Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This report describes the identification of a murine cytomegalovirus (MCMV) G protein-coupled receptor (GCR) homolog. This open reading frame (M33) is most closely related to, and collinear with, human cytomegalovirus UL33, and homologs are also present in human herpesvirus 6 and 7 (U12 for both viruses). Conserved counterparts in the sequenced alpha- or gammaherpesviruses have not been identified to date, suggesting that these genes encode proteins which are important for the biological characteristics of betaherpesviruses. We have detected transcripts for both UL33 and M33 as early as 3 or 4 h postinfection, and these reappear at late times. In addition, we have identified N-terminal splicing for both the UL33 and M33 RNA transcripts. For both open reading frames, splicing results in the introduction of amino acids which are highly conserved among known GCRs. To characterise the function of the M33 in the natural host, two independent MCMV recombinant viruses were prepared, each of which possesses an M33 open reading frame which has been disrupted with the beta-galactosidase gene. While the recombinant M33 null viruses showed no phenotypic differences in replication from wild-type MCMV in primary mouse embryo fibroblasts in vitro, they showed severely restricted growth in the salivary glands of infected mice. These data suggest that M33 plays an important role in vivo, in particular in the dissemination to or replication in the salivary gland, and provide the first evidence for the function of a viral GCR homolog in vivo.
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PMID:Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus. 899 78

The level of LH secretion is determined by both alterations in gonadotrope responsiveness and alterations in GnRH secretion. The molecular mechanisms underlying gonadotrope responsiveness are unknown, but may include G protein-coupled receptor kinases (GRKs). Typically, GRKs phosphorylate the intracellular regions of seven-transmembrane receptors permitting beta-arrestin to bind, which prevents receptor activation of its G protein. Previously, we reported that heterologous expression of GRK2, -3, and -6 in GnRH receptor-expressing COS cells by complementary DNA transfection suppressed GnRH-stimulated inositol trisphosphate production, and that coexpression of GRK2 and beta-arrestin-2 was more inhibitory than either expressed alone. Here, we have investigated the effect of GRK2 on GnRH-stimulated LH secretion using adenovirus-mediated gene transfer in normal pituitary gonadotropes. Pituitary cells were infected with adeno-GRK2 or adeno-beta-galactosidase constructs at a multiplicity of infection of 60 (number of viral particles per cell). Seventy-two hours later, GRK2 expression was measured by enzyme-linked immunosorbent assay, and GnRH-stimulated LH secretion (10(-7) M GnRH-A for 90 min) was assayed by RIA. Adeno-beta-galactosidase infected 96-99% of the cells based on X-Gal staining. Uninfected and adeno-beta-galactosidase-infected cells exhibited endogenous GRK immunoreactivity of about 0.5 (OD405), and LH secretion of 14.8-17.7 ng/ml. Adeno-GRK2-infected cells showed a GRK2 immunoreactivity of about 2.5 (OD405) and LH secretion of 2.5 ng/ml. Therefore, adeno-GRK2 infection resulted in a 5-fold increase in the GRK2 OD405 value, which was accompanied by an 80-85% decrease in GnRH-stimulated LH secretion. GnRH-stimulated inositol trisphosphate production by gonadotropes also was inhibited, suggesting a site of action for GRK2 at phospholipase Cbeta or earlier in the signal transduction pathway. The significance of these findings is 2-fold: 1) adenoviral-mediated gene transfer permits investigation of the regulatory role of gene products in the cell of interest, the gonadotrope, rather than in heterologous cell systems; and 2) additional, stronger evidence is provided that supports a role for GRKs in setting the responsiveness of GnRH receptor signaling.
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PMID:High efficiency method for gene transfer in normal pituitary gonadotropes: adenoviral-mediated expression of G protein-coupled receptor kinase 2 suppresses luteinizing hormone secretion. 1034 43

A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between beta-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX trade mark system, uses a pair of inactive beta-galactosidase (beta-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and beta-arrestin-beta-gal fusion proteins are generated. Following ligand stimulation, beta-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the beta-gal mutant fragments. GPCR activation is measured directly by quantitating restored beta-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the beta2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The beta2-adrenergic receptor cell line was screened with the LOPAC trade mark compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.
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PMID:Cell-based high-throughput screening assay system for monitoring G protein-coupled receptor activation using beta-galactosidase enzyme complementation technology. 1459 61

High-affinity complementation of a small fragment of beta-galactosidase to an inactive deletion mutant of the enzyme forms a stable heteromeric enzyme complex capable of hydrolyzing substrates to produce either chemiluminescent or fluorescent signals. This review describes a series of screening assays in which the small beta-galactosidase fragment, Enzyme Donor or ProLabel, is either chemically conjugated or recombinantly fused to small molecules or proteins, respectively. Chemical conjugation forms the basis of several HitHunter HTS assays in which competitive displacement of the ProLabel conjugate from either a binding protein (receptor or antibody) is induced by the analyte in question. In this manner, a calibration curve is generated, to measure cellular analytes including 3',5'-cyclic AMP. Changes in this second messenger, occurring due to G protein-coupled receptor (GPCR) activation, can thus be easily measured in a homogeneous assay. Similar assays have been developed for tyrosine kinases, serine threonine kinases, nuclear hormone receptors, and proteases. A second form of assay technology involves measurement of cellular protein expression, in which the protein is fused to ProLabel. Analysis can be undertaken in crude cell lysates, or with intact cells, using beta-galactosidase complementation in a microtiter plate. This homogeneous technology is highly sensitive and has been developed to measure protein expression changes occurring in response to pathway activation by targets such as GPCRs, tyrosine kinase receptors, and proteases. In summary, the DiscoveRx technology using beta-galactosidase complementation provides a robust and flexible assay technology for use in cell-free and cell-based HTS.
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PMID:Enzyme fragment complementation: a flexible high throughput screening assay technology. 1509 Jan 61

Adenosine A2a receptor, a member of the G protein-coupled receptor superfamily, has been demonstrated to be an important pharmacological target. It couples to stimulatory G protein and activates adenylate cyclase upon agonist stimulation. Here we attempted to stably transfect Chinese hamster ovary (CHO-K1) cells, which lack any known subtypes of adenosine receptors, with recombinant human adenosine A2a receptors (hA2aR). Rapid down-regulation of hA2aR in a clonal cell line, CHOA2a-2, was observed over a short period of time in culture. This is consistent with other groups' findings of low expression and poor G protein coupling of this receptor in several cell systems. To facilitate pharmacological profiling for hA2aR ligand, we introduced a cyclic AMP response element (CRE)-linked beta-galactosidase reporter gene into CHOA2a-2 cells to generate a stable cell line, CHOA2a-2CREbetagal#26. Robust cyclic AMP signal amplification was obtained using a colorimetric assay measuring beta-galactosidase activity. The EC(50) of 5'-N-ethylcarboxamidoadenosine (NECA), a potent A2a agonist, for inducing beta-galactosidase activity was 23.3 +/- 3.5 nM, similar to 22.7 +/- 3.9 nM, which was the NECA EC(50) in the direct measurement of cyclic AMP of CHOA2a-2 cells in early culture. Subsequently we validated this assay for high throughput screening for hA2aR agonists. The Z' factor for robotic assay performance was 0.79 +/- 0.03, the ratio of signal/noise was 157 +/- 36, and the ratio of signal/background was 10.6 +/- 1.2, demonstrating that this assay is well suitable for quality high throughput screening. High throughput screening of Johnson & Johnson libraries uncovered a couple of distinct series of nonadenosine small molecules, in addition to adenosine analogues, as potential hA2aR agonists with EC(50) values of 2-6 microM. Preliminary characterization of those compounds was presented.
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PMID:Development of a sensitive and HTS-compatible reporter gene assay for functional analysis of human adenosine A2a receptors in CHO-K1 cells. 1528 9

Advances in high throughput screening technologies have led to the identification of many small molecules, "hits", with activities toward the target of interest. And, as the screening technologies become faster and more robust, the rate at which the molecules are identified continues to increase. This evolution of high throughput screening technologies has generated a significant strain on the laboratories involved with the downstream profiling of these hits using cell-based assays. The CellCard System, by enabling multiple targets and/or cell lines to be assayed simultaneously within a single well, provides a platform on which selectivity screening can be quickly and robustly performed. Here we describe two case studies using the beta-lactamase and beta-galactosidase reporter gene systems to characterize G protein-coupled receptor agonist activity. Using these examples we demonstrate how the implementation of this technology enables assay miniaturization without micro-fluidic devices as well as how the inclusion of intra-well controls can provide a means of data quality assessment within each well.
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PMID:The CellCard System: a novel approach to assessing compound selectivity for lead prioritization of G protein-coupled receptors. 1610 Oct 5

The biochemical and genetic tractability of yeasts make them ideal hosts for the analysis of signalling from G protein-coupled receptors (GPCRs). Selected modifications to the strains allow the introduction of non-yeast components, while signal-dependent expression of reporter genes provides growth selection or enzyme read-out as assays for signalling. One issue with such systems is reporter expression in the absence of stimulation, usually because of spontaneous activation of intracellular signalling components and/or incomplete repression of the signal-dependent promoter. This limits the difference between reporter activity in the presence and absence of stimulation, often referred to as the signal:background ratio. In an effort to extend the applicability of the yeast system, we generated a Schizosaccharomyces pombe strain containing pheromone-dependent reporters for both growth selection and beta-galactosidase production. Simultaneous use of the two reporters provided several advantages over strains expressing only one reporter, particularly when coupled to the use of a competitive inhibitor of the nutritional reporter. For example, the beta-galactosidase signal:background ratio following stimulation with 10(-6) M P-factor increased from 35 for a strain containing a single lacZ reporter to almost 2500 for the double reporter. The sensitivity of the system was also improved, with higher signal:background ratios allowing detection of lower concentrations of P-factor. Although we have used Sz. pombe and focused on GPCR-based induction of beta-galactosidase, the principles described can be applied to other yeasts, different signalling pathways and alternative reporters.
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PMID:Combined use of two transcriptional reporters improves signalling assays for G protein-coupled receptors in fission yeast. 1700 18

GIT1 and GIT2 belong to the family of ADP-ribosylation factor GTPase-activating proteins (ARF-GAP) and have been implicated in the regulation of G protein-coupled receptor sequestration, cell migration, T-cell activation, neuronal spine formation, and aggregate formation in Huntington's disease. Examination of endogenous GIT protein expression in tissues, however, has been hampered by the lack of GIT2-specific antibodies. To visualize GIT1 and GIT2 gene expression in mouse tissues, we created mice with beta-galactosidase (beta-Gal) reporters inserted into the two GIT genes. beta-Gal staining confirmed the broad tissue distribution of GIT1 and GIT2 in the mouse but also revealed striking differences. GIT2 is expressed in most cells of the body, whereas GIT1 is restricted to only a subset of cells. For example, GIT2 is uniformly expressed throughout lung and liver, whereas GIT1 is restricted to cells lining blood vessels, bronchi, and bile ducts. Expression of GIT1 and GIT2 is mutually exclusive in the testes, where a developmental expression shift occurs, with GIT2 present in spermatogonia but GIT1 in mature spermatids. In conclusion, analysis of endogenous GIT expression revealed a nearly ubiquitous distribution of GIT2, whereas GIT1 is restricted to specific cell types even in tissues with apparently high GIT1 expression and is entirely absent from some tissues.
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PMID:Differential expression of the ARF GAP genes GIT1 and GIT2 in mouse tissues. 1756 17

The G protein-coupled receptor GPR54, and its peptide ligand kisspeptin (Kp), are crucial for the induction and maintenance of mammalian reproductive function. GPR54 is expressed by GnRH neurons and is directly activated by Kp to stimulate GnRH release. We hypothesized that Kp may be able to act at the GnRH nerve terminals located in the mediobasal hypothalamus (MBH) region. To test this hypothesis, we used organotypic culture of MBH explants challenged with Kp, followed by RIA to detect GnRH released into the cultured medium. Kp stimulation for 1 h induced GnRH release from wild-type male MBH in a dose-dependent manner, whereas this did not occur in MBH explants isolated from Gpr54 null mice. Continuous Kp stimulation caused a sustained GnRH release for 4 h, followed by a decrease of GnRH release, suggesting a desensitization of GPR54 activity. Tetrodotoxin did not alter the Kp-induced GnRH release, indicating that Kp can act directly at the GnRH nerve terminals. To localize Gpr54 expression within the MBH, we used transgenic mice, in which Gpr54 expression is tagged with an IRES-LacZ reporter gene and can be visualized by beta-galactosidase staining. Gpr54 expression was detected outside of the median eminence, in the pars tuberalis. In conclusion, our results provide evidence for a potent stimulating effect of Kp at GnRH nerve terminals in the MBH of the mouse. This study suggests a new point at which Kp can act on GnRH neurons.
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PMID:Kisspeptin can stimulate gonadotropin-releasing hormone (GnRH) release by a direct action at GnRH nerve terminals. 1845 Sep 66

Morphological and biochemical phenotypes of cardiomyocyte hypertrophy are determined by neurohumoral factors. Stimulation of G protein-coupled receptor (GPCR) results in uniform cell enlargement in all directions with an increase in skeletal alpha-actin (alpha-SKA) gene expression, while stimulation of gp130 receptor by interleukin-6 (IL-6)-related cytokines induces longitudinal elongation with no increase in alpha-SKA gene expression. Thus, alpha-SKA is a discriminating marker for hypertrophic phenotypes; however, regulatory mechanisms of alpha-SKA gene expression remain unknown. Here, we clarified the role of SH2-containing protein tyrosine phosphatase 2 (SHP2) in alpha-SKA gene expression. In neonatal rat cardiomyocytes, endothelin-1 (ET-1), a GPCR agonist, but not leukemia inhibitory factor (LIF), an IL-6-related cytokine, induced RhoA activation and promotes alpha-SKA gene expression via RhoA. In contrast, LIF, but not ET-1, induced activation of SHP2 in cardiomyocytes, suggesting that SHP2 might negatively regulate alpha-SKA gene expression downstream of gp130. Therefore, we examined the effect of adenovirus-mediated overexpression of wild-type SHP2 (SHP2(WT)), dominant-negative SHP2 (SHP2(C/S)), or beta-galactosidase (beta-gal), on alpha-SKA gene expression. LIF did not upregulate alpha-SKA mRNA in cardiomyocytes overexpressing either beta-gal or SHP2(WT). In cardiomyocytes overexpressing SHP2(C/S), LIF induced upregulation of alpha-SKA mRNA, which was abrogated by concomitant overexpression of either C3-toxin or dominant-negative RhoA. RhoA was activated after LIF stimulation in the cardiomyocytes overexpressing SHP2(C/S), but not in myocytes overexpressing beta-gal. Furthermore, SHP2 mediates LIF-induced longitudinal elongation of cardiomyocytes via ERK5 activation. Collectively, these findings indicate that SHP2 negatively regulates alpha-SKA expression via RhoA inactivation and suggest that SHP2 implicates ERK5 in cardiomyocyte elongation downstream of gp130.
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PMID:SHP2 mediates gp130-dependent cardiomyocyte hypertrophy via negative regulation of skeletal alpha-actin gene. 2022 89


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