A Case Example of Urotensin II Receptor
Objective: G-protein coupled receptors (GPCRs) represent crucial cell surface receptors in the drug discovery process. Here we present a post-translationally labeling strategy that uses SNAP-tag ® technology to identify stably transfected GPCRs clones exemplified by the human urotensin II (UII) receptor (UT). Materials and methods: In this study, we transfected SNAP-UT into HEK293A cells to generate stable cell clones. Results: In total, 77.5%, 8.6%, 4.7%, and 2.2% of clones had fluorescence intensities that were 2-, 4-, 8-, and 16-fold higher than the control, respectively. In the recombinant clones, the fluorescence intensities were parallel well with the RNA levels of UT. For example, the fluorescence intensity of clone A8 was four times higher than clone A50, and qRT-PCR results showed that the UT mRNA levels in A8 were 3.7 times higher than A50. The binding assay result also suggested that the structure of the recombinant receptor was consistent with wild-type UT. Furthermore, the downstream signals conducted by the recombinant UT were matching with the wild-type. In recombinant clones, both Inositol-1-phosphate (IP1) accumulations and calcium flux levels rose dose-dependently under UII stimulation, and significantly prominent in clone A8 than A50. Conclusion: Our results indicate that SNAP-tag approach on GPCR cell line establishment is fast and simple, furthermore, can quantitatively provide recombinant gene expression levels.
Life Sci J 2013;10(4): 2519-2525