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.
Roy
S F
SF
Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, QC, Canada.
Laporte
S A
SA
Escher
E
E
Leduc
R
R
Guillemette
G
G
D000345:Affinity Labels; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D016466:CHO Cells / Q000378:metabolism / Q000502:physiology; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000276:immunology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010455:Peptides / Q000276:immunology* / Q000378:metabolism; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
D000345:Affinity Labels; D000906:Antibodies; D018076:DNA, Complementary; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide