Fluorescent Corticotropin Releasing Hormone Receptor 2 (CRHR2) Internalization Assay Cell Line
A novel CHOK1/tGFP-CRHR2 cell line has been developed through stable transfection to modulate the internalization of the human corticotropin releasing hormone receptor 2 (CRHR2) complex in living cells and to assay for compounds inducing tGFP-CRHR2 distribution inside the cells.
Fluorescent CRHR2 Internalization Assay Cell Line was obtained by transfection of an expression vector for a fusion protein of tGFP and human CRHR2, as well as Puromycin into CHOK1 cells. Single cells with strong green fluorescence were selected by flow cytometry, and allowed to expand. These cells constitutively express the tGFP-CRHR2 fusion protein.
Assay Details:Innoprot CRHR2 Internalizacion Assay Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate human corticotropin releasing hormone receptor 2 internalization process following and quantifying the fluorescence distribution inside the cells.
This highly reproducible assay allows monitoring CRHR2 receptor internalization process in High Content Analysis and fluorescence microscope applications.
Fig 1: Our expression plasmid containing the coding sequence of human corticotropin releasing hormone receptor 2 tagged in the N-terminal with tGFP protein. Our plasmid was transfected in CHO-K1 cells, using calcium phosphate method. Resistant clones were obtained by limit dilution, and receptor gene expression was tested by RT-PCR.
Fig 2: Internalization of CRHR2-tGFP stimulated with corticotropin hormone (CRF). Cells were treated with 1uM CFR for 6h. Activation and internalization process were detected and analyzed using BD Pathway 855 High-Content Bioimager from BD Biosciences.
Fig 3:CRHR2-tGFP internalization in response to CRF concentrations. Cells were treated with 8 log dilution series (n=8). The Ec50 for the CRF was 38 nM after a treatment of 6h with agonist. Cells were fixed and the nuclei were stained with DAPI. % Activity was calculated relative to positive (1uM). The internalization assay was validated with an average of Z=0.55 +/- 0.05 for High Content Screening.