ChemiSCREEN™ Human Recombinant V_1B Vasopressin Receptor Calcium-Optimized Stable Cell Line

ChemiSCREEN™ Human Recombinant V_1B Vasopressin Receptor Calcium-Optimized Stable Cell Line

• ChemiScreen
• Chemicon (Millipore)

2 vials

Full-length human AVPR_1B cDNA encoding V_1B

Arginine vasopressin (AVP) is a 9 amino acid peptide that functions as an antidiuretic, vasoconstrictor and neurotransmitter. The three vasopressin receptors, V_1A, V_1B and V₂, are GPCRs; V_1A and V_1B couple to Gq and calcium release, whereas V₂ couples to Gs (Birnbaumer, 2000). The V_1B receptor is expressed prominently in the anterior pituitary, where it mediates vasopressin-induced release of ACTH (Tanoue et al., 2004). A selective antagonist of V_1B has recently been developed and shown to reduce depression, anxiety, and aggression in rodents (Blanchard et al, 2005; Griebel et al., 2002). Chemicon's cloned human V_1B-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant V_1B 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 V_1B and its ligands.

Calcium flux assay, ligand binding assays

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

The protein encoded by this gene acts as receptor for arginine vasopressin. This receptor belongs to the subfamily of G-protein coupled receptors which includes AVPR1A, V2R and OXT receptors. Its activity is mediated by G proteins which stimulate a phosphatidylinositol-calcium second messenger system. The receptor is primarily located in the anterior pituitary, where it stimulates ACTH release. It is expressed at high levels in ACTH-secreting pituitary adenomas as well as in bronchial carcinoids responsible for the ectopic ACTH syndrome. A spliced antisense transcript of this gene has been reported but its function is not known.

FUNCTION: SwissProt: P47901 # Receptor for arginine vasopressin. The activity of this receptor is mediated by G proteins which activate a phosphatidyl- inositol-calcium second messenger system.
SIZE: 424 amino acids; 46971 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
SIMILARITY: SwissProt: P47901 ## Belongs to the G-protein coupled receptor 1 family.

Human

EC50 for calcium mobilization by arginine vasopressin: ~ 0.17 nM
Z’ = 0.72 with arginine vasopressin at 2x EC50

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.

V1B

P47901

peptide

Vasopressin

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 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 growth media. Place the flask in a humidified incubator at 37ºC 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 Ca⁺⁺ and Mg⁺⁺ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37ºC 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-1 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-1 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-1 Plating Media for plating for calcium assay.

For special offers Click here http://www.millipore.com/drugdiscovery/dd3/gpcrtargetsolutions

A

2 x 10⁶ cells/vial

• AVPR1B
• VPR3
• V1bR
• AVPR3

GPCRs

ChemiSCREEN™ Human Recombinant V_1B Vasopressin Receptor Calcium-Optimized Stable Cell Line

NM_000707.2

Chem-1 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)
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250g/mL Genetecin/G-418

Chem-1 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-1 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)

2 x 10⁶ cells/mL