Human AVPR2 Reporter Assay Kit

SIZE	SKU	PRICE
1 x-96 well format assays	IB37201	—
3 x-32 assays in-96 well format	IB37201-32	—

SIZE	SKU
3 x-32 assays in-96 well format	IB37201-32
1 x-96 well format assays	IB37201

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Specifications
Data
Target Background
Documentation
Overview
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Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Arginine Vasopressin Receptor 2 (AVPR2). INDIGO’s AVPR1B reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the AVPR2. In addition to AVPR2 Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against AVPR2. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

Clear, Reproducible Results
All-Inclusive Assay Systems
Exceptional Cell Viability Post-Thaw
Consistent Results Lot to Lot

Product Specifications

Target Type		GPCR
Species		Human
Receptor Form		Hybrid
Assay Mode		Agonist, Antagonist
Kit Components		Human AVPR2 Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) Vasopressin, (ref. agonist; in PBS+0.1%BSA) Detection Substrate Detection Buffer White, sterile, cell-culture ready assay plate
Shelf Life		6 months
Shipping Requirements		Dry Ice
Storage temperature		-80C

Data

Activation of AVPR2. Reporter cells were treated with the reference activators polypeptides Vasopressin (provided), Felypressin, Terlipressin and Oxytocin. The absence of signal in Vasopressin treated ‘Mock’ cells (which contain the CRE-Luc reporter vector, but do not express AVPR2) confirms that the observed ligand-dependent response is specific to AVPR2 activation. Luminescence was quantified and values of average (n = 4) RLU, standard deviation (SD), Fold-Activation, and Z’ values were calculated. The least-squares method of non-linear regression was used to plot activity changes vs. Log10 [Compound, nM], and EC50 /IC50 values were determined, using GraphPad Prism software.

Inhibition of AVPR2. Reporter cells were co-treated with an EC80 concentration of the reference activator Vasopressin and varying concentrations of the AVPR2 selective inhibitor Tolvaptan, and the non-selective inhibitor Conivaptan. INDIGO’s Live Cell Multiplex (LCM) Assay confirmed that no treatment concentrations were cytotoxic (data not shown). Luminescence was quantified and values of average (n = 4) RLU, standard deviation (SD), Fold-Activation, and Z’ values were calculated. The least-squares method of non-linear regression was used to plot activity changes vs. Log10 [Compound, nM], and EC50 /IC50 values were determined, using GraphPad Prism software.

Target Background

The Arginine Vasopressin Receptor 2 (AVPR2), also known as the V2 receptor, is a member of the family of G-Protein-coupled receptors (GPCR). There are three subtypes of vasopressin receptors identified in humans, which are AVPR1A, AVPR1B and AVPR2.

The natural ligand of AVPR2 is vasopressin, which is synthesized in the hypothalamus and secreted into the posterior pituitary. Like the closely similar neuropeptide, oxytocin, vasopressin is a nine amino acid neuropeptide that is classified as an antidiuretic hormone (ADH). Release of vasopressin is mediated by stimuli such as hemorrhage or dehydration. When vasopressin binds to AVPR2, signal transduction is mediated through Gas GTP binding proteins, which activates adenylate cyclase to increase intracellular cAMP. This cascade further activates protein kinase A (PKA).

The most abundant expression of AVPR2 is found in the kidney. The main function of AVPR2 is to regulate water homeostasis in the body. Binding of vasopressin to the V2 receptor in the kidney stimulates cellular relocation of aquaporin 2 (AQP2) to the apical plasma membrane. This facilitates water reabsorption from primary urine. The impaired regulation of vasopressin causes a serious condition known as Diabetes insipidus (DI).

Besides as an antidiuretic factor, vasopressin is also known to regulate blood coagulation. Desmopressin has been used as a haemostatic drug for patients with hemophilia A by stimulating production of coagulant factor VIII. Interestingly, drug repurposing of desmopressin revealed the promising treatment for patients with osteosarcoma. In fact, AVPR2 has been detected in several cancers such as kidney, breast, colon and lungs.