ChemiSCREEN™ Human Recombinant MC₅ Melanocortin Receptor Calcium-Optimized Stable Cell Line

ChemiSCREEN™ Human Recombinant MC₅ Melanocortin Receptor Calcium-Optimized Stable Cell Line

Chemicon (Millipore)

2 vials

Full-length human MC5R cDNA encoding MC₅

The melanocortin system consists of five seven-transmembrane spanning G-protein coupled receptors, MC₁-MC₅ (Gantz and Fong, 2003). Among the five members of the melanocortin receptor family, MC₂ and MC₅ are expressed in peripheral tissues. The MC₂ receptor (ACTH receptor) is almost exclusively expressed in the adrenal cortex whereas MC₅ is expressed in several organs including the adrenal cortex. Studies have shown that targeted disruption of the MC5R gene produced mice with a severe defect in water repulsion and thermoregulation caused by decreased production of sebaceous lipids (Chen et al., 1997). In addition, MC₅ is required for secretion from several exocrine glands, indicating that melanocortin peptides integrate the function of exocrine glands (Entwistle et al.,1990). Millipore's cloned human MC₅-expressing cell line is made in the Chem-10 host, which supports high levels of recombinant MC₅ expression on the cell surface and contains high levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between MC₅ and its ligands.

Calcium flux assay, ligand binding assays

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FUNCTION: SwissProt: P33032 # Receptor for MSH (alpha, beta and gamma) and ACTH. The activity of this receptor is mediated by G proteins which activate adenylate cyclase. This receptor is a possible mediator of the immunomodulation properties of melanocortins.
SIZE: 325 amino acids; 36601 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Expressed in the brain but not in the melanoma cells.
SIMILARITY: SwissProt: P33032 ## Belongs to the G-protein coupled receptor 1 family.

Human

EC50 for calcium mobilization by NDP-MSH: ~ 7.35 nM

Chem-10

GPCR Cell Lines

Chemicon

Chem-10 Growth Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020-A)
10% heat-inactivated FBS
1x Nonessential amino acids (from 100x stock, Millipore TMS-001-C)
10mM HEPES (from 1 M HEPES, Millipore TMS-003-C)
100 U/mL Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250μg/mL Genetecin/G-418
250μg/mL Hygromycin

Chem-10 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep

Chem-10 Freezing Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
10% DMSO (cell culture grade)

Chem-10

Cells are frozen at 2 x 10⁶ cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.

MC5

P33032

peptide

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.
4. 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 Chem-10 Growth Media per 1 mL trypsin.
5. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
6. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 10⁶ cells/mL in Chem-10 Freezing Media (cell densities of 2-10 x 10⁶ 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.
7. 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 Chem-10 Plating Media for plating for calcium assay.

Melanocortin

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A

2 x 10⁶ cells/vial

• MC5R
• MC5-R
• MC-2

GPCRs

ChemiSCREEN™ Human Recombinant MC₅ Melanocortin Receptor Calcium-Optimized Stable Cell Line

NM_005913.1