ChemiScreen™ Human Recombinant PTH₂ Parathyroid Hormone Receptor Calcium-Optimized Stable Cell Line

ChemiScreen™ Human Recombinant PTH₂ Parathyroid Hormone Receptor Calcium-Optimized Stable Cell Line

• ChemiScreen
• Chemicon (Millipore)

2 vials

Full-length human PTHR2 cDNA encoding PTH₂.

Parathyroid hormone (PTH) is recognized by three different class B GPCRs; PTH₁, PTH₂, and PTH₃, which couple to G_s to stimulate cAMP production. The PTH₁ receptor is found in high levels in the kidney and bone, where it binds PTH and PTHrP to regulate Ca²⁺ homeostasis (Brown et al, 1996). The PTH₂ receptor, which binds PTH and the neuropeptide tuberoinfundibular peptide 39 (TIP-39) but not PTHrP, has about 50% amino acid sequence identity to the PTH₁ receptor. PTH₂ is found in the greatest volume in the nervous system and at a very low density in the kidney and bone (Usdin et al, 1995). The PTH₃ receptor, which has been recently discovered in the zebrafish, has 61% amino acid identity to the zebrafish PTH₁ receptor and also seems to share its ligand affinity (Rubin et al, 1999). Studies with the selective PTH₂ receptor agonist, TIP-39, and the distribution of the receptor in the superficial dorsal horn of the spinal cord suggest the receptor may play a role in pain perception (Usdin et al, 1999). Chemicon's cloned human PTH₂-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant PTH₂ 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 PTH₂ and its ligands.

Calcium flux assay, ligand binding assays

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The protein encoded by this gene is a member of the G-protein coupled receptor family 2. This protein is a receptor for parathyroid hormone (PTH). This receptor is more selective in ligand recognition and has a more specific tissue distribution compared to parathyroid hormone receptor 1 (PTHR1). It is activated only by PTH and not by parathyroid hormone-like hormone (PTHLH) and is particularly abundant in brain and pancreas.

FUNCTION: SwissProt: P49190 # This is a specific receptor for parathyroid hormone. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase. PTHR2 may be responsible for PTH effects in a number of physiological systems. It may play a significant role in pancreatic function. PTHR2 presence in neurons indicates that it may function as a neurotransmitter receptor (By similarity).
SIZE: 550 amino acids; 62236 Da
SUBUNIT: Binds to TIPF39/TIP39.
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Expressed abundantly in brain and pancreas. Also expressed in the testis.
SIMILARITY: SwissProt: P49190 ## Belongs to the G-protein coupled receptor 2 family.

Human

EC50 for calcium mobilization by TIP-39: ~ 20.3 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.

PTH2

P49190

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.

PTH receptor

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B

2 x 10⁶ cells/vial

• PTH2R
• PTHR2

GPCRs

ChemiScreen™ Human Recombinant PTH₂ Parathyroid Hormone Receptor Calcium-Optimized Stable Cell Line

NM_005048.2

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.