MULTISCREEN™ β-ARRESTIN STABLE CELL LINES
HUMAN RECOMBINANT GPR65 RECEPTOR
1 vial (2 x 106) frozen cells
Cell Banker 2 (Amsbio 11891)
Expression vector containing full-length human GPR65 cDNA (GenBank Accession Number NM_003608) with FLAG tag sequence at N-terminus and ARRB2 cDNA (GenBank Accession Number NM_004313.3)
Liquid nitrogen upon receiving
Propagation Medium: DMEM, 10% FBS, 1 μg/mL puromycin, 250 μg/mL hygromycin
Stable for a minimum of 2 months in continuous culture
Background: GPR65 also known as T cell death-associated gene 8 (TDAG8) is a proton-sensing GPCR and plays a major role in pH homeostasis. This receptor is primarily expressed in lymphoid tissues (spleen, thymus, leukocytes and lymph nodes) and cancer tissues. The major function of this receptor is to reduce immune-mediated inflammation by regulating cytokine production from T cells and macrophages.
Application: Functional assays
Figure 1. Dose-dependent stimulation from arrestin recruitment upon treatment with pH, measured with MultiscreenTM β-Arrestin Assay Kit (Multispan MSBAK01). Figure 2. Dose-dependent increase of intracellular cAMP level upon treatment with ligand, measured with MultiscreenTM TR-FRET cAMP 1.0 No Wash Assay Kit (Multispan MSCM01). Figure 3. Dose-dependent stimulation of calcium flux upon treatment with ligand, measured with MultiscreenTM Calcium 1.0 No Wash Assay Kit (Multispan MSCA01). Figure 4. Receptor expression on cell surface measured by flow cytometry (FACS) using an anti-FLAG antibody. Thin line: parental cells; thick line: receptor- expressing cells.
Ludwig, M.-G., Vanek, M., Guerini, D., Gasser, J. A., Jones, C. E., Junker, U., Hofstetter, H., Wolf, R. M., Seuwen, K. Proton-sensing G-protein-coupled receptors. Nature 425: 93-98, 2003.
Saxena, H., Deshpande, D., Tiegs, B., Yan, H., Battafarano, R., Burrows, W., Penn, R. (2012). The GPCR OGR1 (GPR68) mediates diverse signalling and contraction of airway smooth muscle in response to small reductions in extracellular pH. British Journal of Pharmacology, 166(3), 981–990.
Satoshi Ishii, Yasuyuki Kihara and Takao Shimizu (2005) Identification of T Cell Death-associated Gene 8 (TDAG8) as a Novel Acid Sensing G-protein-coupled Receptor. J Biol Chem 280: 9083- 9087