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 TH. Binding of the myc monoclonal antibody 9E10 to 293-TSHRmyc cells could be detected with [I-125] anti-mouse IgG. No competition was observed between TSH and 9E10 binding to 293-TSHRmyc. Immunoprecipitation by 9E10 of TSHRmyc revealed TSHR forms of similar to 95 and similar to 100 kDa. Endoglycosidase digestion identified the similar to 95 kDa species as the single chain precursor with high mannose carbohydrate. The similar to 100 kDa single chain receptor contained mature, complex carbohydrate. No smaller species of TSHR subunits or proteolytic fragments was observed. Again TSH did nor 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 sire. This epitope-tagged TSHR will be valuable for fur;her studies on the synthesis and trafficking of TSHR. (C) 1996 Academic Press, Inc.