ChemiScreen™ Human Recombinant NK₂ Tachykinin Receptor Calcium-Optimized Stable Cell Line

ChemiScreen™ Human Recombinant NK₂ Tachykinin Receptor Calcium-Optimized Stable Cell Line

• ChemiScreen
• Chemicon (Millipore)

2 vials

Full-length human NK2R cDNA encoding NK2

The tachykinin peptide family in mammals comprises three peptides, substance P, neurokinin A and neurokinin B, which bind to the tachykinin receptor family of GPCRs, NK₁, NK₂ and NK₃ (Severini et al., 2002). Tachykinins have prominent activity in the GI system, in which they stimulate intestinal contraction and salivation; these effects are mediated by NK₁ and NK₂. Experiments with selective antagonists of NK₂, SR 48,968 and MEN 10,376 indicate that NK₂ mediates neurokinin- induced constriction of pulmonary artery and bronchus (Maggi et al., 1993). NK₂ antagonists display efficacy in treatment of irritable bowel disorder (Lecci et al., 2004). Chemicon's cloned human NK₂-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant NK₂ 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 NK₂ and its ligands.

Calcium flux assay, ligand binding assays

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This gene belongs to a family of genes that function as receptors for tachykinins. Receptor affinities are specified by variations in the 5'-end of the sequence. The receptors belonging to this family are characterized by interactions with G proteins and 7 hydrophobic transmembrane regions. This gene encodes the receptor for the tachykinin neuropeptide substance K, also referred to as neurokinin A.

FUNCTION: SwissProt: P21452 # This is a receptor for the tachykinin neuropeptide substance K (neurokinin A). It is associated with G proteins that activate a phosphatidylinositol-calcium second messenger system.
SIZE: 398 amino acids; 44439 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
SIMILARITY: SwissProt: P21452 ## Belongs to the G-protein coupled receptor 1 family.
MISCELLANEOUS: The rank order of affinity of this receptor to tachykinins is: substance K > neuromedin-K > substance P.

Human

EC50 for calcium mobilization by Neurokinin A ~ 0.86 nM
Z’ = 0.92 with neurokinin A 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.

NK2

Cell Line

P21452

peptide

Tackykinin /neurokinin

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.

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A

2 x 10⁶ cells/vial

• TACR2
• SKR
• NKNAR
• NK-2R
• NK2R
• TAC2R

GPCRs

ChemiScreen™ Human Recombinant NK₂ Tachykinin Receptor Calcium-Optimized Stable Cell Line

NM_001057.2

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/vial