Characterization of the promoter of the human farnesyltransferase beta subunit and the impact of the transcription factor OCT-1 on its expression.

Farnesyltransferase (FTase) enables about 100 proteins to interact with cellular membranes by catalyzing the posttranslational addition of a farnesyl group. Farnesylated proteins provide important functions and inhibitors against the ß-subunit of the heterodimer of FTase are intensively studied in clinical and preclinical trials. However, very little is known about the transcriptional regulation of the ß-subunit. The examined promoter region of the human FTase ß-subunit gene (FNTB) showed significant basal promoter activity in HEK-293 and in HeLa cells. We were able to locate the core promoter at -165 to -74. Ten potential binding sites of the transcription factor OCT-1 were detected. Three could be confirmed using EMSA super shift experiments. OCT-1 overexpression and knockdown confirmed it as an important regulator of FNTB expression. Our results provide a basis for further research on FNTB/OCT-1 regulation, its inhibitors and diseases influenced by both such as colon carcinoma or diabetes mellitus.

Characterization of the G protein-coupled receptor kinase 6 promoter reveals a functional CREB binding site.

BACKGROUND: G protein-coupled receptor kinase 6 (GRK6) is part of the G protein-coupled receptor kinase family, whose members act as key regulators of seven-transmembrane receptor signalling. GRK6 seems to play a role in regulation of inflammatory processes, but mechanisms of transcriptional regulation of GRK6 expression in inflammatory cell lines have not been characterized. Protein kinase C (PKC) signalling is also involved in inflammatory regulation and an impact of PKC activation on GRK6 protein expression was described previously. Thus, the aim of this study was to 1) characterize the GRK6 promoter, and 2) investigate a potential influence of PKC on GRK6 expression. METHODS: Five deletion constructs of the GRK6 promoter were cloned. After transient transfection into a human T cell line, promoter activity was assessed using luciferase reporter gene assays. Putative transcription factor binding sites were identified, mutated, and binding was investigated using electrophoretic mobility shift assays (EMSA). Following stimulation with a PKC activator, GRK6 expression on mRNA and protein levels was assessed by reverse transcriptase qPCR and Western blots. RESULTS: Investigation of the GRK6 promoter revealed a putative cAMP responsive element (CRE), whose mutation led to decreased promoter activity (p = 0.0006). Functionality of the CRE binding protein (CREB) binding site was verified in EMSA blots. Stimulation with a PKC activator resulted in decreased GRK6 promoter activity (p = 0.0027), mRNA (p = 0.04) and protein expression. CONCLUSION: We characterized the human GRK6 promoter and identified promoter activity to be influenced by a CREB binding site. PKC might be one determinant contributing to altered GRK6 expression.

Exploring the putative self-binding property of the human farnesyltransferase alpha-subunit.

Farnesylation is an important post-translational protein modification in eukaryotes. Farnesylation is performed by protein farnesyltransferase, a heterodimer composed of an α- (FTα) and a ß-subunit. Recently, homodimerization of truncated rat and yeast FTα has been detected, suggesting a new role for FTα homodimers in signal transduction. We investigated the putative dimerization behaviour of human and rat FTα. Different in vitro and in vivo approaches revealed no self-dimerization and a presumably artificial formation of homotrimers and higher homo-oligomers in vitro. Our study contributes to the clarification of the physiological features of FTase in different species and may be important for the ongoing development of FTase inhibitors.

Decreased expression of miR-133a but not of miR-1 is associated with signs of heart failure in patients undergoing coronary bypass surgery.

OBJECTIVES: Coronary artery disease (CAD)-associated ischemic heart failure is characterized by dysregulated gene expression which is partly mediated by microRNAs (miRNAs). While the muscle-specific miR-1 and miR-133 are involved in cardiac development and hypertrophy, their role in heart failure resulting from CAD is unknown. We, therefore, tested the hypothesis that cardiac miR-1 and miR-133 expression is associated with signs of heart failure in patients undergoing coronary artery bypass grafting. METHODS: 83 patients were included in this prospective study. Cardiac index and vascular pressures were measured under general anesthesia and the miRNA expression was quantified (RNase protection assay and real-time PCR) from samples of the right atrial myocardium. RESULTS: miR-133 expression decreased significantly with increased severity of heart failure, as indicated by a greater New York Heart Association (NYHA) functional class (p = 0.014) and increased pulmonary artery occlusion pressure (p = 0.045). Furthermore, patients with NT-proBNP concentrations >1,800 pg/ml showed a 25% decrease in miR-133 expression compared to patients with concentrations <300 pg/ml (p = 0.023). In contrast, no associations were detected for miR-1 expression. CONCLUSIONS: In surgical CAD patients, a decreased miR-133 expression is associated with variables characteristic of heart failure. This supports a role for miR-133 but not miR-1 in the adaption to and/or remodeling of the ischemic heart.

A functional GNAQ promoter haplotype is associated with altered Gq expression and with insulin resistance and obesity in women with polycystic ovary syndrome.

OBJECTIVES: The G-protein Gq, encoded by GNAQ, is involved in glucose metabolism. The GNAQ promoter harbours three polymorphisms. The TT(-695/-694)GC polymorphism was already shown to affect Gq transcription. Accordingly, we (i) characterized the GNAQ promoter polymorphisms G(-173)A and G(-168)A, (ii) investigated potential influences upon the TT(-695/-694)GC polymorphism and (iii) studied the associations with metabolic abnormalities in polycystic ovary syndrome (PCOS). METHODS: Characterization of the polymorphisms was performed with electrophoretic mobility shift assays and reporter assays. Inhibition of lipolysis and Gq expression were measured in adipocytes isolated from female mammary tissue. We genotyped 266 healthy Caucasians, 265 women with PCOS, and 293 healthy, age-matched female controls to associate GNAQ promoter polymorphisms and haplotypes with anthropometric and metabolic variables. RESULTS: The A(-168) allele was associated with significantly decreased transcriptional activity and altered transcription factor binding, whereas the G(-173)A polymorphism appeared functionally silent. Linkage and haplotype frequencies analysis resulted in four common haplotypes. In adipose tissue, a 44% higher Gq mRNA concentration was observed in homozygous GC(-695/-694)-G(-168) haplotypes compared with homozygous TT(-695/-694)-G(-168) haplotypes (P=0.046). This was associated with increased insulin inhibition of lipolysis in isolated adipocytes. In PCOS patients, the homozygous GC-G haplotype was associated with decreased insulin resistance and body mass index (BMI) compared with the homozygous TT-G haplotype (homeostatic model assessment of insulin resistance: 3.4+/-0.4 vs. 5.6+/-0.7 mmol/l x mmol/l2, P=0.001; fasting insulin: 86.6+/-11.9 vs. 128.8+/-16.5 pmol/l, P=0.003; BMI: 29.3+/-1.2 vs. 33.9+/-1.3 kg/m2, P=0.002). No association with BMI was found in healthy women. CONCLUSION: G(-168)A is functionally relevant and in linkage with TT(-695/-694)GC. GNAQ promoter diplotypes are associated with insulin resistance and obesity in PCOS.

A novel functional haplotype in the human GNAS gene alters Galphas expression, responsiveness to beta-adrenoceptor stimulation, and peri-operative cardiac performance.

AIMS: Cardiac overexpression of the beta-adrenoceptor-coupled G-protein subunit Galphas in mice enhances inotropic responses to sympathetic stimulation, but evokes cardiomyopathy with increasing age. We tested whether functional single nucleotide polymorphisms (SNPs) in the human Galphas (GNAS) gene modulate Galphas expression and assessed functional consequences. METHODS AND RESULTS: Sequencing the promoter and intron 1 of GNAS revealed 11 SNPs resulting in three common haplotypes. Haplotype *3 constructs exhibited significantly higher promoter activity than haplotypes *1 and *2, resulting in a more than 50% higher Galphas mRNA expression in homozygous *3 carriers (*3/*3) than in heterozygous (*3/-) and negative *3 (-/-) carriers (P = 0.002). Basal, Galphas- (via NaF and GTP) and isoproterenol-stimulated adenylyl cyclase (AC) activities were also significantly higher in *3/*3 than in *3/- and -/- carriers. In contrast, direct AC activation via forskolin was independent of GNAS haplotypes. Furthermore, haemodynamic measurements in 137 coronary artery bypass patients revealed a higher cardiac index in *3/*3 carriers than in *3/- and -/- carriers (P = 0.025) associated with a lower NYHA functional class (P = 0.040) and serum NT-proBNP concentrations (P = 0.002). CONCLUSION: SNPs in regulatory regions of GNAS impact upon Galphas expression and stimulated cAMP formation in human hearts in vitro and upon cardiac performance in vivo.

A novel promoter polymorphism in the human gene GNAS affects binding of transcription factor upstream stimulatory factor 1, Galphas protein expression and body weight regulation.

OBJECTIVES: Body weight regulation is under complex control involving the central nervous system and peripheral pathways. The beta-adrenoceptor Galphas protein system plays an important role in heart rate regulation and lipid mobilization suggesting a key role for the stimulatory G protein Galphas in body weight regulation. METHODS: We sequenced the whole GNAS promoter to identify a functional variant which results in altered Galphas expression. We genotyped 110 participants of a randomized placebo-controlled weight loss trial who were under a low calorie diet and were additionally treated with either placebo or 15 mg sibutramine daily for 54 weeks and associated the respective alleles with regard to treatment outcome using an intention-to-treat analysis. RESULTS: A G>A transition at position -1211 the human GNAS promoter (minor allele frequency=0.36) was identified resulting in altered upstream stimulatory factor 1 transcription factor binding, promoter activity, Galphas expression, and lipolysis. Under a low calorie diet -1211GG genotypes lost significantly more weight compared with A-allele carriers (placebo group: 1211GG, 7.5+/-0.4 versus -1211A, 4.5+/-0.3 kg, P=0.020). Sibutramine was effective only in A-allele carriers whereas GG genotypes showed no additional weight loss under sibutramine but showed the strongest increases in resting heart rate (8.5 bpm; 95% confidence interval: 2.7-14.21 bpm; P=0.005) and systolic blood pressure (9.1 mmHg, 95% confidence interval: 3.1-15.1; P=0.004) compared with placebo. CONCLUSIONS: Determination of GNAS promoter alleles may identify obese individuals who lose weight easily under lifestyle changes alone but also those who benefit from adjunct sibutramine therapy.

Quantification of G protein Gaalphas subunit splice variants in different human tissues and cells using pyrosequencing.

The G protein Galphas is derived from four alternatively spliced transcripts, two long variants (Galphas(L)+CAG and Galphas(L)-CAG), which include an extra 45-bp segment, and two short variants (Galphas(S)+CAG and Galphas(S)-CAG). The long and short forms differ in each case by splicing in or out of a serine residue encoded at the 3' end of the variable exon 3. The relative expression of all four variants in human tissues is poorly investigated due to experimental limitations. We therefore established a method for reliable relative mRNA quantification of these splice variants based on the Pyrosequencing technology, and determined Galphas transcript ratios in various human tissues and cells. Galphas(S)/Galphas ratio was highest in blood mononuclear cells (0.84 +/- 0.02, n = 16) and lowest in the brain (0.51 +/- 0.14, n = 3). The different ranges resulted from differences in Galphas(S)+CAG ratios, which ranged from a total Galphas ratio of 0.32 +/- 0.07 (n = 12) in heart tissue to 0.57 +/- 0.03 (n = 16) in blood mononuclear cells (p < 0.0001), whereas the Galphas(S)-CAG ratio was rather constant and ranged from 0.22 +/- 0.04 (n = 7) in retinoblastoma cells to 0.27 +/- 0.04 in lymphocytes (p = 0.19). The Galphas(L)+CAG ratio ranged from 0.02 +/- 0.02 in heart tissue to 0.05 +/- 0.01 in retinoblastoma cells, with a varying proportion of Galphas(L)-CAG, which ranged from 0.14 +/- 0.02 in blood mononuclear cells to 0.41 +/- 0.08 in heart tissue. Stimulation of immortalized B lymphoblasts with isoproterenol resulted in significant changes of splice variant ratios. Our data indicate that changes of long and short ratios of Galphas in different tissues affected Galphas(L)-CAG and Gas(S)+CAG rather than Galphas(L)+CAG and Galphas(S-)CAG. Furthermore, stimulation of cells seemed to affect splice variant ratios. These results are, therefore, suggestive of different biological functions of these variants.

The human G protein beta4 subunit: gene structure, expression, Ggamma and effector interaction.

The aim of this study was the characterization of the human Gbeta4 subunit of heterotrimeric G proteins. Human Gbeta4 is widely expressed. Its gene is located on chromosome 3 with a genomic structure indistinguishable from that of the genes of Gbeta1 to Gbeta3, but entirely different from Gbeta5. In vitro translation co-precipitation analyses revealed that Gbeta4 can form stable dimers with Ggamma1, Ggamma2, Ggamma3, Ggamma4, Ggamma5, Ggamma7, Ggamma10, Ggamma11, Ggamma12, and Ggamma13, dimers which were also able to stimulate phospholipase beta2.

Characterization of the splice variant Gbeta3v of the human G-protein Gbeta3 subunit.

A polymorphism (C825T) in the gene of the G-protein Gbeta3 (GNB3) has been the subject of numerous studies which have shown that the 825T-allele is associated with several cardiovascular and metabolic disorders. Furthermore, the T-allele is associated with the occurrence of the splice variant Gbeta3s which has been implicated in the pathogenesis of these disorders. Here, we characterise a novel splice variant of GNB3, termed Gbeta3v, which is generated by alternative splicing of parts from intron 9 as a novel exon 10a. Gbeta proteins belong to the superfamily of propeller proteins composed of seven regular WD-domains. In Gbeta3v, four of these WD-domains are retained but the protein has a novel C terminus. Gbeta3v forms dimers with Ggamma3 and Ggamma12 but these Gbetagamma complexes do not stimulate phospholipase C-beta2. Thus, a physiological role of Gbeta3v remains to be established. Gbeta3v transcripts are detectable in a wide variety of cells and tissues including fibroblasts, B lymphoblasts, retinoblastoma cells, retina, brain, umbilical cord and colon. However, there is no association with an allele of the GNB3 C825T polymorphism, which suggests that Gbeta3v does not contribute to the complex phenotype observed in association with the 825T-allele.

Identification and characterization of G beta 3s2, a novel splice variant of the G-protein beta 3 subunit.

The T-allele of a polymorphism (C825T) in the gene for the G-protein beta 3 subunit (GNB3) is associated with cardiovascular and metabolic disorders, distinct cellular features and altered drug responses. The molecular mechanisms that give rise to this complex phenotype have been linked to the occurrence of G beta 3s, a splice variant of GNB3. G beta 3s is predominantly expressed in cells with the 825T-allele. In the present study we describe the identification and characterization of an additional G beta 3 splice variant referred to as G beta 3s2. Its mRNA is expressed in heart, blood cells and tumour tissue, and its expression is also tightly associated with the GNB3 825T-allele. G beta 3s2 is generated by alternative splicing using non-canonical splice sites. G beta subunits belong to the family of propeller proteins and consist of seven regular propeller blades. Transcripts for G beta 3s2 are lacking 129 bp of the coding sequence of the wild-type G beta 3 protein. Thus the predicted structure consists of only six propeller blades, which resembles the structure of G beta 3s. Co-immunoprecipitation analyses indicated that G beta 3s2 dimerizes with different G gamma subunits, e.g. G gamma 5, G gamma 8(C) and G gamma 12. In Sf9 insect cells, expression of G beta 3s2 together with G gamma 12 enhances receptor-stimulated activation of G alpha(i2). Expression of G beta 3s2 in mammalian cells activated the mitogen-activated protein kinase cascade. Together, these results suggest that G beta 3s2 is a biologically active G beta variant which may play a role in the manifestation of the complex phenotype associated with the 825T-allele.

Signal transduction of somatostatin in human B lymphoblasts.

Somatostatin (SST) and somatostatin receptors (SSTR) are widely distributed in lymphoid tissues. Here, we report on the stimulatory effects of SST in Epstein-Barr virus-immortalized B lymphoblasts. By RT-PCR, we demonstrated the exclusive expression of the somatostatin receptor isoform 2A (SSTR2A) in B lymphoblasts. Addition of SST rapidly increased the cytosolic free calcium concentration [Ca(2+)](i) maximally by about 200 nM, with an EC(50) of 1.3 nM, and stimulated the formation of inositol phosphates. Furthermore, SST increased binding of guanosine 5'-O-(3-thiotriphosphate) by 50% above basal. These effects were partly inhibited by pertussis toxin (PTX), which indicates the involvement of PTX-sensitive G proteins. We provide further evidence that Galpha(16,) a PTX-insensitive G protein confined to lymphohematopoietic cells, is involved in the otherwise unusual coupling of SSTR2A to phospholipase C activation. In addition, SST activated extracellular regulated kinases and induced a 3.5-fold stimulation of DNA synthesis and a 4.4-fold stimulation of B lymphoblast proliferation, which was accompanied by an enhanced immunoglobulin formation. Thus SST exerts a growth factor-like activity on human B lymphoblasts.

Identification and ethnic distribution of major haplotypes in the gene GNB3 encoding the G-protein beta3 subunit.

The 825T-allele of the gene GNB3 encoding the G protein beta3 subunit is associated with hypertension and obesity, and identifies individuals highly responsive to diuretic therapy. Gbeta3s, a Gbeta3 protein variant generated by alternative splicing in carriers of the 825T-allele, is linked to increased signal transduction and is a potential cause for the observed pathophysiology. Here, we searched the entire GNB3 gene for additional polymorphisms and analysed their prevalence in Caucasian, black African and Asian populations. We detected six novel single nucleotide polymorphisms which were termed according to their location as G76A, G1906T, G2906A, A3882C, G5177A, and G5249A. Furthermore, we found a CACA-insertion-deletion polymorphism at position 6496. Genotyping and association studies resulted in the definition of two major GNB3 haplotypes, termed 'C-haplotype' (alleles 825C, 3882A, 5249G, 6496CACA-) and 'T-haplotype' (alleles 825T, 3882C, 5249A, 6496CACA+). Molecular modelling studies revealed that the pre-mRNA structures of both haplotypes exhibit marked differences which may account for the alternative splicing predominantly observed with the T-haplotype. The prevalence of these haplotypes in major ethnic populations differs considerably. Furthermore, we detected additional frequent GNB3 polymorphisms. These variants were restricted to one or two major ethnic populations. Our results will aid future studies on population-specific effects of the GNB3 variants on risk and course of frequent diseases, including hypertension, obesity, stroke and myocardial infarction. Furthermore, they will contribute to the understanding of GNB3-related population-specific pharmacogenetic differences in the response to major drugs, as already shown for diuretics and antidepressants.