isopropyl-thiogalactoside and fura-2-am

Coupling of the group I metabotropic glutamate receptors, mGlu1a and mGlu5a, to the cAMP response element binding protein (CREB) has been studied in Chinese hamster ovary cell lines where receptor expression is under the control of an inducible promoter. Both receptors stimulate CREB phosphorylation with similar time courses, and agonist potency was also comparable between the two receptors. Stimulation of cells in Ca(2+)-free medium containing EGTA (100 microm), with or without the additional depletion of intracellular stores, caused marked decreases in agonist-mediated responses in both cell lines. Down-regulation of protein kinase C (PKC) activity by phorbol ester treatment, or treatment with the broad spectrum PKC inhibitor Ro 31-8220, partially attenuated both mGlu1a and mGlu5a receptor-mediated responses. Furthermore, stimulation of cells in the absence of extracellular Ca(2+) following prior PKC down-regulation resulted in additive inhibitory effects. The involvement of extracellular signal-regulated kinases (ERK1/2), Ca(2+)/calmodulin or Ca(2+)/calmodulin-dependent protein kinases was assessed using pharmacological inhibitors. Results indicated that coupling of the group I mGlu receptors to CREB phosphorylation occurs independently of these pathways. Thus, although the [Ca(2+)](i) signatures activated by these mGlu receptors differ, they couple to CREB with comparable potency and recruit similar downstream components to execute CREB phosphorylation.

Topics: Analysis of Variance; Animals; Blotting, Western; Calcium; Calcium Channel Blockers; CHO Cells; Colforsin; Cricetinae; Cricetulus; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Fura-2; Gene Expression Regulation; Glutamic Acid; Indoles; Ionomycin; Ionophores; Isopropyl Thiogalactoside; Pertussis Toxin; Phosphorylation; Protein Kinase C; Quisqualic Acid; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Signal Transduction; Teprotide; Time Factors

We recently discovered a novel gene on chromosome 19p13.1 and its product, an integral endoplasmic reticulum (ER) membrane protein, termed CHERP (calcium homoeostasis endoplasmic reticulum protein). A monoclonal antibody against its C-terminal domain inhibits Ins(1,4,5) P (3)-induced Ca(2+) release from ER membrane vesicles of many cell types, and an antisense-mediated knockdown of CHERP in human erythroleukemia (HEL) cells greatly impaired Ca(2+) mobilization by thrombin. In the present paper, we explore further CHERP's function in Jurkat T-lymphocytes. Confocal laser immunofluorescence microscopy showed that CHERP was co-localized with the Ins(1,4,5) P (3) receptor throughout the cytoplasmic and perinuclear region, as previously found in HEL cells. Transfection of Jurkat cells with a lac I-regulated mammalian expression vector containing CHERP antisense cDNA caused a knockdown of CHERP and impaired the rise of cytoplasmic Ca(2+) (measured by fura-2 acetoxymethyl ester fluorescence) caused by phytohaemagglutinin (PHA) and thrombin. A 50% fall of CHERP decreased the PHA-induced rise of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)), but Ca(2+) influx was unaffected. Greater depletion of CHERP (>70%) did not affect the concentration of Ins(1,4,5) P (3) receptors, but diminished the rise of [Ca(2+)](i) in response to PHA to

Topics: Calcium; Calcium Signaling; Cell Division; Culture Media, Serum-Free; DNA-Binding Proteins; Fluorescent Dyes; Fura-2; Humans; Immunohistochemistry; Inositol 1,4,5-Trisphosphate; Isopropyl Thiogalactoside; Jurkat Cells; Leukemia, Erythroblastic, Acute; Lymphocyte Activation; Membrane Proteins; NFATC Transcription Factors; Nuclear Proteins; Oligonucleotides, Antisense; Phytohemagglutinins; RNA-Binding Proteins; T-Lymphocytes; Transcription Factors; Tumor Cells, Cultured