MULTIMERIC COMPLEX-FORMATION BY THE THYROTROPIN RECEPTOR IN SOLUBILIZED THYROID MEMBRANES

The TSH receptor (TSHR) has a large glycosylated ectodomain comprising the amino-terminal half of the molecule (394 of 743 residues) implica ted in TSH binding, as well as autoantibody recognition in Graves' dis ease. In this study we employed antibodies specific for the amino-term inus (Ab1), midportion (Ab2), and carboxyl-terminus (Ab3) of the TSHR- ectodomain, previously mapped using recombinant receptor proteins, to detect the natural receptor present in detergent-solubilized porcine t hyroid cell membranes via immunoblotting. Several forms of the recepto r were detected. In reduced samples Abl detected full-length holorecep tors present in both nonglycosylated and glycosylated forms of apparen t molecular masses 80 and 90 kDa, respectively, as well as apparent di meric nonglycosylated and dimeric glycosylated holoreceptor forms resi stant to reduction. Also detected by Ab1 were a glycosylated amino-ter minal 47- to 52-kDa fragment of the holoreceptor (gly alpha-subunit), reduced to 42 kDa (alpha-subunit by Endo F deglycosylation. Ab2 detect ed all of the same forms. Ab3 detected primarily a carboxy-terminal, n onglycosylated fragment of 35 kDa (beta-subunit). In unreduced samples , the recognition pattern was unchanged with Ab1. Ab2 detected cleaved and disulfide-linked gly alpha-beta holoreceptors. Ab3 detected monom eric and dimeric beta-subunits, as well as higher order complexes. The different TSHR forms present in unreduced preparations were resolved by ammonium sulfate precipitation, confirming their autonomy. The data demonstrate the presence of multiple forms of the natural TSHR. Their roles in TSH action and TSHR autoimmunity require further exploration .