Fluorescent Human Pituitary Adenylate Cyclase-Activating Polypeptide Type I Receptor (ADCYAP1R1) Internalization Assay Cell Line
A novel U2OS/tGFP-PAC1 cell line has been developed through stable transfection to modulate the internalization of the Human Pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1) complex in living cells and to assay for compounds inducing tGFP-PAC1 redistribution inside the cells.
Fluorescent PAC1 Internalization A assay cell line was obtained by stable transfection of an expression vector for a fusion protein of tGFP and human Pituitary adenylate cyclase-activating polypeptide type I receptor, as well as G418 into U2OS cells. Single cells with strong green fluorescence were selected by flow cytometry, and allowed to expand. These cells constitutively express the tGFP-PAC1 fusion protein.
Assay Details: Innoprot PAC1 Internalization Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate Pituitary adenylate cyclase-activating polypeptide type I receptor activation and the following redistribution proce Beta inside the cells. This highly reproducible assay allows monitoring PAC1 receptor activation and redistribution process in High Content Analysis and fluorescence microscope applications.
This internalization assay was validated with an average of Z=0.84 +/- 0.02 for High Content Screening.
Fig 1: Our expression plasmid containing the coding sequence of human Pituitary adenylate cyclase-activating polypeptide type I receptor tagged in the N-terminal with tGFP protein. Our plasmid was transfected in U2OS cells. Resistant clones were obtained by limit dilution, and receptor gene expression was tested by RT-PCR.
Fig 2: Internalization of ADCYAP1R1 stimulated with PACAP-38.Concentrations from 0 to 10M were tested for 24h. Activation and internalization processes were detected and analyzed using BD Pathway 855 High-Content Bioimager from BD Biosciences.
Fig 3:Concentration response curve for PACAP-38in ADCYAP1R1 cell line. Cells were treated with 11 log dilution series (n=5). The Ec50 for PACAP-38 was 1.06 x 10-7 M after a treatment of 24 h with the agonist. Cells were fixed and the nuclei were stained with DAPI. % Activity was calculated relative to positive (10M). The internalization assay was validated with an average of Z =0.73+/- 0.02 for High Content Screening.