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)

Retinal photoreceptor rods and pinealocytes contain well-characterized proteins such as arrestin and phosducin whose expression is highly restricted to these cell types. Transgenic mice having a LacZ gene under the control of an arrestin promoter expressed beta-galactosidase (beta-Gal) in the photoreceptor rods and pinealocytes. In addition, it was expressed in very small numbers of discrete cells in the habenular commissura, amygdala, ventral tegmental area and superior colliculus of the brain. Immunocytochemical studies with antibody probes revealed that high level of arrestin and phosducin were also found in the same cell types. Furthermore melatonin was found in those cells of the habenula commissura. The results indicate that novel cell types are present in the brain tissues. Since high levels of arrestin and phosducin expression are generally restricted to photoreceptor rod cells and pinealocytes, these data suggest that certain brain cells may have functions similar to pinealocytes.
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PMID:Arrestin and phosducin are expressed in a small number of brain cells. 945 Jun 83

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

Neural progenitor cells (NPCs) have shown ability to repair injured CNS, and might provide precursors to retinal neurons. NPCs were isolated from the brains of 14 day murine embryos of transgenic mice that express beta-galactosidase (beta-gal) on the arrestin promoter, which specifically directs expression to retinal photoreceptor cells. NPCs were transferred to adult, syngeneic mice via inoculation into the anterior chamber of the eye, the peritoneal cavity, or the brain. At 14 weeks postgrafting, tissues were collected and examined to determine if differentiated NPC progeny were present in retina based on histochemical detection of beta-gal. Four of six anterior chamber-inoculated recipients showed Bluo-gal-stained cells in retina, indicating the presence of transferred NPCs or their progeny. Because the progenitor cells do not express beta-gal, positive staining indicates differentiation leading to activation of the arrestin promoter. Two recipients inoculated by the intraperitoneal route also exhibited Bluo-gal staining in retina. The NPCs did not express beta-gal if inoculated into brain, but survived and dispersed. Most recipients, regardless of inoculation route, were PCR positive for beta-gal DNA in extraocular tissues, but no Bluo-gal staining was found outside of the retina. Injury to the retina promoted, but was not required, for progenitor cell engraftment. beta-Gal-positive cells were concentrated in the outer layers of the retina. In summary, a reporter gene specifically expressed in differentiated retinal photoreceptor cells due to the activity of the arrestin promoter was expressed in recipient mouse retina following transfer of NPCs prepared from the beta-gal transgenic mice. The presence of beta-gal DNA, but not Bluo-gal staining, in spleen and other tissues revealed that the cells also migrated elsewhere and took up residence in other organs, but did not undergo differentiation that led to beta-gal expression.
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PMID:Engrafted neural progenitor cells express a tissue-restricted reporter gene associated with differentiated retinal photoreceptor cells. 1671 48

G-protein coupled receptors (GPCRs) are a versatile and ubiquitous family of membrane receptors that transmit extracellular signals to mammalian cells and constitute the most important class of drug targets. Yet, sensitive and specific methods are lacking that would allow quantitative comparisons of pharmacologic properties of these receptors in physiological or pathological settings in live animals. We sought to overcome these limitations by employing low affinity, reversible beta-galactosidase complementation to quantify GPCR activation via interaction with beta-arrestin. A panel of cell lines was engineered expressing different GPCRs together with the reporter system. In vitro evaluation revealed highly sensitive, dynamic, and specific assessment of GPCR agonists and antagonists. Following implantation of the cells into mice, it was possible for the first time to monitor pharmacological GPCR activation and inhibition in their physiological context by noninvasive bioluminescence imaging in living animals. This technology has unique advantages that enable novel applications in the functional investigation of GPCR modulation in live animals in biological research and drug discovery.
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PMID:A universal technology for monitoring G-protein-coupled receptor activation in vitro and noninvasively in live animals. 1794 28

G-protein-coupled receptor (GPCR) signaling is involved in a wide range of physiological processes and diseases, and around one-half of currently used drugs target GPCRs. Assays for the signaling of GPCRs have suffered from drawbacks, including low signal-to-noise, temporally transient signals, and difficulty in applying a single assay to a wide range of GPCRs. We have developed a set of assays for G-protein-coupled receptor signaling based on beta-galactosidase enzyme complementation in live mammalian cells. We previously described an assay for GPCR activation by monitoring the binding of beta-arrestin to the receptor. Here we describe a second assay that monitors the internalization of GPCRs to endosomes, an event that follows receptor activation and is critical in desensitizing and resensitizing the receptor. We show that both assays display high signal-to-noise ratios with low variability and are quantitative for a wide range of GPCRs. EC50s obtained with these assays closely match results reported in the literature. Finally, we show that these assays are readily adapted to high-throughput chemical screens. Thus, these two assays for monitoring G-protein-coupled receptor activation and internalization should prove valuable in basic biological studies as well as in high-throughput screens.
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PMID:A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization. 1794 29

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between beta-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (approximately 4 kDa), optimized alpha fragment peptide (termed ProLink) derived from beta-galactosidase, and beta-arrestin is fused to an N-terminal deletion mutant of beta-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the beta-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active beta-galactosidase enzyme, and thus GPCR activation can be determined by quantifying beta-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Galphai-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified.
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PMID:A homogeneous enzyme fragment complementation-based beta-arrestin translocation assay for high-throughput screening of G-protein-coupled receptors. 1866 Apr 57

beta-Arrestin recruitment assays provide a generic assay platform for drug discovery on G-protein-coupled receptors (GPCRs). The PathHunter assay technology developed by DiscoveRx (Fremont, CA) uses enzyme fragment complementation of beta-galactosidase to measure receptor-beta-arrestin proximity by chemiluminescence. This study describes an agonistic screen on the human endothelial differentiation sphingolipid GPCR 1 (EDG1), also known as S1P1, using PathHunter beta-arrestin recruitment technology. Screening of a collection of 345,052 compounds yielded 2157 agonistic hits. Only 10 of these compounds showed beta-arrestin recruitment activity on a nonrelated receptor, indicating high accuracy and specificity of the assay. The authors show that receptor activation with reference agonists can be detected within the same EDG1 PathHunter cell line at the level of beta-arrestin recruitment, G(i/o) protein-mediated inhibition of cyclic adenosine monophosphate (cAMP), and activation of downstream phosphorylation of extracellular signal-regulated protein kinases. The degree of beta-arrestin recruitment was largely unaffected upon blockade of G(i/o) protein signaling with pertussis toxin, whereas kinetic studies demonstrated a lower rate of beta-arrestin-receptor association. In contrast, inhibition of cAMP and phosphorylation of extracellular signal-regulated protein kinases were fully G(i/o) protein regulated. The data indicate that the beta-arrestin enzyme fragment complementation cell line can be used not only for agonistic screening of GPCRs but also for the identification of "biased ligands" (i.e., compounds that differ in G-protein coupling and beta-arrestin-mediated cellular effects).
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PMID:beta-Arrestin recruitment assay for the identification of agonists of the sphingosine 1-phosphate receptor EDG1. 1903 7

The contribution of peripheral expression of tissue-specific CNS Ags to the generation of tolerance is uncertain. To study this question, we examined mice transgenic (Tg) for expression of beta-galactosidase (beta gal) on the retinal photoreceptor cell arrestin promoter, in conjunction with TCR Tg mice producing CD4(+) T cells specific for beta gal (beta galTCR). Several strategies were used to test the hypothesis that betagal expressed in the retina supported thymus-independent tolerance and regulatory T cell development. Retinal expression generated an immunoregulatory response that depressed development of immune responses to beta gal following systemic immunization with beta gal. This regulation was transferable to naive mice by CD3(+)4(+)25(+) T cells from naive retinal beta gal(+) donors. Experiments that removed the beta gal(+) retina by enucleation showed that subsequent development of a regulatory response was lost. Adoptive transfer of CD25(-) beta galTCR T cells into retinal beta gal Tg mice on the Rag(-/-) background led to regulatory activity that limited lymphopenia-induced proliferation of beta galTCR T cells in mice with retinal expression of beta gal and inhibited the ear-swelling assay for delayed type hypersensitivity. These results show that retinal expression of very small amounts of a tissue-specific Ag can generate tolerance that includes regulatory T cells.
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PMID:Peripheral induction of tolerance by retinal antigen expression. 1954 66

C5L2 (or GPR77) is a high-affinity receptor for the complement fragment C5a and its desarginated product, C5a-desArg. Unlike the classical C5a receptor CD88, C5L2 does not couple to intracellular G-protein-signaling pathways but is thought to function as a decoy receptor. The authors show that stimulation of C5L2 with C5a and C5a-desArg induces redistribution of green fluorescent protein-labeled beta-arrestin2 to cytoplasmic vesicles. C3a and C3a-desArg were inactive in the beta-arrestin translocation assay. Direct interaction of ligand-stimulated C5L2 with beta-arrestin was confirmed using a novel beta-galactosidase fragment complementation assay. In this assay, C5L2 was labeled with a mutationally altered peptide fragment of beta-galactosidase, whereas beta-arrestin2 was labeled with a corresponding deletion mutant of the enzyme. Stable transfection of the modified C5L2 and subsequent stimulation with C5a or C5a-desArg restored beta-galactosidase activity in a dose-dependent manner. The subnanomolar potency of beta-arrestin coupling in the beta-galactosidase fragment complementation assay is in agreement with the affinity of the receptor-ligand interaction. C5L2 is the first example of a 7-transmembrane decoy receptor that couples to beta-arrestin in a ligand-dependent manner. This observation supports the notion that G-protein-signaling and beta-arrestin coupling can be 2 separate activities of 7-transmembrane receptors. Furthermore, the beta-arrestin assays described in this article provide methods of screening for selective C5L2 modulators.
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PMID:C5a-stimulated recruitment of beta-arrestin2 to the nonsignaling 7-transmembrane decoy receptor C5L2. 1964 Dec 21


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