Regulation of thyrotropin receptor protein expression in insect cells

Expression of large quantities of conformationally intact thyrotropin recep tor (TSHR) is essential to understand the structure-function relationship o f the receptor. We expressed three different constructs of full-length huma n TSHR in insect cells: (a) a TSHR cDNA lacking signal sequence (TSHR-ns), (b) a TSHR cDNA containing human TSHR signal sequence (TSHR-hs) and (c) a T SHR cDNA with baculovirus envelope protein encoded signal sequence gp-67 (T SHR-gp). No unique protein band, corresponding to any or these recombinant proteins, was visible upon Coomassie Blue staining after SDS-PAGE. However, Western blot using TSHR specific monoclonal antibody showed unique bands a round 80, 100 and 100 kDa in TSHR-ns, TSHR-hs and TSHR-gp virus infected in sect cells respectively. All three full-length TSHR proteins could neutrali ze the TSH binding inhibitory immunoglobulin (TBII) activity from sera of e xperimental animals. However, only glycosylated proteins (TSHR-hs and TSHR- gp) neutralized the TBII activity of sera from autoimmune thyroid patients, confirming the importance of glycosylation for patient autoantibody reacti vity. Expression levels of full-length TSHR proteins were much lower than t he levels of similarly produced corresponding ectodomains of TSHR proteins. Southern blot and Northern blot analyses showed that DNA and RNA levels in full-length TSHR virus infected insect cells were comparable to the levels found in cells infected with viruses encoding only the ectodomain of TSHR. These data suggest that full-length TSHR expression is very low and is reg ulated at the translational level.