ChemiScreen™ Human Recombinant OX₁ Orexin Receptor Calcium-Optimized Stable Cell Line

ChemiScreen™ Human Recombinant OX₁ Orexin Receptor Calcium-Optimized Stable Cell Line

• ChemiScreen
• Chemicon (Millipore)

2 vials

Full-length human HCRTR1 cDNA encoding OX1.

The orexin/hypocretin peptides orexin A and orexin B (also known as hcrt-1 and hcrt-2) are 33- and 28-amino acid peptides, respectively, that are preferentially expressed in hypothalamus. The orexins bind to two GPCRs, OX₁ and OX₂, to increase intracellular calcium. Experiments with OX₁ selective antagonists have shown the receptor plays a role in food consumption and also in acquisition and consolidation of spatial memory.(Haynes et al., 2000; Akbari et al, 2006) In addition, the orexinergic system plays a role in energy metabolism and stress responses (Kukkonen et al., 2002). Chemicon's cloned human OX1-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant OX₁ expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between the OX₁ and its ligands.

Calcium flux assay, ligand binding assays

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HCRTR1, or orexin 1 receptor (OX1R), is a G-protein coupled receptor expressed in the hypothalamus and involved in the regulation of feeding behaviour. HCRTR1 selectively binds the orexin A neuropeptide. It shares 64% identity with HCRTR2.

FUNCTION: SwissProt: O43613 # Moderately selective excitatory receptor for orexin-A and, with a lower affinity, for orexin-B neuropeptide. Seems to be exclusively coupled to the G(q) subclass of heteromeric G proteins, which activates the phospholipase C mediated signaling cascade (By similarity).
SIZE: 425 amino acids; 47522 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
SIMILARITY: SwissProt: O43613 ## Belongs to the G-protein coupled receptor 1 family.

Human

SPECIFICATIONS: EC50 for calcium mobilization by Orexin A: ~ 1 nM

Chem-1

GPCR Cell Lines

Chemicon

Chem-1

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.

OX1

O43613

peptide

Place cells in liquid nitrogen immediately upon receipt. Maintain frozen in liquid nitrogen for up to 5 years. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37ºC 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 20 mL growth media, and place in a humidified 37°C incubator with 5% CO2.. After 8-24 h, cells will adhere to the plate, at which time the media should be replaced to remove residual DMSO. Cells are passaged by washing with Ca++ and Mg++-free HBSS (10 mL/T75), incubating with 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) for 5-10 minutes at 37°C, and rapping the side of the flask to dislodge the cells. Neutralize the trypsin by addition of 4 volumes growth media. Cells are typically passaged 1:10 with every 3-4 days, and should be passaged at least once after thawing prior to use in calcium flux assays.

Orexin

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A

2 x 10⁶ cells/vial

• HCRTR1
• Ox1r
• OX1R

GPCRs

ChemiScreen™ Human Recombinant OX₁ Orexin Receptor Calcium-Optimized Stable Cell Line

NM_001525.1

DMEM containing 4.5 g/L glucose/10% heat inactivated fetal bovine serum/1x nonessential amino acids/10 mM HEPES/0.25 mg/ml Geneticin (G418)/100 U/ml each penicillin and streptomycin.