ChemiSCREEN™ Human Recombinant Y₄ Neuropeptide Y Receptor Calcium-Optimized Stable Cell Line

ChemiSCREEN™ Human Recombinant Y₄ Neuropeptide Y Receptor Calcium-Optimized Stable Cell Line

Chemicon (Millipore)

2 vials

Full-length human PPYR1 cDNA encoding Y₄

The NPY family consists of three 36-amino acid peptides, neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), which bind to the NPY receptor family of G protein-coupled receptors. Five NPY receptors, Y₁, Y₂, Y₄, Y₅ and y₆, have been defined at the molecular level, and each signals primarily through Gi/o. Binding of NPY family peptides to NPY receptors mediates a variety of physiological effects, including promotion of food intake, decreased anxiety, inhibition of neurotransmitter and hormone release, vasoconstriction, and gut motility. Y₄ binds preferentially to PP, with significant binding to NPY and PYY (Michel et al., 1998). Y₄ plays a role in control of reproduction, as Y₄-null, ob/ob double knockout mice have increased fertility relative to ob/ob mice with functional Y₄ (Sainsbury et al., 2002). In addition, mice lacking Y₄ display resistance to weight gain induced by high fat diet, and mice lacking both Y₂ and Y₄ are resistant to the enhanced weight gain exhibited by Y₁-null mice fed a high fat diet (Sainsbury et al., 2006). Millipore's cloned human Y₄-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant Y₄ 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 Y₄ 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.

FUNCTION: SwissProt: P50391 # Receptor for neuropeptide Y and peptide YY. The rank order of affinity of this receptor for pancreatic polypeptides is PP, PP (2-36) and [Ile-31, Gln-34] PP > [Pro-34] PYY > PYY and [Leu-31, Pro-34] NPY > NPY > PYY (3-36) and NPY (2-36) > PP (13- 36) > PP (31-36) > NPY free acid.
SIZE: 375 amino acids; 42195 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Highest levels found in brain, coronary artery and ileum. Low levels in pancreas and kidney. Detected in colon and small intestine.
SIMILARITY: SwissProt: P50391 ## Belongs to the G-protein coupled receptor 1 family.

Human

EC50 for calcium mobilization by Pancreatic Polypeptide: ~ 17.6 nM

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.

Y4

P50391

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 Ca++ and Mg++ (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-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.

Neuropeptide Y

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A

2 x 10⁶ cells/vial

• PPYR1
• Y4
• PP1
• MGC116897
• NPY4R
• NPY4-R

ChemiSCREEN™ Human Recombinant Y₄ Neuropeptide Y Receptor Calcium-Optimized Stable Cell Line

NM_005972.4

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)
100 U/mL Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250µg/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)