GABA(A) receptor beta isoform protein expression in human alcoholic brain: interaction with genotype

Chronic alcohol misuse by human subjects leads to neuronal loss in regions such as the superior frontal cortex. Reduced GABA transmission may mediate this. The expression of GABA(A) receptor beta(1), beta(2), and beta(3) isoform proteins was analyzed by western blotting in vulnerable (superior frontal cortex) and spared (primary motor cortex) cortical tissue obtained at autopsy from Caucasian subjects, and the effect of genotypes of candidate genes for alcoholism assessed. There was a significant regional difference in global isoform expression, but no significant overall group difference in beta(2) or beta(3)expression between controls and alcoholics undifferentiated by genotype in either cortical region. There were significant, regionally selective, interactions of DRD2B, SLC1A2 and APOE genotypes with beta protein expression when alcoholics were compared with controls. In each instance possession of the alcoholism-associated allele increased the beta(2):beta(3) ratio in the pathologically vulnerable region, by two distinct mechanisms. The SFC beta(2):beta(3) ratio in DRD2B-B2,B2 alcoholics was 22% higher than that in DRD2B-B1,B1 alcoholics, and 17% higher than that in DRD2B-B2,B2 controls. The SFC beta(2):beta(3) ratio in SLC1A2A603 homozygote alcoholics was 25% higher than that in alcoholics with at least one 603G allele, and 75% higher than that in SLC1A2A603 homozygote controls. The SFC beta(2):beta(3) ratio in alcoholics lacking an APOE epsilon3 allele was 73% higher than that in alcoholics with at least one epsilon3 allele, and 70% higher than that in controls without an epsilon3 allele. ADH1C genotype also differentiated cases and controls, but the effect was not localized. GABRB2 and GRIN2B genotypes were associated with significant regional differences in the pattern of beta subunit expression, but this was not influenced by alcoholism status. DRD2A and SLC6A4 genotypes were without significant effect. A restricted set of genotypes may influence subunit expression in this group of high-consumption alcoholics.     

Association of estrogen receptor beta gene polymorphism with bone mineral density

Chromosomal mapping of the human estrogen receptor beta (ERbeta) gene by fluorescence in situ hybridization (FISH) reveals that ERbeta is located at human chromosome 14, region q23-24.1, where the aberration of DNA copy number in the bone disorders is frequently involved. Then, we investigated the association between dinucleotide (cytosine-adenine; CA) repeat polymorphism located in the flanking region of ERbeta gene and bone mineral density (BMD) in 204 healthy postmenopausal Japanese women. The genotype was classified into "A" through "O" according to the number of the repeats, from 18 to 32. BMD was expressed in Z score (a deviation from the weight-adjusted average BMD of each age using the standard deviation as a unit). When we separate the subjects into two groups bearing at least one I allele (26 CA repeats) and those who did not, the former subjects had significantly higher Z score of L2-4 BMD (mean +/- standard deviation; 0.674 +/- 1.53 vs 0.128 +/- 1.38; P = 0.027). These data suggest that genetic variation at the ERbeta locus may be associated with some determinants for BMD and the possible involvements of this polymorphism in the cause of postmenopausal osteoporosis in Japanese women.     

Characterization of estrogen receptor beta2 and expression of the estrogen receptor subtypes alpha, beta1, and beta2 in the protandrous black porgy (Acanthopagrus schlegeli) during the sex change process

Estrogens play an important role in many physiological processes in both female and male vertebrates, mediated by specific nuclear receptor, estrogen receptors (ERs). We have isolated a third ER (ERbeta2), which was found to contain 2004 nucleotides including an open reading frame that encodes 667 amino acids. We have also cloned ERalpha and ERbeta1 from the published information (GenBank accession nos. AY074780 and AY074779) and investigated the expression pattern of these ER subtypes in the gonads during gonad sex change of black porgy by quantitative polymerase chain reaction. Maturity stages can be divided into five stages during the sex change process from immature male to female (immature male, mature male, male of mostly testis, male of mostly ovary and mature female). The expression of ERalpha mRNA was highest in the ovary of mature female, followed by the testis of mature male and testicular portion of mostly testis. ERbeta1 expression was higher in the mature testis and ovary than in the gonads of other maturity stages. In contrast to that, ERbeta2 was highest in the ovary of mature female, and significantly lower levels of ERbeta2 expression were observed in the gonads of the other maturity stages. The present study describes the molecular characterization of ERbeta2, and documents the expression changes of three ER subtypes during sex change process of the protandrous black porgy.     

Positive selection within the Schizophrenia-associated GABA(A) receptor beta(2) gene

The gamma-aminobutyric acid type-A (GABA(A)) receptor plays a major role in inhibitory neurotransmissions. Intronic SNPs and haplotypes in GABRB2, the gene for GABA(A) receptor beta(2) subunit, are associated with schizophrenia and correlated with the expression of two alternatively spliced beta(2) isoforms. In the present study, using chimpanzee as an ancestral reference, high frequencies were observed for the derived (D) alleles of the four SNPs rs6556547, rs187269, rs1816071 and rs1816072 in GABRB2, suggesting the occurrence of positive selection for these derived alleles. Coalescence-based simulation showed that the population frequency spectra and the frequencies of H56, the haplotype having all four D alleles, significantly deviated from neutral-evolution expectation in various demographic models. Haplotypes containing the derived allele of rs1816072 displayed significantly less diversity compared to haplotypes containing its ancestral allele, further supporting positive selection. The variations in DD-genotype frequencies in five human populations provided a snapshot of the evolutionary history, which suggested that the positive selections of the D alleles are recent and likely ongoing. The divergence between the DD-genotype profiles of schizophrenic and control samples pointed to the schizophrenia-relevance of positive selections, with the schizophrenic samples showing weakened selections compared to the controls. These DD-genotypes were previously found to increase the expression of beta(2), especially its long isoform. Electrophysiological analysis showed that this long beta(2) isoform favored by the positive selections is more sensitive than the short isoform to the inhibition of GABA(A) receptor function by energy depletion. These findings represent the first demonstration of positive selection in a schizophrenia-associated gene.     

Identification of novel ryanodine receptor 1 (RyR1) protein interaction with calcium homeostasis endoplasmic reticulum protein (CHERP)

The ryanodine receptor type 1 (RyR1) is a homotetrameric Ca(2+) release channel located in the sarcoplasmic reticulum of skeletal muscle where it plays a role in the initiation of skeletal muscle contraction. A soluble, 6×-histidine affinity-tagged cytosolic fragment of RyR1 (amino acids 1-4243) was expressed in HEK-293 cells, and metal affinity chromatography under native conditions was used to purify the peptide together with interacting proteins. When analyzed by gel-free liquid chromatography mass spectrometry (LC-MS), 703 proteins were identified under all conditions. This group of proteins was filtered to identify putative RyR interacting proteins by removing those proteins found in only 1 RyR purification and proteins for which average spectral counts were enriched by less than 4-fold over control values. This resulted in 49 potential RyR1 interacting proteins, and 4 were selected for additional interaction studies: calcium homeostasis endoplasmic reticulum protein (CHERP), endoplasmic reticulum-Golgi intermediate compartment 53-kDa protein (LMAN1), T-complex protein, and phosphorylase kinase. Western blotting showed that only CHERP co-purified with affinity-tagged RyR1 and was eluted with imidazole. Immunofluorescence showed that endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle. A combination of overexpression of RyR1 in HEK-293 cells with siRNA-mediated suppression of CHERP showed that CHERP affects Ca(2+) release from the ER via RyR1. Thus, we propose that CHERP is an RyR1 interacting protein that may be involved in the regulation of excitation-contraction coupling.     

Rab11 regulates the recycling of the beta2-adrenergic receptor through a direct interaction

The beta2ARs (beta(2)-adrenergic receptors) undergo ligand-induced internalization into early endosomes, but then are rapidly and efficiently recycled back to the plasma membrane, restoring the numbers of functional cell-surface receptors. Gathering evidence suggests that, during prolonged exposure to agonist, some beta2ARs also utilize a slow recycling pathway through the perinuclear recycling endosomal compartment regulated by the small GTPase Rab11. In the present study, we demonstrate by co-immunoprecipitation studies that there is a beta2AR-Rab11 association in HEK-293 cells (human embryonic kidney cells). We show using purified His(6)-tagged Rab11 protein and beta2AR intracellular domains fused to GST (glutathione transferase) that Rab11 interacts directly with the C-terminal tail of beta2AR, but not with the other intracellular domains of the receptor. Pull-down and immunoprecipitation assays revealed that the beta2AR interacts preferentially with the GDP-bound form of Rab11. Arg(333) and Lys(348) in the C-terminal tail of the beta2AR were identified as crucial determinants for Rab11 binding. A beta2AR construct with these two residues mutated to alanine, beta2AR RK/AA (R333A/K348A), was generated. Analysis of cell-surface receptors by ELISA revealed that the recycling of beta2AR RK/AA was drastically reduced when compared with wild-type beta2AR after agonist washout, following prolonged receptor stimulation. Confocal microscopy demonstrated that the beta2AR RK/AA mutant failed to co-localize with Rab11 and recycle to the plasma membrane, in contrast with the wild-type receptor. To our knowledge, the present study is the first report of a direct interaction between the beta2AR and a Rab GTPase, which is required for the accurate intracellular trafficking of the receptor.     

Homologous desensitization of signalling by the beta (beta) isoform of the human thromboxane A2 receptor

Thromboxane (TX) A(2) is a potent stimulator of platelet activation/aggregation and smooth muscle contraction and contributes to a variety of pathologies within the vasculature. In this study, we investigated the mechanism whereby the cellular responses to TXA(2) mediated through the TPbeta isoform of the human TXA(2) receptor (TP) are dynamically regulated by examining the mechanism of agonist-induced desensitization of intracellular signalling and second messenger generation by TPbeta. It was established that TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA(2) mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser(145) within intracellular domain (IC)(2) has been identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser(239) and Ser(357) within its IC(3) and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser(239) or Ser(357), through site directed mutagenesis, impaired desensitization while mutation of both Ser(239) and Ser(357) almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser(145) partially impairs. TPbeta signalling while GRK2/3 phosphorylation at both Ser(239) and Ser(357) within its IC(3) and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization. Thromboxane (TX) A(2) is a potent stimulator of platelet aggregation and smooth muscle contraction and contributes to a variety of vascular pathologies. Herein the mechanism whereby the cellular responses to TXA(2) mediated through the TPbeta isoform of the human TXA(2) receptor (TP) are dynamically regulated was investigated by examining the mechanism of its agonist-induced desensitization of intracellular signalling and second messenger generation. TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA(2) mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser(145) within intracellular domain (IC)(2) was identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser(239) and Ser(357) within its IC(3) and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser(239) or Ser(357), through site directed mutagenesis, impaired desensitization while mutation of both Ser(239) and Ser(357) almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser(145) partially impairs TPbeta signalling while GRK2/3 phosphorylation at both Ser(239) and Ser(357) within its IC(3) and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization.     

HIV-1 interaction with human mannose receptor (hMR) induces production of matrix metalloproteinase 2 (MMP-2) through hMR-mediated intracellular signaling in astrocytes

Astrocytes are susceptible to HIV-1 infection. We have recently demonstrated that human mannose receptor (hMR) is directly involved in CD4-independent HIV-1 infection of astrocytes. The apparent paradox between the vivid binding affinity of HIV-1 gp120 protein to hMR and the low efficiency of hMR-mediated HIV-1 infection raises the possibility that HIV-1 binding to hMR alone may negatively affect astrocyte function. In this study, we examined the relationship between HIV-1 interaction with hMR and the production of matrix metalloproteinases (MMPs) in astrocytes. We took advantage of an astroglial cell line U87.MR stably expressing hMR as an in vitro astrocyte model system and human primary astrocytes, and demonstrated that HIV-1 binding to astrocytes induced the production of MMP-2. This induction appeared to be most potent with M-tropic HIV-1 viruses. Increased MMP-2 production was not due to hMR-mediated HIV-1 entry and/or HIV-1 viral gene expression, as the transfection of HIV-1 proviral DNA did not result in MMP-2 production, and the infection of AT-2-treated HIV-1 viruses did not inhibit MMP-2 production. Direct involvement of hMR in HIV-induced MMP-2 production was confirmed by the inhibition of the yeast mannan, an hMR ligand antagonist, and an anti-hMR serum. Furthermore, HIV-induced MMP-2 production in astrocytes was shown to involve hMR-mediated intracellular signaling. Taken together, these results suggest that HIV-1 binding to astrocytes in the absence of HIV-1 viral entry is sufficient to alter astrocyte function through hMR-mediated intracellular signaling. In addition, these results provide new evidence to support the notion that hMR is capable of eliciting intracellular signaling upon ligand binding.     

The dinucleotide (CA) repeat polymorphism of estrogen receptor beta but not the dinucleotide (TA) repeat polymorphism of estrogen receptor alpha is associated with venous ulceration

Venous ulcers are the predominant form of chronic wound in the elderly, accounting for around 70% of all cases. The steroid sex hormone estrogen plays a crucial role in normal human skin maintenance and during cutaneous wound repair following injury. Estrogen can reverse age-related impaired wound healing by dampening the inflammatory response and increasing matrix deposition at the wound site. The molecular actions of estrogen are mediated through two nuclear sex steroid hormone receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta). We have conducted a case-control study to investigate whether dinucleotide repeat polymorphisms in the estrogen receptor genes are associated with venous ulceration in the UK Caucasian population. Genomic fragments containing the ERalpha dinucleotide (TA)(n) repeat polymorphism or the ERbeta dinucleotide (CA)(n) repeat polymorphism were amplified by polymerase chain reaction in subject DNA samples and genotyped according to fragment length by capillary electrophoresis. There was no evidence to suggest that the TA repeat polymorphism of ERalpha was associated with venous ulceration. However, the CA*18 allele of the ERbeta CA repeat polymorphism was significantly associated with venous ulceration (n = 120, OR = 1.8, 95% CI = 1.1-2.8, P = 0.02). When the CA repeats alleles were grouped together into either low (L < or = 18) or high (H > 18) numbers of CA repeats, the low (L) repeat allele was significantly associated with venous ulceration (OR = 1.5, 95% CI = 1.0-2.2, P = 0.03). Our results show that a specific ERbeta variant is associated with impaired healing in the elderly, predisposing individuals to venous ulceration.     

Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.

Mitogen-activated protein (MAP) kinases bind tightly to many of their physiologically relevant substrates. We have identified a new subfamily of murine serine/threonine kinases, whose members, MAP kinase-interacting kinase 1 (Mnk1) and Mnk2, bind tightly to the growth factor-regulated MAP kinases, Erk1 and Erk2. MNK1, but not Mnk2, also binds strongly to the stress-activated kinase, p38. MNK1 complexes more strongly with inactive than active Erk, implying that Mnk and Erk may dissociate after mitogen stimulation. Erk and p38 phosphorylate MNK1 and Mnk2, which stimulates their in vitro kinase activity toward a substrate, eukaryotic initiation factor-4E (eIF-4E). Initiation factor eIF-4E is a regulatory phosphoprotein whose phosphorylation is increased by insulin in an Erk-dependent manner. In vitro, MNK1 rapidly phosphorylates eIF-4E at the physiologically relevant site, Ser209. In cells, Mnk1 is post-translationally modified and enzymatically activated in response to treatment with either peptide growth factors, phorbol esters, anisomycin or UV. Mitogen- and stress-mediated MNK1 activation is blocked by inhibitors of MAP kinase kinase 1 (Mkk1) and p38, demonstrating that Mnk1 is downstream of multiple MAP kinases. MNK1 may define a convergence point between the growth factor-activated and one of the stress-activated protein kinase cascades and is a candidate to phosphorylate eIF-4E in cells.     

Estrogen receptor alpha interaction with estrogen response element half-sites from the rat prolactin gene

Estrogen regulation of the rat prolactin gene requires sequences within the DNase I hypersensitive site II (HSII). We have used overexpressed mouse estrogen receptor alpha (ERalpha) protein to study interactions of ERalpha with an imperfect estrogen response element (ERE) and four ERE half-site sequences from HSII. We confirmed that ERalpha has higher affinity for ERE half-sites than for the imperfect ERE. As expected, the imperfect ERE formed a complex with ERalpha similar to that between mERalpha and a consensus ERE in gel shift assays. The ERalpha complex with half-sites, however, had faster mobility on a 4% polyacrylamide gel than the ERalpha complex with a consensus ERE, indicating that the complexes had different compositions. Ferguson analysis revealed that the ERalpha/half-site complex had a larger molecular weight and higher negative charge than the ERalpha/consensus ERE complex. Similar results were observed with purified human ERalpha, showing that the ERalpha/half-site complex contained only ERalpha and oligonucleotides. These results are best explained by a model in which a dimer of ERalpha is bound to two half-site oligonucleotides. We propose that two ERalpha dimers may interact with the four ERE half-sites in HSII to influence estrogen regulation of this gene.