Increased receptor binding by bovine (b) TSH bound to monoclonal antibody to bTSHbeta-subunit

TSH receptor (R) binding and cAMP production by bovine (b) TSH-bound to a monoclonal antibody (MoAb) or polyclonal antibody (Ab) to bTSH were examined, using TSH receptor (R) coating tube and porcine thyroid cells. (125) I-bTSH bound-to MoAbs to bTSH(alpha) or discontinuous type MoAb showed TSHR binding (10%) similar to intact (125) I-bTSH. TSHR binding was completely decreased (<2%) when (125) I-bTSH was bound by polyclonal Abs to bTSH(alpha) in Graves' patient or rabbit polyclonal Abs to bTSH. When either of the two MoAb (No. 1 and 2) to bTSH(beta) was bound to (125) I-bTSH, TSHR binding was 4 times higher (40%) compared to intact (125) I-bTSH. Binding of another MoAb (No. 3) caused no increased binding. TSHR binding of intact (125) I-bTSH was decreased from 10% to 2% by excess amounts of bTSH. Binding of (125) I-bTSH bound to MoAb to bTSH(beta) (No. 1 and 2) decreased from 40% to 30% by excess amounts of bTSH. When (125) I-bTSH bound-Fab of MoAb was used, the binding was reduced from 30 to 10% (No. 1) and from 25 to 6% (No. 2), respectively. In contrast, cAMP production by bTSH was decreased by pre-binding of all MoAbs and polyclonal Abs. Binding of (125) I-MoAb to bTSH (beta) to a synthetic peptide array of bTSH (beta) sequence was examined by the radioautography. The epitope of MoAb to bTSH(beta) was suggested to be LPK (beta 42-44) for No. 1, KLF (beta 39-41) for No. 2 and PKYA (beta 43-46) for No. 3, respectively, although the existence of discontinuous epitope could not be ruled out. The increased TSHR binding and the decreased cAMP production by bTSH bound to MoAbs may be due to the conformational change of TSH molecule or TSHR by binding of both bTSH and MoAb.     

Interference with thyrotropin receptor antibody determination by a spuriously occurring anti-bovine TSH antibody

Abnormally negative values of thyrotropin binding inhibitor immunoglobulin (TBII) were found in the sera from a patient with Graves' disease. This was due to the presence of potent bovine TSH (bTSH) binding activity in the sera. This activity was demonstrated to be in immunoglobulin G (IgG) with a lambda light chain isotype, which was shown to have an affinity for bTSH with a Ka value of 3.5 X 10(10) M-1 and a maximum binding capacity of 1.1 X 10(-14) M/mg IgG. F(ab')2 fragments obtained through pepsin digestion from the patient's IgG retained bTSH binding activity. [125I] bTSH binding to this IgG was inhibited by the TSH receptor. The inhibition was not completely competitive, suggesting the presence of different binding sites for this IgG and the TSH receptor on the TSH molecule. This IgG, however, could not bind labelled human TSH (hTSH). Since neither TSH nor other pituitary derivatives had ever been given to the patient, this bTSH binding activity was considered to be due to a spuriously occurring anti-bTSH antibody.     

Activities of deglycosylated thyrotropin at the thyroid membrane receptor-adenylate cyclase system

A bovine thyrotropin (bTSH) preparation was deglycosylated by treatment with anhydrous hydrogen fluoride (HF) in the presence of anisole. The resulting material consisted of TSH derivatives that exhibited different molecular sizes, all smaller than the native hormone. The majority (62%) of the deglycosylated TSH derivatives did not bind to the lectin concanavalin A, while 98% of the native TSH was able to bind. The deglycosylated TSH derivatives bound to the high affinity-high specificity TSH binding sites in human thyroid membranes with a potency more than twice that of equivalent immunological amounts of the native bTSH. Despite the enhanced binding affinity for the TSH receptor, the deglycosylated TSH derivatives were unable to stimulate adenylate cyclase fully. Maximal stimulation achieved with bTSH derivatives was only 9 to 17% of the maximal stimulation achieved with native bTSH. Further, the deglycosylated derivatives competitively inhibited stimulation of the thyroidal adenylate cyclase by native bTSH. We conclude that HF treatment of bTSH results in partially deglycosylated TSH derivatives that exhibit enhanced ability to bind to the TSH receptor and markedly diminished adenylate cyclase-stimulating activity.     

Microheterogeneous forms of radioiodinated bovine thyrotropin: discrimination of different receptor-active components by gel permeation chromatography

The products of the radioiodination and subsequent receptor adsorption of bovine TSH (bTSH) radiolabeled by the lactoperoxidase method have been further investigated. After receptor adsorption, [125I]bTSH was resolved by gel permeation chromatography on Sephadex G-100 (superfine) under low ionic strength conditions into three peaks of radioactivity (tracers 2a, 2b, and 2c, respectively). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions demonstrated that each tracer component was radiolabeled on both the alpha- and beta-subunits. Analysis of the three tracers by TSH radioreceptor assay (under different radioreceptor assay conditions) showed that tracers 2b and 2c exhibited saturable rebinding to crude thyroid membranes containing functional TSH receptors. However, tracer 2c exhibited a maximum binding 2-fold greater than tracer 2b. This difference has been attributed to the abundance of an apparently low affinity binding component in tracer 2c. Rechromatography of tracers 2b and 2c on Sephadex G-100 (superfine) under high ionic strength conditions yielded tracer profiles that were coincident, demonstrating that the initial separation under low ionic strength conditions was not based on differences in molecular volume. The data indicate that radioiodination of highly purified bTSH yields multiple tracer components. Further, receptor adsorption, commonly used to purify freshly iodinated bTSH before radioreceptor assay, purifies at least two species of receptor-active [125I] bTSH.     

Characterization and purification of iodinated bovine thyrotropin by high performance liquid chromatography

Reversed-phase (RP) HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography have been used to study the incorporation of 125I into bovine (b) TSH by the lactoperoxidase-catalyzed radioiodination procedure. Two preparations of [125I]bTSH were studied, being freshly iodinated bTSH and tracer purified from this preparation by the method of receptor adsorption. It is demonstrated with these methods that both the alpha- and beta-subunits of bTSH are labeled with 125I, and that the tracer purified by receptor adsorption retains this incorporation pattern. However, the implied theoretical specific activity of (at least) 2 I atoms per TSH molecule (or approximately 140 muCi/micrograms) suggested by this result was not achieved, with observed tracer specific activity being 30-60 muCi/micrograms, indicating that hormone molecules with varying extents of labeling must exist. Evidence to support this was provided by comparison of the MIT/DIT ratios for the 2 tracer preparations. Receptor adsorption decreased the MIT/DIT ratio from 75:25 in the freshly iodinated bTSH to 93:7, indicating the selection of particular iodinated species. Tryptic mapping by RP-HPLC was used to study both tracer preparations, and it is shown that at least 14 iodine-containing tryptic peptides may be resolved for each preparation, which is greater than the theoretical maximum of 13 peptides if every tyrosine was labeled and tryptic cleavage occurred at all possible lysine and arginine residues. Tracer heterogeneity was also studied by purification using RP-HPLC. Selection of peak fractions demonstrated that intact [125I]bTSH may be recovered from RP-HPLC which in TSH radioreceptor assay exhibit increased assay sensitivity, increased saturable binding, and decreased nonsaturable binding.     

Characteristics of auto-antibodies to bovine TSH in the serum of two patients with Graves' disease

In this report we describe the characteristics of auto-antibodies to bovine TSH (bTSH) detected in the serum of 2 females among 102 patients with Graves' disease. These patients had never been injected with bTSH. One patient had high LATS activity and high bTSH binding activity after isotope therapy. The other patient showed no detectable LATS activity. Interestingly, the antibody showed a specifically high binding activity for the labelled TSH preparation purified by receptor. The auto-antibody could be demonstrated by the double antibody method, polyethylene glycol method, and by gel-filtration. The antibody was polyclonal immunoglobulin G (IgG). Because the binding of [125I]bTSH with the patient's antibody was inhibited by pituitary extracts from mammalian species other than human, this antibody may cross-react with bovine, rat, dog, rabbit and whale TSH. Although the incidence of the antibody in Graves' disease is low and the pathological significance remains obscure, the existence of this antibody in the serum of patients may suggest that autoimmune mechanisms may involve not only the thyroid but also the pituitary in Graves' disease.     

ANTI-TSH ANTIBODY WITH HIGH SPECIFICITY TO HUMAN TSH IN SERA FROM A PATIENT WITH GRAVES'' DISEASE: ITS ISOLATION FROM, AND INTERACTION WITH, TSH RECEPTOR ANTIBODIES

A patient with thyrotoxic Graves' disease had an apparent measurable level of serum TSH (2.5 microU/ml) by double-antibody radioimmunoassay (RIA). The serum IgG bound with both [125I]human(h)TSH and [125I]bovine(b)TSH. The [125I]hTSH binding was more effectively displaced by human than bovine TSH, whereas [125I]bTSH binding was displaced exclusively by bTSH. Scatchard analyses revealed that [125I]hTSH binding showed two components, whereas [125I]bTSH binding had only one component. Serum TSH determined by RIA became undetectable 21 months after antithyroid drug treatment with a parallel decrease of [125I]hTSH binding IgG activity. Four thyrotrophin binding inhibitory immunoglobulins (TBII) from other patients did not interfere with the binding of the patient's serum to [125I]h- or bTSH. Furthermore, the in-vitro thyroid stimulating activities of three thyroid stimulating antibodies (TSAb) were not affected by the addition of this patient's IgG. On the other hand, this patient's Ig (3 mg/ml) abolished the in-vitro thyroid stimulation by bTSH (100 microU/ml), but did not affect that by hTSH (100 microU/ml). The anti-hTSH antibody, TSH receptor antibody and anti-bTSH antibody in the serum, which contains TSAb as well as anti-TSH antibodies, could be partially purified by hTSH-agarose and subsequently by guinea pig fat cell membrane affinity absorptions. However, the anti-hTSH antibody fraction obtained had both hTSH binding activity and thyroid stimulating activity, and this fraction did not show any inhibitory effect on the in-vitro thyroid stimulation of autologous TSH receptor antibody or hTSH. The possible significance of anti-TSH antibodies is discussed.     

Positive regulation of the guinea pig thyrotropin receptor

Subcutaneous administration of bovine (b) TSH (up to 10 IU) to 8-week-old male guinea pigs was followed by a transient elevation in serum thyroid hormone levels (T4 and T3) and an increase in thyroid weight (approximately 25-40%), which returned to control levels by 3 days. Total thyroid TSH receptor content was assessed by the binding of receptor-purified [125I]bTSH to 15,000 X g fractions of thyroid homogenate. The TSH receptor content paralleled the increase in thyroid weight, with no detectable change in the TSH-binding capacity per mg tissue. Intraperitoneal minipump infusions of bTSH (1 IU/day) over 6 days produced marked and persistent increases in thyroid hormone levels and thyroid weight (greater than 300%) and a similar increase in TSH receptor content. There was no change in the single site equilibrium association constant for bTSH [1.1 X 10(9) M-1 +/- (SE) 9.6 X 10(7) M-1] and no alteration in the binding capacity per mg tissue (67.2 +/- 6.4 pg/mg). Investigation of the in vitro adenylate cyclase response to bTSH (10 mU/ml) and the production of immunoassayable cAMP showed no difference between thyroid tissue obtained from bTSH-treated animals and that obtained from untreated control animals. These observations demonstrated that TSH exerted a positive regulatory effect on its receptor and, under the in vivo conditions used, failed to induce TSH receptor loss or physiologically important desensitization. Such data may explain how TSH receptor antibodies are able to act as TSH agonists and maintain increased thyroid hormone output in human disease.     

Comparison of binding of bovine and human thyroid-stimulating hormone to receptor sites on human thyroid membranes

The binding of 125I-labelled bovine TSH (bTSH) to a wide range of human thyroid membrane preparations was compared with that of 125I-labelled human TSH (hTSH). Much higher binding percentages were obtained with the 125I-labelled bTSH. This was because the receptors had a higher binding affinity for bTSH than for hTSH. No differences in tracer purity, nor differences in optimal conditions for the binding of bTSH or hTSH, nor tracer degradation contributed significantly to the better binding of 125I-labelled bTSH. Good correlation was found between binding percentages for 125I-labelled bTSH and 125I-labelled hTSH over the range of thyroid specimens. Useful information on human TSH receptors is, therefore, obtainable from binding studies with 125I-labelled bTSH. The TSH displacement curves yielded linear Scatchard plots whenever the tracer and displacing hormones were of the same species. The data were, therefore, consistent with a simple binding reaction between TSH and a single set of independent receptor sites.     

Interaction of epidermal growth factor with receptors on human and porcine thyroid membranes

Recent evidence suggests that epidermal growth factor (EGF) may play an important role in the regulation of thyroid growth and function. We have examined the interaction of murine EGF (mEGF) with human and porcine thyroid membranes and compared this with the binding of bovine TSH (bTSH) using 125I-labelled hormones as tracers. The characteristics of the binding of mEGF were found to be similar for human and porcine thyroid membranes. Epidermal growth factor bound with high affinity (affinity constant = 1.4 X 10(9) l/mol); the density of binding sites was low compared with the TSH receptor. At 37 degrees C, the binding of 125I-labelled EGF was maximal at 1 h and was saturable in the presence of unlabelled EGF; half-maximal inhibition was at 1 ng EGF/tube (0.5 nmol/l) using 0.5 mg membrane protein/tube. Unlabelled bTSH had no effect on the binding of labelled EGF. Similarly, unlabelled EGF did not affect the binding of labelled TSH; hence it was concluded that mEGF and bTSH bound to independent sites. Epidermal growth factor had no effect on adenylate cyclase activity in membranes prepared from human non-toxic goitre; increasing concentrations of EGF did not affect basal, TSH-stimulated or fluoride-stimulated enzyme activity.     

Growth hormone binding sites in tilapia (Oreochromis mossambicus) liver.

125I-labeled bovine and tilapia growth hormones were used to assess the presence of growth hormone receptors in membranes prepared from tissues of the tilapia Oreochromis mossambicus. The highest level of specific binding was detected in liver membranes from animals of both sexes and the binding was protein-dependent. Tilapia growth hormone, bovine growth hormone, and ovine prolactin, but not tilapia prolactin, potently inhibited the hepatic binding of 125I-labeled bovine growth hormone. Scatchard analysis of the 125I-labeled bovine growth hormone binding data revealed a Bmax (maximum binding) value of 180 fmol/mg protein and a Kd (dissociation constant) value of 13 nM. Tilapia growth hormone potently inhibited hepatic binding of 125I-labeled tilapia growth hormone. Scatchard analysis revealed a single class of binding sites with Bmax and Kd values of 390 fmol/mg protein and 2.5 nM, respectively. Bovine growth hormone and ovine prolactin were less potent while tilapia prolactin was inactive in inhibiting hepatic 125I-labeled tilapia growth hormone binding.     

Relative localization of the prolactin receptor binding sites for lactogenic hormones.

A monoclonal antibody termed MAb R7B4, directed to an epitope present in prolactin receptors (PRLRs), was used as a tool to map the receptor binding sites for human growth hormone (hGH), ovine prolactin (oPRL) and human placental lactogen (hPL). Although the three hormones completely inhibited the binding of each other to Nb2 cells or rat liver receptors, MAb R7B4 behaviour was different depending on the hormone tested and the receptor source. According to the MAb effects, PRLR from Nb2 cells would locate both hGH and oPRL close to R7B4 epitope, whereas hPL would bind far from the MAb binding site. On the other hand, PRLR from rat liver should bind hGH close to the R7B4 epitope but oPRL and hPL would be recognized by a separate region of the same receptor. Thus, results presented in this paper suggest that PRLR binding sites for hGH, oPRL and hPL do not exactly overlap in spite of full competition between ligands.     

Different binding of stimulatory-type and blocking-type TSH receptor antibody with guinea-pig testis membrane.

A receptor assay using [125I]bTSH-binding to guinea-pig testis membrane was developed. Unlabelled hCG and FSH inhibited [125I]bTSH binding. In patients with Graves' disease and in untreated hyperthyroid patients, almost all long-acting thyroid stimulators and thyroid-stimulating antibodies, respectively did not inhibit [125I]bTSH binding, which on the other hand was inhibited by thyroid stimulation blocking antibodies in patients with primary hypothyroidism. When the inhibitory effect on the binding of [125I]hCG and 125I-synthetic alpha-subunit peptide (alpha 26-46) of hCG to testis membrane was examined, bTSH resulted in a significant inhibition. However, all three kinds of TSH receptor antibodies had no inhibitory effect. This study demonstrated 1. interaction of alpha-subunit of TSH and hCG with the testicular receptor; 2. binding of thyroid stimulation-blocking antibody and lack of binding of thyroid-stimulating antibody to the testicular TSH receptor in spite of binding of these TSH receptor antibodies to the thyroidal TSH receptor, and 3. lack of binding of thyroid-stimulating antibody and thyroid stimulation-blocking antibody to the testicular gonadotropin receptor.     

Plasma immunoreactive TSH: spurious elevation due to antibodies to bovine TSH which cross-react with human TSH

A patient with thyroid carcinoma treated by thyroidectomy who received multiple injections of bovine (bTSH) exhibited elevated and nonsuppressible levels of plasma "immunoreactive TSH." Antibodies to TSH of the IgG class which bound bTSH and human TSH (hTSH) were demonstrated using specific radioimmunoassay and radioimmunoelectrophoretic techniques. Antibodies were present for more than one year and binding of bTSH was greater than that of hTSH throughout this period. Characterization of the antibodies with respect to the binding of human and bovine glycoprotein hormones and subunits revealed two populations of antibodies, one of which bound both bTSH and hTSH and the other which bound only bTSH. Both antibodies appeared to be directed toward antigenic sites on the beta subunit of TSH as both hTSH-beta and bTSH-beta displaced the binding of intact TSH from antibodies whereas the alpha subunits were virtually unreactive. The binding studies suggest that the cross-reactivity of the antibody to hTSH occurred on the basis of common antigenic determinants on the beta subunits of the two species. Documentation of a true elevation of plasma hTSH was achieved by gel filtration of plasma in which a peak of immunoreactivity co-eluting with [125I]-hTSH could be shown. Evidence for suppression of TSH secretion by thyroxine administration in the presence of interfering antibody was obtained by demonstrating that the level of plasma "immunoreactive TSH" did not change in response to TRH administration.     

Demonstration of anti-TSH antibody in TSH binding inhibitory immunoglobulin-positive sera of patients with Graves' disease

OBJECTIVES: The presence of anti-TSH antibodies in Graves' patients with unusually low TSH binding inhibitory immunoglobulin (TBII) has been reported. Recently, we found the first case of an anti-TSH antibody in TBII-positive sera of patients with Graves' disease. The prevalence and immunological specificity of this anti-TSH antibody were examined. DESIGN: The presence of 125I-bovine(b) TSH binding antibody in TBII positive serum was examined by prolonged incubation of more than 1 day because only weak binding occurred after 1 h incubation at 37 degrees C. The clinical course of these patients and binding characteristics of anti-bTSH antibody were examined. RESULTS: The corrected method-TBII activity (%)[1 - (a - b)/(c - d)] x 100 and the standard method-TBII activity (%) [1 - (a - d)/(c - d)] x 100 [a, 125I-bTSH binding with TSH receptor (R) in the presence of test serum; b, 125I-bTSH binding with test serum; c, 125I-bTSH binding with TSH R in the presence of normal serum; d, 125I-bTSH binding with normal serum] were calculated. The corrected method-TBII activity was always higher than the standard method-TBII activity in anti-bTSH antibody-positive serum. Anti-bTSH antibody-positive cases in TBII-positive Graves' disease were found in approximately 1% of Graves' patients. Anti-bTSH antibodies were confirmed as IgG from the increase of precipitated radioactivity by adding rabbit antihuman IgG antibody after the incubation of 125I-bTSH with test serum. These antibodies bind with not only bTSH, bTSH(alpha) and bLH, but also porcine (p)TSH, pTSH(alpha) and pFSH. However, these antibodies did not bind with human TSH. Binding of 125I-bTSH with patient's serum was neither inhibited by other Graves' thyroid stimulating antibody (TSAb), nor thyroid blocking antibody (TBAb) in primary hypothyroidism. CONCLUSIONS: The presence of anti-bTSH antibody in TBII-positive serum of high titre means that TBII-positive sera cannot rule out the absence of anti-bTSH. Thus, determination of 125I-bTSH binding with test serum in TSH receptor assays is necessary to determine the precise TBII activity and to detect anti-bTSH antibody.     

Identification of key amino acid residues in a thyrotropin receptor monoclonal antibody epitope provides insight into its inverse agonist and antagonist properties

CS-17 is a murine monoclonal antibody to the human TSH receptor (TSHR) with both inverse agonist and antagonist properties. Thus, in the absence of ligand, CS-17 reduces constitutive TSHR cAMP generation and also competes for TSH binding to the receptor. The present data indicate that for both of these functions, the monovalent CS-17 Fab (50 kDa) behaves identically to the intact, divalent IgG molecule (150 kDa). The surprising observation that CS-17 competes for TSH binding to the human but not porcine TSHR enabled identification of a number of amino acids in its epitope. Replacement of only three human TSHR residues (Y195, Q235, and S243) with the homologous porcine TSHR residues totally abolishes CS-17 binding as detected by flow cytometry. TSH binding is unaffected. Of these residues, Y195 is most important, with Q235 and S243 contributing to CS-17 binding to a much lesser degree. The functional effects of CS-17 IgG and Fab on constitutive cAMP generation by porcinized human TSHR confirm the CS-17 binding data. The location of TSHR amino acid residues Y195, Q235, and S243 deduced from the crystal structure of the FSH receptor leucine-rich domain provides valuable insight into the CS-17 and TSH binding sites. Whereas hormone ligands bind primarily to the concave surface of the leucine-rich domains, a major portion of the CS-17 epitope lies on the opposite convex surface with a minor component in close proximity to known TSH binding residues.     

The hinge region of the TSH receptor stabilizes ligand binding and determines different signaling profiles of human and bovine TSH

The hinge region (HinR) is a variable structure of glycoprotein hormone receptors. Its amino acid composition and length is different for glycoprotein hormone receptors and connects the ligand binding domain with the serpentine domain. The role of the HinR of the receptors for TSH, follitropin (FSH), and LH/choriogonadotropin (LHCG) in receptor and signaling specificity is unknown. To investigate the role of the HinR for ligand binding, signal generation, and for the transmission of the signal towards the serpentine domain, we replaced the HinR of the TSH receptor (TSHR) by those of LHCG receptor and FSH receptor and introduced constitutively activating mutations and one mutation deficient for bovine (b)TSH binding in these chimeras. Functional characterization of the TSHR variants was carried out after transient transfection of COS-7 cells by determination of the cell surface expression, ligand binding, and recombinant human (rh)TSH or bTSH activation of second messengers. We show that the HinR of the TSHR stabilizes hormone binding regarding ligand affinity and retention time of the bound ligand as determined by dissociation experiments. Introduction of a constitutively activating extracellular loop mutation in these constructs lead to partially restored binding patterns. These findings indicate that the HinR-extracellular loop interface is besides signaling also important for bTSH binding. Furthermore, data for G protein signaling reveal that the activity of bTSH, but not of rhTSH, depends on the TSHR HinR, which was indicated by a significant right shift in the dose-response curves for G(s) and G(q) activation for TSHR chimeras harboring the LHCG receptor and FSH receptor HinR, respectively. Moreover, we identified different G protein signaling profiles for bTSH and rhTSH, which cannot be explained by the characterized HinR. For future studies regarding structure and function of the TSHR, it will be necessary to characterize TSHR variants with both or more ligands.     

Identification of novel TSH interaction sites by systematic binding analysis of the TSHR hinge region

In which ways the binding of the thyroid stimulating hormone to the extracellular domain of its receptor leads to activation of the thyroid-stimulating hormone receptor (TSHR) is currently only incompletely understood. It is known that TSH binding to the TSHR depends on the interaction with the leucine-rich repeat and sulfation at Y385 of the hinge region. Recently it was also shown that electrostatic interactions between positive charges of bovine (b) TSH and the residues E297, E303, and D382 of the hinge region contribute to hormone-TSHR binding. After the identification of these first TSH binding sites in the hinge region, it was apparent that multiple positions in this region remained to be characterized for their roles in hormone binding. The goal of this study was therefore to clarify whether further contact points of TSH exist in the structurally undefined hinge region. Therefore, we systematically analyzed 41 uncharacterized residues of the TSHR hinge region as single mutants regarding differences between cell surface expression and bTSH binding. Indeed, we identified further amino acids of the hinge region with influence on bTSH binding. Some of these contribute to a new binding domain from human TSHR position F381 to D386. These hinge mutants with influence on bTSH binding were also analyzed for binding of the superagonistic human TSH analog TR1401 demonstrating that these positions also have an impact on TR1401 binding. Moreover, side chain variations revealed that different amino acid properties like the negative charge, aromatic as well as hydrophilic characteristics, contribute to maintain the hormone-TSHR hinge interaction.     

Monoclonal antibody mapping of the antigenic surface of human thyrotropin and its subunits

Although the amino acid sequence of the alpha- and beta-subunits of glycoprotein hormones in various species has been deciphered, data on their tertiary structure are not abundant. This impedes correlation between structure and function. The availability of monoclonal antibodies to human TSH (hTSH) offers the opportunity to enumerate the antigenic determinants present on the surface of hTSH and its subunits and to examine their spatial relationships. Twenty-eight monoclonal antibodies to hTSH were obtained from several fusions, and screens carried out separately in the laboratories involved in this study. Affinities for hTSH ranged from 10(8)-10(11) M-1. Cross-reactivity with bovine TSH (bTSH), human gonadotropins (hLH, hFSH, and hCG), and the alpha- and beta-subunits of hTSH distinguished 10 groups of monoclonal antibodies (mAb) according to their main cross-reactions: 1) hTSH alpha, hLH, hFSH, and hCG; 2) hTSH alpha, bTSH, hLH, hFSH, and hCG; 3) hFSH; 4) bTSH and hFSH; 5) bTSH, hLH, and hFSH; 6) bTSH, hLH, hFSH, and hCG; 7) hTSH beta; 8) hTSH beta and bTSH; 9) hTSH beta and hFSH; and 10) hTSH beta, hLH, hFSH, and hCG. mAb were incorporated into 2-site binding assays to probe hTSH by a 28 X 28 matrix, the free alpha-subunit by a 4 X 4 matrix, and the free beta-subunit by a 18 X 18 matrix. Regarding intact hTSH, 12 different clusters of mAb were distinguished and interpreted as reflecting 12 distinct antigenic regions on the surface of the hTSH molecule. Two of them were localized on the alpha-subunit, and 6 on the beta-subunit; 4 were only expressed by the holo-hormone and, thus were designated conformational antigenic regions (alpha beta). Surface mapping of the free alpha- and beta-subunits was virtually identical to that observed with the holo-hormone. Modification of the operative conditions of mAb reacting only with holo-hTSH shows that they recognize the alpha-subunit, but not the beta-subunit of hTSH. These results indicate that 1) hTSH beta presents epitopes that are evolutionary conserved; 2) hTSH alpha presents several epitopes that are species specific and 2 that are not hormone specific; 3) dissociation of hTSH does not modify the antigenic surface expressed by both subunits when they are associated; and 4) some of the conformational determinants expressed only by holo-hTSH are more likely derived from the alpha-subunit than from the beta-subunit.     

Localization and synthesis of the hormone-binding regions of the human thyrotropin receptor.

Two regions of human thyrotropin (thyroid-stimulating hormone, TSH) receptor (TSHR) (residues 12-44 and 308-364) were selected on the basis that they exhibit no sequence resemblance to luteinizing hormone/chorionic gonadotropin receptor. Five synthetic overlapping peptides (12-30, 24-44, 308-328, 324-344, and 339-364) were studied for their ability to bind 125I-labeled human TSH (hTSH), its isolated alpha and beta subunits, bovine TSH, ovine TSH, human luteinizing hormone, and human follicle-stimulating hormone. The human TSHR peptides 12-30 and 324-344 exhibited remarkable binding activity to human, bovine, and ovine TSH and to the beta chain of hTSH. Lower binding activity resided in the adjacent overlapping peptides, probably due to the contribution of the shared overlap to the binding. The specificity of TSH binding to these peptides was confirmed by their inability to bind human luteinizing hormone, human follicle-stimulating hormone, and the alpha chain of hTSH. Thyrotropins did not bind to bovine serum albumin or to peptide controls unrelated to the TSHR system. Furthermore, the binding of hTSH to TSHR peptides 12-30 and 324-344 was almost completely (approximately 90%) inhibited by rabbit antibodies against hTSH but not by antisera against unrelated proteins. It is concluded that the binding of TSH to its receptor involves extensive contacts and that the TSHR peptides 12-30 and 324-344 contain specific binding regions for TSH that might be either independent sites or two faces (subsites) within a large binding site.