Epitope-tagging of a functional thyrotropin receptor: detection of the native receptor on intact cells.
To facilitate immunological detection of thyrotropin receptor (TSHR), we inserted a c-myc epitope within the unique, 50 amino acid segment of the ectodomain (TSHRmyc). When stably expressed in 293 human embryonal kidney (HEK) cells, TSHRmyc demonstrated high affinity TSH binding and the ability to produce cAMP in response to TSH. Binding of the myc monoclonal antibody 9E10 to 293-TSHRmyc cells could be detected with [125I] anti-mouse IgG. No competition was observed between TSH and 9E10 binding to 293-TSHRmyc. Immunoprecipitation by 9E10 of TSHRmyc revealed TSHR forms of approximately 95 and approximately 100 kDa. Endoglycosidase digestion identified the approximately 95 kDa species as the single chain precursor with high mannose carbohydrate. The approximately 100 kDa single chain receptor contained mature, complex carbohydrate. No smaller species of TSHR subunits or proteolytic fragments was observed. Again TSH did not inhibit immunoprecipitation of TSHRmyc by 9E10. These data demonstrate that the normally functioning c-myc epitope-tagged TSHR can be detected directly and in native form with a readily available anti-myc 9E10 and without the need for prior affinity capture. Lack of competition between 9E10 and TSH suggests that at least part of the 50 amino acid segment in TSHR ectodomain is not a TSH binding site. This epitope-tagged TSHR will be valuable for further studies on the synthesis and trafficking of TSHR.
Department of Pharmacology, Nagasaki University School of Medicine, Japan.
K
K
Tanaka
Y
Y
Nagayama
H
H
Yamasaki
H
H
Hayashi
H
H
Namba
S
S
Yamashita
M
M
Niwa
D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D000939:Epitopes; D016259:Genes, myc; D006801:Humans; D011233:Precipitin Tests; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000276:immunology; D011989:Receptors, Thyrotropin / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D016271:Proto-Oncogene Proteins c-myc; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP