An investigation of the properties of TSH receptors on FRTL, cells using affinity labelling with a rz51-labelled photoactive derivative of TSH is described. Our studies suggest that FRTL, cells contain 2 principal types of cell surface TSH receptors. One form, probably a precursor, consists of a single polypeptide chain (Mr 120000) with an intrachain loop of amino acids formed by a disulphide bridge. The other type of receptor consists of a water-soluble A chain (MT 55000) linked to an amphiphilic B chain (Mr 35000) by a disulphide bridge. The 2 chain structure is probably derived from the single chain 120000 protein by enzymatic cleavage of peptide sequences within the loop of amino acids formed by the intrachain disulphide bridge.

Porcine thyrotropin (TSH) receptors have been purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labelled photoactive derivative (N-hydroxysuccinimidyl 4-azidobenzoate; HSAB) of TSH (125I-HSAB-TSH). Purification of the crosslinked complexes on Sephacryl S-300 followed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that the receptor contained two subunits. One subunit (A) with Mr 45 000 was crosslinked to TSH and the other (B) subunit, Mr 25 000, was linked to the A subunit by a disulphide bridge(s). Other, as yet unidentified, subunits may have been non-covalently associated with the A and B subunits. Analysis of reduced and non-reduced crosslinked TSH receptor-125I-HSAB-TSH on Sephacryl S-300 in the presence and absence of detergent indicated that the A subunit was a hydrophilic peptide. This was confirmed in studies of the release into aqueous solution by reducing agent treatment of 125I-HSAB-TSH crosslinked to the TSH receptor A subunit in thyroid membranes. Similar results were obtained with TSH receptors in human thyroid and guinea pig fat cell membranes. These studies suggest that the hydrophilic A subunit of the receptor forms a binding site for TSH on the outside surface of the cell membrane and that the A subunit is linked to the cell membrane by way of a disulphide bridge to the receptor B subunit.

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The ability of Graves' sera to interact with the TSH receptor crosslinked to a 125I-labelled photoactive derivative of TSH has been investigated. Crosslinked complexes were prepared using non-purified detergent solubilized human thyroid and guinea pig fat TSH receptors. Affinity purified porcine TSH receptor preparations wee also used. After crosslinking, the crosslinked TSH-TSH receptor complexes were separated from aggregates and free TSH on Sephacryl S-300, incubated with test sera followed by immunoprecipitation using anti-IgG or Protein A. Using non-purified human TSH receptors crosslinked to TSH, a mean +/- SD of 12.1 +/- 4.9% of the crosslinked complex was immunoprecipitated with Graves' sera (n = 7) compared with 10.3 +/- 2.6% with Hashimoto sera (n = 6; P greater than 0.14) and 3.8 +/- 1.0% with normal sera (n = 6; P less than 0.004). These values were markedly reduced when TSH receptor preparations free of other thyroid autoantigens (guinea pig fat TSH receptors) were used. Under these conditions immunoprecipitation with Graves' sera (n = 24) was 1.6 +/- 1.3% compared with 0.8 +/- 0.6% for Hashimoto sera (n = 13) and 0.8 +/- 0.4% for normal sera (n = 12; P less than 0.003). In addition complexes formed between TSH and affinity purified porcine TSH receptors gave low immunoprecipitation values for Graves' (1.44 +/- 0.73%; n = 20) and Hashimoto sera (1.7 +/- 0.94; n = 11) which were not significantly different (P greater than 0.4). Overall, therefore, the effects of Graves' and Hashimoto sera were similar and the amounts of material immunoprecipitated were markedly reduced when TSH receptor preparations containing reduced amounts of other autoantigens were used. Consequently the Graves' sera did not appear to interact specifically with crosslinked TSH-TSH receptor complexes. However the Graves' sera studied did contain TSH receptor antibodies which could inhibit the binding of labelled TSH to TSH receptors in the preparations used and our results suggest that the binding of TSH and these antibodies to the receptor is mutually exclusive. There is considerable evidence that serum from patients with Graves' disease contains antibodies to the TSH receptor (Rees Smith, 1981). Several studies have suggested that binding of the receptor antibody and TSH to the TSH receptor is mutually exclusive (Manley et al., 1977; Petersen et al., 1977; Rickards et al., 1981) but recently the formation of termolecular complexes consisting of detergent solubilized receptors, labelled TSH and Graves' IgG has been reported (Konishi et al., 1982; De Bruin et al., 1984).(ABSTRACT TRUNCATED AT 400 WORDS)

Studies of the TSH receptor using affinity labelling with photoactive derivatives of TSH and analysis by SDS-PAGE have shown that the receptor contains 2 subunits (A and B), linked by a disulphide bridge. Similar results are obtained with TSH receptors from human, porcine and guinea pig thyroid tissue and from guinea pig fat. Analysis of affinity labelled receptors under non-denaturing conditions suggest that subunits additional to the A and B subunits are not present.Hydrodynamic measurements indicate that the receptor A subunit has an approximately spherical structure (Stokes' radius 70\ l =A%o\ )and when this interacts with TSH (an elongated structure with Stokes' radius 56\ l =A%o\ ) a very elongated complex (Stokes' radius 104\l =A%o\)is formed.Isoelectric focusing studies of the TSH receptor A subunit, TSH and TSH receptor antibodies indicate that charge-charge interactions are of considerable importance in the binding of hormone and antibody to the receptor.The TSH receptor is an integral membrane glyco¬ protein which forms a binding site for TSH on the outside surface of thyroid follicular cells. The binding of TSH to the receptor causes the hor¬ mone-receptor complex to interact with the regu¬ latory subunits of adenylate cyclase. This interac¬ tion takes place in the lipid bilayer or at the cytoplasmic surface of the cell membrane and results in production of increased amounts of cyclic AMP which mediate most, if not all, of the actions of the hormone. In Graves' disease, a relatively common autoimmune disorder, auto¬ antibodies to the TSH receptor are formed. They bind to the receptor in such a way as to mimic the effects of TSH and this results in the development of hyperthyroidism (Rees Smith 1981;Rees Smith et al. 1985).This short review considers the properties of the receptor and the nature of its interaction with TSH and TSH receptor antibodies. In addition, the relationship of the TSH receptor-receptor antibody system to other autoantibody-autoantigen system will be considered. Photoaffinity labelling of the TSH receptorThe TSH receptor is present on the cell surface in very small amounts (in the region of 103 sites per cell; Rees Smith et al. 1985), and consequently special techniques are often required to study the receptor. One of the most powerful techniques currently available for studying trace amounts of cell surface receptor is affinity labelling (Ji 1979). In the case of the TSH receptor, we have found TSH coupled to photoactive cross-linking rea¬ gents to be particularly useful (Buckland et al. 1986). These studies have indicated that both subunits of TSH form part of the hormone's receptor binding site (Buckland et al. 1986). Analysis by SDS-PAGE of porcine TSH recep¬ tors cross-linked to 125I-labelled TSH have shown that the receptor contains 2 subunits linked by a disulphide bridge. One of the subunits (A; mol wt 50 K) is water soluble and forms the binding site for TSH on the outside surface of the cell mem¬ brane. The other (B) subunit (mol wt 30 ) penetrates the lipid bilayer (Ka...