Human GHR Reporter Assay Kit

SIZE	SKU	PRICE
1 x-96 well format assays	IB14001	—
3 x-32 assays in-96 well format	IB14001-32	—
1 x-384 well format assays	IB14002	—

SIZE	SKU
1 x-96 well format assays	IB14001
3 x-32 assays in-96 well format	IB14001-32
1 x-384 well format assays	IB14002

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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 Growth Hormone Receptor (GHR). INDIGO’s GHR reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of GHR. In addition to GHR 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 human GHR. 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		Growth Factor Receptor
Species		Human
Receptor Form		Native
Assay Mode		Antagonist
Kit Components		GHR Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) Growth Hormone (GH), (ref. activator; in PBS/0.1% BSA ) Detection Substrate Detection Buffer White, sterile, cell-culture ready assay plate
Shelf Life		6 months
Orthologs Available		No
Shipping Requirements		Dry Ice
Storage temperature		-80C

Data

GHR Activation assay. Dose-response analyses were performed according to the protocol provided in the assay Technical Manual. 200 µl / well of GHR Reporter Cell suspension was dispensed into the 96-well assay plate, which was then incubated for 4 hours. The concentrated stock of the peptide GH (provided) was further diluted using CSM to produce treatment media at the desired assay concentrations. The pre-culture media were discarded from the assay wells and 200 µl per well of the prepared treatment media were dispensed (n = 3/conc.), including ‘untreated’ control wells. ‘Mock’ reporter cells, which contain the STAT5-Luc reporter gene, but lack expression of GHR, were similarly treated with GH. Following a 22 hr incubation period treatment media were discarded, Luciferase Detection Reagent was added, and luminescence intensity per well was quantified. Values of average relative light units (RLU) and corresponding values of standard deviation (SD), percent coefficient of variation (%CV), Fold-Activation and Z’ were determined for each treatment concentration. Non-linear regression analyses of Fold Activation vs. Log10[ng/mL] and EC50 determination was performed using GraphPad Prism software.

Target Background

The Growth Hormone Receptor (GHR) is a single-pass transmembrane receptor that functions as a homo-dimer. Growth Hormone (GH, aka Somatotropin) activates GHR to initiate signal transduction by a variety of pathways, including Ras/ERK, PI3K/Akt, and JAK2/STAT1,3,5,6. The activation of these various pathways may culminate in the activation of cytosolic targets, or in the activation of specific transcription factors and the induction of their respective target genes.

JAK2 dependent phosphorylation and activation of the transcription factor STAT5 is a prominent outcome of GHR activation, and it is the signaling pathway exploited by the reporter cells in this assay. Specifically, INDIGO’s Reporter Cells contain the luciferase reporter gene functionally linked to an engineered minimal promoter sequence with upstream tandem STAT5 genetic response element (GRE) sequences. Growth hormone activates the GHR in a dose-dependent manner, thereby triggering the JAK2/STAT signal transduction cascade. Activated STAT5 binds to its consensus GREs to initiate the formation of a complete transcription complex that drives expression of the Luc reporter gene. Therefore, quantifying changes in luciferase activity from peptide-, drug-, or antibody-treated reporter cells relative to that of “untreated” cells provides a sensitive, dose-dependent surrogate measure of changes in the activity of GHR.

GH/GHR play critical roles in the physiological processes of early bone and muscle development, and the regulation of lipid and carbohydrate metabolism. Also important is the role of GH-dependent activation of GHR in modulating the production and secretion of insulin-like growth factor-1 (IGF-1) within the liver, known as the GH-IGF1 axis. Dysregulation of GH production, either up or down, has significant physiological consequences. The development of body mass and size, insulin sensitivity, oncogenesis, and the onset of obesity, liver disease, and age-related diseases are greatly impacted by a chronic imbalance of GH production and/or function.

Not surprisingly, the development of novel GH-like peptide therapeutics, as well as small molecule and antibody modulators of Growth Hormone Receptor activity, continues to command much interest. Accordingly, the primary application of this assay is to screen test materials for any functional activity, either agonist or antagonist, that they may exert against the human GHR.

This assay utilizes proprietary human cells that provide constitutive expression of the Human Growth Hormone Receptor, isoform 1 (GHR).