A common haplotype in the G-protein-coupled receptor gene GPR74 is associated with leanness and increased lipolysis

The G-protein-coupled receptor GPR74 is a novel candidate gene for body weight regulation. In humans, it is predominantly expressed in brain, heart, and adipose tissue. We report a haplotype in the GPR74 gene, ATAG, with allele frequency ~4% in Scandinavian cohorts, which was associated with protection against obesity in two samples selected for obese and lean phenotypes (odds ratio for obesity 0.48 and 0.62; nominal P=.0014 and .014; n=1,013 and 1,423, respectively). In a population-based sample, it was associated with lower waist (P=.02) among 3,937 men and with obesity protection (odds ratio 0.36; P=.036) among those selected for obese or lean phenotypes. The ATAG haplotype was associated with increased adipocyte lipid mobilization (lipolysis) in vivo and in vitro. In human fat cells, GPR74 receptor stimulation and inhibition caused a significant and marked decrease and increase, respectively, of lipolysis, which could be linked to catecholamine stimulation of adipocytes through beta -adrenergic receptors. These findings suggest that a common haplotype in the GPR74 gene protects against obesity, which, at least in part, is caused by a relief of inhibition of lipid mobilization from adipose tissue. The latter involves a cross-talk between GPR74 and beta -adrenoceptor signaling to lipolysis in fat cells.     

The alpha 3 subunit gene of the nicotinic acetylcholine receptor is a candidate gene for ethanol stimulation

Alcohol and nicotine are coabused, and preclinical and clinical data suggest that common genes may influence responses to both drugs. A gene in a region of mouse chromosome 9 that includes a cluster of three nicotinic acetylcholine receptor (nAChR) subunit genes influences the locomotor stimulant response to ethanol. The current studies first used congenic mice to confirm the influential gene on chromosome 9. Congenic F₂ mice were then used to more finely map the location. Gene expression of the three subunit genes was quantified in strains of mice that differ in response to ethanol. Finally, the locomotor response to ethanol was examined in mice heterozygous for a null mutation of the α3 nAChR subunit gene ( Chrna3 ). Congenic data indicate that a gene on chromosome 9, within a 46 cM region that contains the cluster of nAChR subunit genes, accounts for 41% of the genetic variation in the stimulant response to ethanol. Greater expression of Chrna3 was found in whole brain and dissected brain regions relevant to locomotor behavior in mice that were less sensitive to ethanol-induced stimulation compared to mice that were robustly stimulated; the other two nAChR subunit genes in the gene cluster (α5 and β4) were not differentially expressed. Locomotor stimulation was not expressed on the genetic background of Chrna3 heterozygous (+/−) and wild-type (+/+) mice; +/− mice were more sensitive than +/+ mice to the locomotor depressant effects of ethanol. Chrna3 is a candidate gene for the acute locomotor stimulant response to ethanol that deserves further examination.     

The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.     

Association study of the nicotinic acetylcholine receptor alpha4 subunit gene, CHRNA4, in attention-deficit hyperactivity disorder

Attention-deficit hyperactivity disorder (ADHD) is a common childhood-onset psychiatric condition with a strong genetic component. Evidence from pharmacological, clinical and animal studies has suggested that the nicotinic system could be involved in the disorder. Previous studies have implicated the nicotinic acetylcholine receptor α4 subunit gene, CHRNA4 , in ADHD. Particularly, a polymorphism in the exon 2–intron 2 junction of CHRNA4 has been associated with severe inattention defined by latent class analysis. In the current study, we used the transmission disequilibrium test (TDT) to investigate four polymorphisms encompassing this region of CHRNA4 for association with ADHD in a sample of 264 nuclear families from Toronto. No significant evidence of biased transmission was observed for any of the marker alleles for ADHD defined as a categorical trait (all subtypes included), although one haplotype showed marginal evidence of under-transmission. No association was found with the ADHD predominantly inattentive subtype or with symptom dimension scores of inattention. On the contrary, nominally significant evidence of association of individual markers was obtained for the ADHD combined subtype and with teacher-rated hyperactivity–impulsivity scores, with the same haplotype being under-transmitted. Based on our results and others, CHRNA4 may be involved in ADHD; however, its role in ADHD symptomatology remains to be clarified.     

CD4+ T cell response to the human acetylcholine receptor alpha subunit in myasthenia gravis. A study with synthetic peptides

The production of antinicotinic acetylcholine receptor (AcChR) antibodies in myasthenia gravis (MG) is modulated by specific Th (CD4+) lymphocytes that can recognize epitopes on the denatured AcChR alpha subunit. Thirty-two overlapping synthetic peptides corresponding to the complete sequence of human AcChR alpha subunit were used to investigate the anti-alpha subunit response of unselected lymphocytes and of CD8(+)-depleted, CD4(+)-enriched lymphocytes from the blood of nine MG patients and from four healthy controls. One subject was a newly diagnosed MG patient that was tested three times after the development of the disease. An anti-AcChR response of the CD4(+)-enriched cells was present that could be detected only after removal of the CD8+ population and that seems to be related to the clinical conditions of the patient. The high basal rate of the cell proliferation of the unselected unstimulated blood lymphocytes and the normal basal rate observed for the CD8(+)-depleted population suggested the presence of activated CD8+ cells. The study of surface markers of the T cells confirmed the existence of activated CD8+ and CD4+ cells in numbers correlated with the severity of the disease and the results of the in vitro response of the T cells. The anti-AcChR activity of the CD4+ cells in MG may be a useful marker of the activity of the disease and it seems to be influenced by activated CD8+ cells present in the patients' blood.     

A common haplotype of the TNF receptor 2 gene modulates endotoxin tolerance

Endotoxin tolerance is characterized by the suppression of further TNF release upon recurrent exposure to LPS. This phenomenon is proposed to act as a homeostatic mechanism preventing uncontrolled cytokine release such as that observed in bacterial sepsis. The regulatory mechanisms and interindividual variation of endotoxin tolerance induction in man remain poorly characterized. In this paper, we describe a genetic association study of variation in endotoxin tolerance among healthy individuals. We identify a common promoter haplotype in TNFRSF1B (encoding TNFR2) to be strongly associated with reduced tolerance to LPS (p = 5.82 × 10(-6)). This identified haplotype is associated with increased expression of TNFR2 (p = 4.9 × 10(-5)), and we find basal expression of TNFR2, irrespective of genotype and unlike TNFR1, is associated with secondary TNF release (p < 0.0001). Functional studies demonstrate a positive-feedback loop via TNFR2 of LPS-induced TNF release, confirming this previously unrecognized role for TNFR2 in the modulation of LPS response.     

Identification of a brain acetylcholine receptor alpha subunit able to bind alpha-bungarotoxin

Peptides corresponding to sequence segments homologous to an alpha-bungarotoxin (alpha-BGT) binding region on the alpha subunit of the Torpedo nicotinic cholinergic receptor (nAChR) were synthesized for each identified nAChR alpha subunit of the rat nervous system (alpha 1, which is expressed in muscle, and alpha 2, alpha 3, alpha 4, and alpha 5, which are expressed by neurons). The peptides were tested for their ability to directly bind 125I-alpha-BGT and to compete for 125I-alpha-BGT with Torpedo nAChR and with the alpha-BGT-binding component expressed by PC12, a sympathetic neuronal cell line. In addition to peptides of the muscle alpha 1 subunit, peptides corresponding to the sequence of a neuronal subunit, alpha 5, were able to bind 125I-alpha-BGT. Peptides containing the sequence segments 182-201 of the alpha 1 subunit and 180-199 of the alpha 5 subunit competed with Torpedo nAChR for 125I-alpha-BGT binding with IC50 values of 0.5 and 3.5 microM, respectively. Both of these peptides were also able to compete for 125I-alpha-BGT binding with native Torpedo nAChR and with the alpha-BGT-binding protein(s) expressed on PC12 cells. To determine if other sequence segments contribute to form the neuronal alpha-BGT-binding site, overlapping peptides corresponding to the putative extracellular domain of the alpha 5 subunit were synthesized and used both in direct binding assays and in competition experiments. Peptides corresponding to amino acids 16-35 and 180-199 of the alpha 5 subunit directly bound 125I-alpha-BGT and inhibited the binding of toxin to both Torpedo nAChR and PC12 cells. The results of these studies strongly support identification of the alpha 5 subunit as a component of a neuronal alpha-BGT-binding nAChR.     

CHRNB2 is the second acetylcholine receptor subunit associated with autosomal dominant nocturnal frontal lobe epilepsy

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is an uncommon, idiopathic partial epilepsy characterized by clusters of motor seizures occurring in sleep. We describe a mutation of the beta2 subunit of the nicotinic acetylcholine receptor, effecting a V287M substitution within the M2 domain. The mutation, in an evolutionary conserved region of CHRNB2, is associated with ADNFLE in a Scottish family. Functional receptors with the V287M mutation are highly expressed in Xenopus oocytes and characterized by an approximately 10-fold increase in acetylcholine sensitivity. CHRNB2 is a new gene for idiopathic epilepsy, the second acetylcholine receptor subunit implicated in ADNFLE.     

Bone mineral density is associated with estrogen receptor gene polymorphism in men

In order to identify genetic effects of allelic variation on bone mineral density (BMD), association studies have been performed recently. Examining the relation between PvuII and XbaI restriction fragment length polymorphism (RFLPs) at the estrogen receptor (ER alpha) gene and BMD, in women or men, have yielded conflicting results. We analyzed the association between this polymorphism and BMD Z score values of cancellous bone at the 3rd finger in 344 members of nuclear families of European population, Chuvasha, living in Russia. The population sample included 183 males, aged 18-84, and 161 females, aged 23-79. The analysis has been performed separately for both sexes and for both generations (parents and offspring). We used a novel direct haplotyping method, which determines simultaneously each of the PvuII and XbaI RFLPs and their relation to each other. The haplotypes were represented as the combination of both polymorphic sites on the same chromosome, by using P/p and X/x for PvuII and XbaI restriction sites, respectively. The subjects were classified into 3 groups of genotypes: A = PXPX (homozygote for the PX haplotype); B = PXPx, PXpx (the heterozygotes for the PX haplotype); C = PxPx, Pxpx, pxpx (genotypes that are lacking the PX haplotype). The PXPX genotype (A) was associated with higher BMD Z score values in comparison to the genotypes that are lacking the PX haplotype (C), in total males [0.618 vs. -0.133 (p = 0.004)] and for the "sons" generation [0.724 vs. -0.198 (p = 0.02)]. Similar tendency was observed for the "fathers" generation (0.539 vs. -0.085), though the difference did not approach statistical significance (p = 0.087). These findings were not found in the female samples, nor in the "mothers" or "daughters" generations. The question if there are differences in the mode of action of estrogen through its receptor on bone mass, between the genders or between the males' generations, have to be further investigated.     

Agonist- and competitive antagonist-induced movement of loop 5 on the alpha subunit of the neuronal alpha4beta4 nicotinic acetylcholine receptor

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that rapidly convert a chemical signal into an electrical signal. Although the structure of the nAChR is quite well described, the coupling between agonist binding and channel gating is still under debate. In this study, we probed local conformational transitions on the neuronal α4β4 nAChR by specifically tethering a conformation-sensitive fluorescent dye on αG98C located on loop 5 (L5), and simultaneously monitoring fluorescence intensity and current after expression in Xenopus oocytes. The potency of acetylcholine (ACh) was significantly higher in the cysteine mutant and further increased upon tetramethylrhodamine-6-maleimide labeling, suggesting a role of L5 in binding or gating. Structural reorganizations of L5 were shown to occur upon activation, as revealed by the fluorescence intensity increase during ACh exposure. Fluorescence changes were also detected at ACh concentrations lower than needed for current activation, suggesting a movement of L5 for a closed, resting or desensitized state. The competitive antagonist dihydro-β-erythroidine also induced a movement of L5 although at concentrations significantly higher than needed for current inhibition. Consequently L5, located inside the lumen of the pentamer, plays a role in both activation and inhibition of the nAChR.     

The T cell receptor V alpha 3 gene segment is associated with reactivity to p-azobenzenearsonate

The murine T cell receptor V alpha 3 gene segment is associated with reactivity to p-azobenzenearsonate, as indicated by several independent lines of evidence. First, three out of four arsonate-reactive T cell clones tested (two I-Ad-and one I-Ak-restricted) utilized V alpha 3. Second, bulk splenic cultures enriched for arsonate/H-2d and arsonate/H-2k responsive T cells showed increased expression of V alpha 3 mRNA. Third, a V alpha 3-containing alpha chain (Ar-5: arsonate/I-Ad) transferred arsonate responsiveness to an appropriate recipient T cell (O3: ovalbumin/I-Ad). Fourth, an independently derived V alpha 3-expressing T cell clone (2C: alloreactive to Ld) showed a response to arsonate/Ld. Thus, a V alpha 3 gene segment, in conjunction with at least two different J alpha segments (J alpha 20'(Ar-5) and J alpha pHDS58(2C)) and at least three different beta chains (V beta 2(Ar-5), V beta 6(O3), and V beta 8(2C], confers reactivity to arsonate in association with at least three different MHC proteins (I-Ad, I-Ak, and Ld). We suggest that V alpha 3 encodes a protein sequence with a binding site for the arsonate hapten.     

Chromosome location and characterization of the human nicotinic acetylcholine receptor subunit alpha (alpha) 9 (CHRNA9) gene

The human nicotinic acetylcholine receptor (nAChR) subunit alpha9 gene (CHRNA9) codes for a component of the AChR in hair cells of the inner ear. While no direct evidence presently links this gene to known hearing disorders, it may underlie individual susceptibility to acoustic inner ear injury, and is associated with the autoimmune skin disorder Pemphigus vulgaris. Future studies will depend upon a thorough characterization of the nAChR alpha9 gene. CHRNA9 was localized to chromosome 4p15.1-->p14 by FISH analysis. Radiation hybrid mapping further localized the gene between markers D4S405 and D4S496 (Stanford G3 panel), and between markers WI-3875 and D4S1231 (Genebridge 4 panel), representing a distance of approximately 3.1 cR. The D4S405 marker has been linked to a non-syndromic form of hereditary hearing loss, DFNB-25. The gene contains five exons, separated by four introns. Exons 1-5 are 78, 145, 154, 532 and 877 bases, respectively. Introns 1-4 are 294, 1239, 11517, and 4571 bases, respectively. The intron-exon splice junction sites correlate identically with those of the rat alpha9 gene and are nearly identical to those of the human alpha10 gene. Sequence promoter analysis reveals a number of potential regulatory elements, including several in common with the nAChR alpha10 gene, whose expressed protein is assumed to combine with alpha9 in the inner ear.     

A novel integrin beta subunit is associated with the vitronectin receptor alpha subunit (alpha v) in a human osteosarcoma cell line and is a substrate for protein kinase C.

We demonstrate that a novel integrin beta subunit is present in association with the vitronectin receptor (VNR) alpha subunit on the surface of MG-63 human osteosarcoma cells. This beta subunit and the glycoprotein IIIa beta subunit (beta 3) were both found complexed with VNR alpha on MG-63 cells and in at least two other human cell types we examined. Tryptic peptide mapping indicated that the two beta subunits are related but distinct. The novel beta chain, referred to here as beta s, was not recognized by the monoclonal antibody AP3, which recognizes GPIIIa, nor by an antiserum raised against a peptide from the COOH-terminal cytoplasmic domain of beta 3. Both receptor complexes bound to and were specifically eluted from a column containing the cell adhesion peptide GRGDSP. The unique beta subunit became phosphorylated at high stoichiometry when MG-63 cells or AG1523 human fibroblasts were treated with the phorbol-ester tumor promoter phorbol 12-myristate 13-acetate. This phosphorylation occurred mainly on serine and probably at one major site, as determined by phosphotryptic peptide mapping. Protein kinase C phosphorylated the beta s subunit of intact receptor in vitro, at the same site phosphorylated in treated cells, indicating that protein kinase C is likely to be responsible for this phosphorylation in vivo.     

Neuronal nicotinic acetylcholine receptor beta-subunit is coded for by the cDNA clone alpha 4

Acetylcholine receptors (AChRs) with high affinity for nicotine but no affinity for alpha-bungarotoxin, which have been purified from rat and chicken brains by immuno-affinity chromatography, consist of two types of subunits, alpha and beta. The beta-subunits form the ACh binding sites. Putative nicotinic AChR subunit cDNAs alpha 3 and alpha 4 have been identified by screening cDNA libraries prepared from rat PC12 cells and rat brain with cDNA probes encoding the mouse muscle AChR alpha-subunit. Here we determine the amino-terminal amino acid sequence of the rat brain AChR beta-subunit by protein microsequencing to be the same as amino acid residues 27-43 of the protein which could be coded by alpha 4. Further, we present evidence consistent with a subunit stoichiometry of alpha 3 beta 2 for this neuronal nicotinic AChR.     

The alpha4beta2alpha5 nicotinic cholinergic receptor in rat brain is resistant to up-regulation by nicotine in vivo

We used immunoprecipitation with subunit-specific antibodies to examine the distribution of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) that contain the α5 subunit in the adult rat brain. Among the regions of brain we surveyed, the α5 subunit is associated in ∼37% of the nAChRs in the hippocampus, ∼24% of the nAChRs in striatum, and 11–16% of the receptors in the cerebral cortex, thalamus, and superior colliculus. Sequential immunoprecipitation assays demonstrate that the α5 subunit is associated with α4β2* nAChRs exclusively. Importantly, in contrast to α4β2 nAChRs, which are increased by 37–85% after chronic administration of nicotine, the α4β2α5 receptors are not increased by nicotine treatment. These data thus indicate that the α4β2α5 nAChRs in rat brain are resistant to up-regulation by nicotine in vivo , which suggests an important regulatory role for the α5 subunit. To the extent that nicotine-induced up-regulation of α4β2 nAChRs is involved in nicotine addiction, the resistance of the α4β2α5 subtype to up-regulation may have important implications for nicotine addiction.