Epitope tagging and immunoreactivity of the human angiotensin II type 1 receptor

Structural analysis of G-protein-coupled receptors has largely been limited to photoaffinity labeling and site-directed mutagenesis. This is primarily due to the difficulty in the production of antibodies against this class of receptors. We were therefore interested in tagging the amino-terminal side of the human angiotensin II (AngII) type 1 receptor (AT1) with the FLAG epitope DYKDDDDK. Competitive binding experiments with [125I][Sar1,Ile8]AngII revealed that stably transfected Chinese hamster ovary (CHO) cells express 37,300 hAT1-FLAG receptors/cell with a high affinity of 0.53 nM, comparable with that of the wild-type hAT1. After photolabeling and solubilization, a significant proportion of hAT1-FLAG specifically immunoprecipitated with anti-FLAG M5 and M2 antibodies. The immunoprecipitated receptor comigrated on SDS-PAGE with photolabeled wild-type hAT1. Immunofluorescence studies by FACS scan analysis revealed that 11.9% of CHO cells expressing hAT1-FLAG receptor significantly increased their fluorescence level as a result of M5 specific reactivity. Western blot analysis failed to show any specific interaction between M5 antibody and denatured hAT1-FLAG receptor. These results demonstrate the efficiency of the epitope tagging approach for specific immunoreactivity against AT1 receptor. Appropriate refinements of this approach could improve the level of immunoreactivity.