ChemiScreen™ Human Recombinant D₅ Dopamine Receptor Calcium-Optimized Stable Cell Line

ChemiScreen™ Human Recombinant D₅ Dopamine Receptor Calcium-Optimized Stable Cell Line

• ChemiScreen
• Chemicon (Millipore)

2 vials

Full-length human DRD5 cDNA encoding D₅

Dopamine is a catecholamine neurotransmitter that functions in the CNS to control locomotor, cognitive, emotional and neurendocrine processes, and in the periphery to modulate cardiovascular, renal and gastrointestinal processes. The biological activities of dopamine are mediated by a family of five GPCRs. The D₁ and D₅ subtypes couple to Gs to increase intracellular cAMP, whereas the D₂, D₃ and D₄ subtypes couple to Gi to reduce cAMP (Missale et al., 1998). The hypertensive phenotype of mice with a targeted deletion of D₅ indicates that D₅ regulates central control of sympathetic vascular tone (Hollon et al., 2002). In addition, D₅ modulates locomotion and corticostriatal long-term depression (Centonze et al., 2003). Chemicon's cloned human D₅-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant D₅ 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 D₅ and its ligands.

Calcium flux assay, ligand binding assays

Human

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This gene encodes the D5 subtype of the dopamine receptor. The D5 subtype is a G-protein coupled receptor which stimulates adenylyl cyclase. This receptor is expressed in neurons in the limbic regions of the brain. It has a 10-fold higher affinity for dopamine than the D1 subtype. Pseudogenes related to this gene reside on chromosomes 1 and 2.

FUNCTION: SwissProt: P21918 # This is one of the five types (D1 to D5) of receptors for dopamine. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase.
SIZE: 477 amino acids; 52951 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Neuron-specific, localized primarily within limbic regions of the brain.
DISEASE: SwissProt: P21918 # Defects in DRD5 may be a cause of schizophrenia, but no proof has yet been found. & Defects in DRD5 are a cause of blepharospasm [MIM:606798]. It is a primary focal dystonia affecting the orbicularis oculi muscles, usually beginning in middle age. Initial symptoms include eye irritation and frequent blinking, progressing to involuntary spasms of eyelid closure. In severe cases, this can lead to functional blindness.
SIMILARITY: SwissProt: P21918 ## Belongs to the G-protein coupled receptor 1 family.

Human

Chemicon

D5

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.

non-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.

Dopamine

A

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• DRD5
• DRD1B
• DBDR
• DRD1L2
• D
• MGC10601

GPCRs

ChemiScreen™ Human Recombinant D₅ Dopamine Receptor Calcium-Optimized Stable Cell Line

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.

EC50 for calcium mobilization by Dopamine: ~ 33 nM

Chem-1

GPCR Cell Lines

Chem-1

P21918

2x10⁶ cells/vial

NM_000798.3