"MC2R" Related Products

Human MC2R/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0696
Cell Line Description: This human MC2-MRAP double stable cell line is made in the Chem-1 host, which supports high levels of recombinant MC2 expression on the cell surface and contains high levels of the endogenous promiscuous G protein Galpha15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between MC2 and its ligands.
Background: The melanocortins, alpha-, beta- and gamma-melanocyte-stimulating hormones (MSHs) and adrenocorticotropin (ACTH), are peptides derived from a precursor protein POMC. The MSH peptides and ACTH bind to a family of five Class 1 Gs -coupled seven transmembrane receptors (MC1-5) and play important roles in energy balance, reproductive function, pigmentation and inflammation. MC2, the ACTH receptor, is a member of this family but is the only one that does not bind the MSHs, it instead binds ACTH exclusively. MC2 is expressed mainly in cells of the adrenal cortex, where it signals cells in the adrenal cortex to synthesize and secrete glucocorticoids. Mutations in MC2 lead to familial glucocorticoid deficiency, or ACTH insensitivity. Familial glucocorticoid deficiency can lead to increased pigmentation and increased longitudinal bone growth. In addition to mutations in MC2 leading to Familial glucocorticod deficiency, it has also been shown that mutations in MRAP also lead to this disorder. Expression of MC2 on the cell surface has been found to be dependent on the interaction of MC2 with a protein called MC2-R accessory protein (MRAP). This interaction helps to move MC2 from the endoplasmic reticulum to the cell surface.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of MC2R. 2. Protein expression: MC2R in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by ACTH (1-24): ~ 4 nM; Signal/Noise ratio = 16.3.
Application: Calcium flux assay, ligand binding assays
Sub-type: Melanocortin
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: MC2R melanocortin 2 receptor (adrenocorticotropic hormone) [ Homo sapiens ]
Official Symbol: MC2R
Synonyms: MC2R; melanocortin 2 receptor (adrenocorticotropic hormone); adrenocorticotropic hormone receptor; ACTHR; MC2 receptor; ACTH receptor; corticotropin receptor; adrenocorticotropin receptor; MGC125798;
Gene ID: 4158
mRNA Refseq: NM_000529
Protein Refseq: NP_000520
MIM: 607397
UniProt ID: Q01718
Chromosome Location: 18p11.2
Pathway: Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (s) 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; corticotropin receptor activity; melanocortin receptor activity; protein binding; receptor activity; signal transducer activity;

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