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Human GHSR/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0663
Cell Line Description: This human Ghrelin Receptor-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant Ghrelin Receptor expression on the cell surface and contains high levels of the promiscuous G protein to enhance coupling of the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for agonists and antagonists at Ghrelin Receptor.
Background: Ghrelin is a 28 amino acid peptide, containing a unique octanoyl moiety added post-translationally to Ser3, with diverse activities in the CNS, gastrointestinal tract, and cardiovascular system. In the CNS, ghrelin stimulates release of growth hormone from the anterior pituitary and increases appetite by binding to neurons within the arcuate nucleus. Circulating concentrations of ghrelin increase with preprandially and decrease post-prandially, and thus counterbalances the effects of leptin to coordinate energy balance, appetite and food intake. Ghrelin is also expressed in the cardiovascular system where it acts as a potent vasodilator; receptors are up-regulated in patients with atherosclerosis, suggesting that it plays a role in compensating for increased vasoconstriction. The effects of ghrelin are mediated by a Gq-coupled receptor, originally designated GHSR (growth hormone secretogogue receptor), and more recently termed the Ghrelin Receptor or GRLN. Two splice variants have been described; type 1a (GHS-R1a) is the functional receptor, whereas type 1b (GHS-R1b) encodes a truncated, inactive protein with only 5 transmembrane domains. As administration of agonists of the ghrelin receptor to rats leads to increased food intake and antagonists reduce food intake, antagonism and inverse agonism of the ghrelin receptor may reduce food intake in certain types of obesity, and agonists of the ghrelin receptor are potentially useful for treatment of anorexia and cachexia.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of GHSR. 2. Protein expression: GHSR in this cell line has been validated by immunocytochemical staining.
Application: Calcium flux assay, ligand binding assays
Sub-type: Ghrelin
Propagation: Complete growth medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x Nonessential amino acids + 10mM HEPES + 1x Pen-Strep + 250μg/mL Genetecin/G-418 Plating medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-StrepAtmosphere: air, 95%; carbon dioxide (CO2), 5%Temperature: 37 °C
Starting Cells From Frozen Cell Stock: 1. Immediately upon receipt, thaw cells or place cells in liquid nitrogen. Maintain frozen in liquid nitrogen for up to 5 years. 2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37°C water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37°C with 5% CO2. 3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
Subculturing: 1. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca2+ and Mg2+ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37°C with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Growth Media per 1 mL trypsin. 2. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Freezing medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 20% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-Strep + 10% DMSO
Storage: Liquid nitrogen
Preservation: 1. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Subculture-Step 1). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 10^6 cells/mL in Freezing Media (cell densities of 2-10 x 10^6 are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at –70°C overnight. Store the vials in liquid nitrogen. 2. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Plating Media for plating for calcium assay.
Safety Considerations: The following safety precautions should be observed.1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum.2. No eating, drinking or smoking while handling the stable line.3. Wash hands after handling the stable line and before leaving the lab.4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells.5. All waste should be considered hazardous.6. Dispose of all liquid waste after each experiment and treat with bleach.
Ship: Dry ice
Gene Name: GHSR growth hormone secretagogue receptor [ Homo sapiens ]
Official Symbol: GHSR
Synonyms: GHSR; growth hormone secretagogue receptor; growth hormone secretagogue receptor type 1; GHRP; GHS-R; ghrelin receptor; GH-releasing peptide receptor;
Gene ID: 2693
mRNA Refseq: NM_004122
Protein Refseq: NP_004113
MIM: 601898
UniProt ID: Q92847
Chromosome Location: 3q26.31
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (q) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class A Rhodopsin-like, organism-specific biosystem; Neuroactive ligand-receptor interaction, organism-specific biosystem; Neuroactive ligand-receptor interaction, conserved biosystem;
Function: G-protein coupled receptor activity; growth hormone secretagogue receptor activity; growth hormone-releasing hormone receptor activity; peptide hormone binding; receptor activity; signal transducer activity;

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Optional requirements on this protein    +Expand
C-fusion    N-fusion   Non-tagged
His    GST   Fc   Others
<1.0 eu/μg    <0.1 eu/μg   <0.01 eu/μg   Not required
Monomer Isolation    Dimer Isolation    Not required
>80% by SDS-PAGE    >90% by SDS-PAGE   >95% by SDS-PAGE   Others

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