FOLDING-DEPENDENT BINDING OF THYROTROPIN (TSH) AND TSH RECEPTOR AUTOANTIBODIES TO THE MURINE TSH RECEPTOR ECTODOMAIN

The mouse TSH receptor ectodomain (mTSHR-ecd) was amplified from murin e thyroid complementary DNA and ligated into the pAcGP67B insect cell vector, and the nucleotide sequence was confirmed. Employing a baculov irus-insect cell system, the mTSHR-ecd (amino acids 22-415) was expres sed as a fusion protein with the gp67 insect cell signal sequence at t he NH2-terminus and a C-terminal six-histidine tag. Protein expression was assessed by Western blot using a murine monoclonal antibody (reco gnizing amino acids 22-35) and a rabbit antipeptide antibody (recogniz ing amino acids 397-415). These antibodies detected two principal spec ies of mTSHR-ecd, one glycosylated (66 kDa) and one nonglycosylated (5 2 kDa), in cell lysates of infected insect cells. More than 10% of the se species were present in a water-soluble (cytosolic) fraction. This fraction was then used to purify, under native conditions, 100-mu g am ounts of mTSHR-ecd using nickel-nitrilo-triacetic (Ni-NTA) resin chrom atography. The purified cytosolic mTSHR-ecd migrated as a homogeneous 66-KDa band visible on Coomassie blue-stained gels and was confirmed b y Western blotting. We also purified the mTSHR-ecd from total cell lys ates under denaturing conditions, followed by ''in vitro'' refolding o n the Ni-NTA column. Under these conditions, milligram amounts of solu ble mTSHR-ecd were obtained. This material consisted primarily of the 66-kDa glycosylated form, but in addition contained four or five lower molecular mass, partially glycosylated intermediates and the 52-kDa n onglycosylated form. Deglycosylation with either endoglycosidase F or H, reduced all mTSHR-ecd glycosylated species to a 52-kDa nonglycosyla ted form. Both the cytosolic and refolded mTSHR-ecd preparations inhib ited the binding of [I-125]TSH to the full-length human TSHR expressed in Chinese hamster ovary cells in a dose-dependent manner, with simil ar affinities. The affinity of such interactions was 3 orders of magni tude less than observed with native porcine TSHR and was further reduc ed by unfolding the mTSHR-ecd preparations. The cytosolic and refolded mTSHR-ecd were also recognized by hT-SHR autoantibodies in the serum of patients with hyperthyroid Graves' disease. Such autoantibody bindi ng to mTSHR-ecd was also markedly reduced by unfolding the antigen. Th ese results demonstrated the successful production of large quantities of well characterized, biologically active, mTSHR-ecd antigen. In add ition, the data showed that although the ectodomain of the mTSHR bound TSH, intact holoreceptor may be required for high affinity Ligand bin ding. Whether the transmembrane region is required for direct ligand b inding, as seen for other G protein-linked receptors, or whether it is needed to stabilize the ligand binding to the ectodomain and maintain a correctly folded state, remains unclear.