Technology – Nuclear Hormone Receptors (NHRs)
PathHunter® Cell-Based Nuclear Hormone Receptor (NHR) assays are whole cell, functional assays that measure NHR translocation and NHR-coactivator interaction. Unlike standard reporter gene assays, these assays are chemiluminescent and do not require lengthy 16-24 hour compound incubations making them ideal assays for screening and profiling.
Assay Principle
Nuclear Translocation NHR assays measure the activation status of a cytosolic NHR following binding of an agonist or an antagonist by monitoring its translocation to the nucleus. In this system, the ProLabel tag is fused to the NHR of interest and co-expressed in cells stably expressing the larger, N-terminal deletion mutant of β-gal called Enzyme Acceptor (EA) which is sequestered in the nucleus. Activation of the receptor and translocation to the nucleus forces complementation of the two enzyme fragments and leads to an increase in enzyme activity that can be measured using chemiluminescent PathHunter® Detection Reagents.
NHRPRO Protein Interaction assays detect NHR interaction with a co-activator fusion protein. In this system, the ProLink™ tag is fused to the C-terminus of the NHR of interest and the Enzyme Acceptor (EA) is attached to SRCP (Steroid response co-activator protein) that resides in the nucleus. These components interact only when in close proximity following activation of the receptor and translocation to the nucleus, forming active β-gal enzyme that converts substrate to detectable signal that can be measured using chemiluminescent PathHunter® Detection Reagents.
The PathHunter® NHR Advantage
- Single functional readout – interrogate the same receptor in 2 different formats
- Stable cell lines - increased reliability and reproducibility
- Full length receptors – more physiologically relevant data
- Short assay time (6 hours) - fewer off-target effects versus other reporter gene assays
- Specific signal generation – tagged receptor eliminates background from endogenous receptors/proteins
- Chemiluminescent detection - minimizes false positives from autofluorescence
Clonal Cell Lines
A non-imaging whole cell nuclear translocation assays to analyze translocation of NHR upon ligand stimulation.
Whole cell functional assays, based on the interaction between NHR and Steroid Receptor Coactivator Peptides (SRCP). An ideal assayl for inhibitor screening and pre-toxicity analysis.
Sample profiling data using NHRPRO
Profiling data for known ligands of the Androgen Receptor showing the expected pharmacology in the PathHunter® NHRPRO assay format [click graph to enlarge].
Inverse agonist data generated with NHRPRO Protein Interaction Assay demonstrating that multiple types of pharmacology can be detected with PathHunter® technology [click graph to enlarge].