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) implicated in TSH binding, as well as autoantibody recognition in Graves' disease. In this study we employed antibodies specific for the amino-terminus (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 thyroid cell membranes via immunoblotting. Several forms of the receptor were detected. In reduced samples Ab1 detected full-length holoreceptors present in both nonglycosylated and glycoslylated forms of apparent molecular masses 80 and 90 kDa, respectively, as well as apparent dimeric nonglycosylated and dimeric glycosylated holoreceptor forms resistant to reduction. Also detected by Ab1 were a glycosylated amino-terminal 47- to 52-kDa fragment of the holoreceptor (gly alpha-subunit), reduced to 42 kDa (alpha-subunit) by Endo F deglycosylation. Ab2 detected all of the same forms. Ab3 detected primarily a carboxy-terminal, nonglycosylated fragment of 35 kDa (beta-subunit). In unreduced samples, the recognition pattern was unchanged with Ab1. Ab2 detected monomeric 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.
Graves
P N
PN
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA.
Vlase
H
H
Bobovnikova
Y
Y
Davies
T F
TF
D000645:Ammonium Sulfate; D000818:Animals; D000911:Antibodies, Monoclonal; D005591:Chemical Fractionation; D004220:Disulfides / Q000378:metabolism; D015151:Immunoblotting; D008566:Membranes / Q000378:metabolism; D051379:Mice; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D013552:Swine; D013961:Thyroid Gland / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004220:Disulfides; D011989:Receptors, Thyrotropin; D000645:Ammonium Sulfate