have also been reported to alter properties of receptor function, studied using in vitro expression systems (Bond et al. 1998;Koch et al. 2000;Befort et al. 2001;Margas et al. 2007).The A118G variant of the human mu opioid receptor gene (OPRM1) is in the coding region of the first exon and is the single nucleotide polymorphism (SNP) with the highest overall allelic frequency of any OPRM1 coding region variant reported, although its heterozygosity varies widely across populations, from 1% to 2% frequencies of the minor (118G) allele reported in African Americans up to 50% in Japanese (e.g. Bond et al. 1998;Gelernter et al. 1999;Szeto et al. 2001;Tan et al. 2003;Bart et al. 2004;Kim et al. 2004). This SNP encodes an amino acid substitution of asparagine to

Rat genome U34A (Affymetrix) oligonucleotide microarrays were used to analyze changes in gene expression in the caudate putamen (CPu) of Fischer rats induced by 1 and 3 days of "binge" cocaine (or saline) administration. A triplicate array assay of pooled RNA of each treatment group was used to evaluate the technical variability and sensitivity of microarrays. Cocaine-regulated genes were identified using the Affymetrix MAS 5.0 and Data Mining Tool v. 3. Eighty-nine upregulated and eight downregulated genes/ESTs were found after 1 day of "binge" cocaine. Following 3 days of cocaine treatment we identified 21 upregulated and 17 downregulated genes/ESTs. RNase protection assays of selected genes confirmed reliability of changes identified by the microarrays at the level of > or =1.40-fold increase. Many genes upregulated in the CPu by cocaine were immediate early genes for transcription factors and for "effector" proteins (e.g., vesl/Homer1a, Arc, synaptotagmin IV). Acute "binge" cocaine also increased mRNA levels for glutamate receptor GluR2, dopamine receptor D1, and a number of phosphatases. Genes downregulated by cocaine include several genes associated with energy metabolism in mitochondria, as well as the phosphatydylinositol-4 kinase and the regulator of G-protein signaling protein 4 (RGS4). A differential expression of somatostatin receptor SSTR2, not known to be a cocaine-responsive gene, as well as the clock gene Per2, were found by microarrays and confirmed by RNase protection assay. These results demonstrate the potential of microarrays in profiling gene expression with > or =40% increase or > or =14% decrease in mRNA levels for discovery of novel cocaine-responsive genes.

In this study we report that human phosphatidylethanolamine-binding protein (hPBP) facilitates heterotrimeric G protein-coupled signaling. In Xenopus laevis oocytes, coexpression of hPBP with human opioid receptor, human ␦ opioid receptor, or human somatostatin receptor 2 evoked an agonist-induced increase in potassium conductance of G protein-activated inwardly rectifying potassium channels. This activation of heterotrimeric G protein signaling in oocytes could also be elicited by injection of bacterially overexpressed and purified hPBP. Stimulatory effect was pertussis toxinsensitive and present even in the absence of coexpressed receptors. Additionally, an increase in G protein-mediated inhibition of adenylate cyclase activity, measured by the inhibition of forskolin-mediated cAMP accumulation, could be detected in HEK293 and NIH3T3 cells after expression of hPBP and in Xenopus oocytes after injection of hPBP. As

The rat 1z-opioid receptor (rMOR1), expressed in human embryonic kidney 293 (HEK293) cells, showsa desensitization to the inhibitory effect of the js agonist DAMGO on adenylate cyclase activity within 4 h of DAMGO preincubation. To investigate the role of calcium/calmodulin-dependent protein kinase Il (CaM kinase Il) on 1s-opioid receptor desensitization, we coexpressed rMOR1 and constitutively active CaM kinase Il in HEK293 cells. This coexpression led to a faster time course of agonist-induced desensitization of the~-opioid receptor. The increase ofdesensitization could not be observed with a te-opioid receptor mutant (S261A!S266A) that lacks two putative CaM kinase Il phosphorylation sites in the third intracellular loop. In addition, injection of CaM kinase Il in Xenopus oocytes led only to desensitization of expressed rMOR1, but not of an S261 A! S266A receptor mutant. These results suggest that phosphorylation of Ser 251 and Ser266 by CaM kinase II is involved in the desensitization of the 1s-opioid receptor. Key Words:~t-Opioid receptor -Desensitization -Calcium! calmodulin -dependent protein kinase phosphorylation-G protein-activated channelXenopus oocytes. J. Neurochem. 69, 1767-1770 (1997).Extensive physiological, behavioral, and pharmacological studies have defined three major types of opioid receptors, designated~.t, 6, and K (Wood and Iyengar, 1988;Corbett et al., 1993). The activation of all three opioid receptor types can inhibit adenylate cyclase. In addition, opiates have been shown to open K~channels and to close Ca 2~channels (North, 1993).In Xenopus oocytes expressing opioid receptors and the G protein-gated, inwardly rectifying K channel (known as KGA or GIRK1), application of the~.t agonist [D-Ala2, MePhe4,G1y5-ol] enkephalin (DAMGO) evoked a dose-dependent increase in K + conductance (Chen and Yu, 1994). Chen and Yu (1994) demonstrated that repeated agonist stimulation of the~i-opioid receptor coexpressed with GIRK 1 in Xenopus oocytes resulted in desensitization of the u-activated current response. Using the Xenopus expression system, it has also been reported that phosphorylation by protein kinase C and Ca2~/calmodulin-dependentprotein kinase II (CaM kinase II) modulates desensitization of the human p~-opioidreceptor (Mestek et al., 1995), whereas receptor desensitization of the 6-opioidreceptor is influenced by protein kinase C (Ueda et al., 1994(Ueda et al., , 1995. Due to the experimental conditions, it is not clearly shown in these experiments whether the target of these kinases is the opioid receptor, the G protein, the K + channel, or some intermediary factors. Moreover, Kovoor et al. (1995) suggested that the desensitization of the rat~t-opioid receptor in the Xenopus oocyte system is receptor-independent and caused by an inactivation of the K + channel itself.To investigate further the role of CaM kinase II in opioid receptor desensitization, we used, in addition to the Xenopus oocyte expression system, the human embryonic kidney 293 (HEK293) cell line for expressing the ...

To fully understand human biology and link genotype to phenotype, the phase of DNA variants must be known. Here we present a comprehensive analysis of haplotype-resolved genomes to assess the nature and variation of haplotypes and their pairs, diplotypes, in European population samples. We use a set of 14 haplotype-resolved genomes generated by fosmid clone-based sequencing, complemented and expanded by up to 372 statistically resolved genomes from the 1000 Genomes Project. We find immense diversity of both haploid and diploid gene forms, up to 4.1 and 3.9 million corresponding to 249 and 235 per gene on average. Less than 15% of autosomal genes have a predominant form. We describe a ‘common diplotypic proteome’, a set of 4,269 genes encoding two different proteins in over 30% of genomes. We show moreover an abundance of cis configurations of mutations in the 386 genomes with an average cis/trans ratio of 60:40, and distinguishable classes of cis- versus trans-abundant genes. This work identifies key features characterizing the diplotypic nature of human genomes and provides a conceptual and analytical framework, rich resources and novel hypotheses on the functional importance of diploidy.

The decrease in mu-opioid receptor activity after chronic agonist exposure (1 microM [D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin) is largely due to kinase-mediated phosphorylation of intracellular receptor domains. We have recently shown that the substitution of two putative Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) phosphorylation sites, S261 and S266, by alanines in the third intracellular loop of the rat mu-opioid receptor (rMOR1) confers resistance to CaMK II-induced receptor desensitization. In the present study, we show that the injection of active CaMK II in Xenopus laevis oocytes led to the desensitization of S261A but not S266A receptor mutant, indicating that S266 is the primary CaMK II phosphorylation site of the rMOR1. For the corresponding phosphorylation site in the human mu-opioid receptor (hMOR), an allelic variation S268P has been recently identified. After expression in X. laevis oocytes and human embryonic kidney 293 cells, this human S268P receptor and a corresponding rat S266P receptor mutant revealed a loss of CaMK II-induced receptor desensitization and a decreased G protein coupling compared with the wild-type receptors. Our results suggest that serines 266 (rMOR1) and 268 (hMOR) play crucial role in receptor desensitization and signaling and that the allelic variation S268P results in a human receptor type with a weaker but persistent G protein coupling after agonist treatment.

Alterations in expression of a cannabinoid receptor (CNR1, CB1), and of fatty acid amide hydrolase (FAAH) that degrades endogenous ligands of CB1, may contribute to the development of addiction. The 385C>A in the FAAH gene and six polymorphisms of CNR1 were genotyped in former heroin addicts and control subjects (247 Caucasians, 161 Hispanics, 179 African Americans and 19 Asians). In Caucasians, long repeats (≥14) of 18087–18131 (TAA)8–17 were associated with heroin addiction (P = 0.0102). Across three ethnicities combined, a highly significant association of long repeats with heroin addiction was found (z = 3.322, P = 0.0009). Point-wise significant associations of allele 1359A (P= 0.006) and genotype 1359AA (P= 0.034) with protection from heroin addiction were found in Caucasians. Also in Caucasians, the genotype pattern, 1359G>A and −6274A>T, was significantly associated with heroin addiction experiment wise (P= 0.0244). No association of FAAH 385C>A with heroin addiction was found in any group studied.

The analysis of genetic variation in candidate genes is an issue of central importance in pharmacogenomics. The specific approaches taken will have a critical impact on the successful identification of disease genes, the molecular correlates of drug response, and the establishment of meaningful relationships between genetic variants and phenotypes of biomedical and pharmaceutical importance in general. Against a historical background, this article distinguishes different approaches to candidate gene analysis, reflecting different stages in human genome research. Only recently has it become feasible to analyze genetic variation systematically at the ultimate level of resolution, ie, the DNA sequence. In this context, the importance of haplotype-based approaches to candidate gene analysis has at last been recognized; the determination of the specific combinations of variants for each of the two sequences of a gene defined as a haplotype is essential. An up-to-date summary of such maximum resolution data on the amount, nature, and structure of genetic variation in candidate genes will be given. These data demonstrate abundant gene sequence and haplotype diversity. Numerous individually different forms of a gene may exist. This presents major challenges to the analysis of relationships between genetic variation, gene function, and phenotype. First solutions seem within reach. The implications of naturally occurring variation for pharmacogenomics and "personalized" medicine are now evident. Future approaches to the identification, evaluation, and prioritization of drug targets, the optimization of clinical trials, and the development of efficient therapies must be based on in-depth knowledge of candidate gene variation as an essential prerequisite.