Thyroid Membrane Preparations Research Articles

Autoantibody stimulation of the human thyrotropin receptor: regulation of adenylate cyclase activity, thyroglobulin and thyroid peroxidase mRNA levels in primary cultures of Graves’ thyroid tissue

The effects of immunoglobulin preparations from hyperthyroid Graves' disease patients on primary cultures of thyroid cells have been studied at the mRNA level. Autoantibodies to the thyrotropin (TSH) receptor from these patients, which had been initially characterized by their ability to stimulate adenylate cyclase and inhibit the binding of radiolabelled TSH to thyroid membrane preparations, were studied for their effects on thyroglobulin and thyroid peroxidase mRNA levels. Incubation of thyroid cells with TSH receptor autoantibodies from different Graves' disease patients for 48 h led to time- and dose-dependent increases in the levels of thyroid peroxidase and thyroglobulin mRNA in primary cultures of thyrocytes. The incomplete correlation between G protein-linked adenylate cyclase activation and thyroid mRNA elevation indicates the possibility of the involvement of alternative second messenger pathways in the regulation of thyroid cell function and differentiation.

Severe but not mild alterations of thyroid function modulate the density of thyroid-stimulating hormone receptors in the rat thyroid gland.

TSH initiates its action by binding to specific membrane receptors' thyroid cells and induces activation of the adenylate cyclase-cAMP cascade. The factors involved in the regulation of TSH receptors are poorly known, except for the TSH dose-dependent regulatory effect. The fact that the thyroid gland of Graves' patients has a normal density of TSH receptors with suppressed TSH and high T4 and T3 levels suggests a modulatory role of thyroid hormones on TSH receptors. To evaluate this hypothesis, the density of TSH receptors and the activity of adenylate cyclase were determined in the thyroid membranes from hyperthyroid and hypothyroid adult male rats; they were rendered hyperthyroid either with bovine TSH, TRH, or T3 for 7 days and hypothyroid by propylthiouracil treatment or by hypophysectomy. NaCl was given to the control group. Plasma T4, T3, and TSH were also quantified. Bovine TSH and TRH treatments induced mild hyperthyroidism with a small goiter and a 50% reduction in the density of TSH receptors due to hyperstimulation of the gland by either exogenous or endogenous high TSH levels. Severe hyperthyroidism caused by T3 treatment resulted in low T4, high T3, and suppressed TSH thyrocyte stimulation; it was associated with a significant increase in the number of TSH receptors (29.6 +/- 2.3 vs. control 17.9 +/- 1.7 mU TSH/mg protein). These last results suggest a putative positive effect of T3 on TSH receptors. To confirm this effect, hypothyroid rats were investigated. Severe primary hypothyroidism due to propylthiouracil treatment was associated with a large goiter, high plasma TSH levels (11.8 +/- 1.2 vs. control 1.5 +/- 0.1 mU TSH/ml), low plasma T4 and T3, and a 70% reduction in TSH receptors, confirming the down-regulatory effect of high TSH on the thyroid cell. However, in hypophysectomized rats, a 45% reduction in the density of TSH receptors was also observed in the absence of TSH. Injections of either TSH or T3 to these hypophysectomized rats restored a normal number of TSH-binding sites, and simultaneous TSH and T3 treatments resulted in a mildly additive effect in the number of TSH receptors, which was slightly greater than that of the controls. No important changes were found in the adenylate cyclase activity in the thyroid membrane preparations from hyperthyroid and hypothyroid rats despite variations in the density of TSH receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Delineation of amino acid residues within hTSHr 256-275 that participate in hormone binding.

The amino acid sequence 256-275 of the human thyrotropin (TSH) receptor extracellular domain has previously been shown to participate in a high affinity TSH binding site by a synthetic peptide approach as well as by site-directed mutagenesis. To further investigate this binding site, we synthesized a series of peptides with alanine substitutions for each residue in the native sequence. Peptides were also synthesized containing truncations or deletions of the native sequence. Each peptide was tested for its ability to inhibit 125I-bTSH binding to porcine thyroid membrane preparations, and the concentration at which 50% inhibition of binding occurred was determined (EC50). Alanine substitution at residues Tyr258, Cys262, Cys263, Phe265, Lys266, Asn267, Lys269, Lys270, and Arg272 all resulted in statistically significant decreases in activity when compared to the native sequence (p < 0.05). Alanine substitution of the remaining residues did not alter their activity. Comparison of this sequence with the corresponding sequences of the remaining glycoprotein hormone receptors (human lutropin and human follitropin receptors) reveals that these residues lie within one of the most highly conserved regions of the extracellular domain. We conclude that 9 specific amino acids within the sequence 256-275 of hTSHr (-Y--CC-FKN-KK-R--) participate in the interaction of the hTSHr-extracellular domain with TSH. This may represent a site in which the nonconserved residues are involved in the binding of the beta-subunit and the conserved residues are involved in the binding of the common alpha-subunit or a region of the beta-subunit that is common to all glycoprotein hormones.

Production of Ab2β, Anti-idiotypic Monoclonal Antibodies, Specific for Human Thyrotropin Receptor

We had previously selected five monoclonal antibodies (mAb1) upon their specific binding to human thyrotropin (hTSH). These mAbs1, which are specific for two distinct epitopes of the hTSH β-chain, also inhibit the hTSH binding to human thyroid membrane preparations. We then immunized BALB/c mice with the various mAbs1 in attempt to generate anti-idiotypic Abs (Ab2) directed to the hTSH receptor (hTSHR). Five hybridomas secreting monoclonal antibodies (mAb2) to the hTSHR were selected from one mouse immunized with the mixture of the five mAbs1. These monoclonal antibodies to the hTSHR were selected upon their ability to specifically bind to the hTSHR. Moreover, we showed that binding of mAbs2 to the individual mAbs1 used for immunization was specifically inhibited by hTSH and not by insulin or by human chorionic gonadotrophin, a highly related glycoprotein hormone. The five mAbs2 inhibited the binding of iodine-labeled hTSH to its receptor by 8 to 41%: moreover, two of them stimulated the adenylatecyclase system of the thyroid cells. With respect to their properties, mAbs2 were classified as mAb2-β.

Significance of anti-eye muscle antibody in patients with thyroid-associated ophthalmopathy by quantitative western blot.

To investigate the prevalence of antibody against rat eye muscle membrane antigen, as determined from SDS-polyacrylamide gel electrophoresis and western blotting, in sera from patients with thyroid-associated ophthalmopathy (TAO), we quantitatively analyzed the binding activity with a rat eye muscle membrane 64 kDa protein using chromato-scanner. Eye muscle antibody activity was expressed as ratio of density of the 64 kDa band to that at 66 kDa found with all normal sera and phosphate buffered saline. The mean (+/- SD) eye muscle antibody activity was 2.7 +/- 2.7 in TAO (P < 0.01 v.s. normal), 1.5 +/- 1.7 in Graves' disease without evident eye disease, 1.6 +/- 2.5 in Hashimoto's thyroiditis and 0.45 +/- 0.26 in normal subjects. A positive band at 64 kDa was found in 71% of patients with TAO, 36% of those of Graves' disease without evident eye disease and in 35% of patients with Hashimoto's thyroiditis without eye disease. The prevalence of this antibody activity tended to correlate to the severity of ophthalmopathy. Furthermore, the level of eye muscle antibody activity decreased in parallel with the improvement of eye signs in two patients. Sera reactive with rat eye muscle membrane 64 kDa protein reacted also with a human eye muscle membrane 64 kDa protein but not with human thyroid, liver, spleen or pancreas membrane preparations. In conclusion, antibody to rat eye muscle membrane 64 kDa protein is present in TAO and may be a useful clinical marker of ophthalmopathy.

Thyrotrophin increases the α1b-adrenergic receptors in rat thyroid gland in vivo

Using chlorethylclonidine (CEC), an alpha 1b-adrenergic receptor-selective antagonist, we characterized alpha 1-adrenoceptor subtypes in rat thyroid gland, and investigated the effect of methimazole (MMI)-induced high TSH levels on alpha 1 receptor subtypes and noradrenaline-induced iodide organification. The density of thyroid alpha 1-adrenergic receptors was increased about sixfold in rats treated with MMI for 3 weeks compared with controls. Pretreatment of thyroid membrane preparations with CEC (10 mumol/l) caused an 83% decrease in specific 2-[beta-(hydroxy-3-[125I]iodophenyl) ethylaminomethyl]tetralone binding sites in MMI-treated rats, but only a 43% decrease in control rats. The density of CEC-insensitive alpha 1 receptors (alpha 1a) was similar in MMI-treated and control rats, so MMI was shown to increase CEC-sensitive alpha 1 receptors (alpha 1b). Noradrenaline-stimulated iodide organification was threefold greater in MMI-treated rats than in control rats when values were expressed as a per cent increase over basal levels. Pretreatment of thyroid lobes with 10 mumol CEC/l for 30 min caused a 66% decrease in maximal noradrenaline-induced iodide organification in MMI-treated rats, but a significantly lower decrease (49%) in control rats. These results suggest that the rat thyroid gland contains both alpha 1a and alpha 1b receptors, both of which mediate noradrenaline-induced iodide organification, and also that TSH enhances noradrenaline-induced iodide organification by increasing alpha 1b receptor density.

Synthetic α-Subunit Peptides Stimulate Testosterone Productionin Vitroby Rat Leydig Cells*

The glycoprotein hormones (LH, hCG, FSH, and TSH) have a common 92-amino acid alpha-subunit which is noncovalently linked to a hormone-specific beta-subunit. Synthetic peptides of the alpha-subunit have been shown to inhibit binding of [125I]iodo-hCG to rat ovarian membrane and [125I]iodo-TSH to human thyroid membrane preparations. Synthetic overlapping peptides of the alpha-subunit of hCG were prepared by solid phase techniques and tested in a standard in vitro rat Leydig cell bioassay. Three regions in the alpha-subunit (alpha 1-15, alpha 30-45, and alpha 71-85) were found to stimulate testosterone production. All three regions correlate with inhibition of hCG binding to ovarian receptors, but subtle differences exist between the binding sites and effector sites. These data indicate that the glycoprotein alpha-subunit has intrinsic bioactivity.

Epidermal growth factor receptors and adenylate cyclase activity in human thyroid tissues

Thyroid stimulating hormone (TSH) and epidermal growth factor (EGF) are growth factors for some thyroid cells in cultures. We have previously found more EGF receptors in neoplastic human thyroid tissues than in normal thyroid tissues. We have also found a higher TSH-stimulated adenylate cyclase (AC) activity in neoplastic human thyroid tissues than in normal thyroid tissues. To clarify the relationship between the effect of EGF and TSH on thyroid tissue, we measured the binding of EGF and TSH and the basal, TSH-stimulated and forskolin-stimulated adenylate cyclase activity in 49 normal, hyperplastic and neoplastic human thyroid tissues (5 normal, 2 Hashimoto thyroiditis, 5 Graves' disease, 14 multinodular goiters, 9 follicular adenomas, 5 follicular carcinomas, 8 papillary carcinomas, and 1 undifferentiated carcinoma). Specific binding of EGF and TSH were measured by radioreceptor assays using competitive inhibition of radio-labeled ligand by unlabeled ligand. Basal, maximally (300 mU/ml) TSH-stimulated, and maximally (100 mM) forskolin-stimulated adenylate cyclase activities were also measured in the same membrane particulate fractions from the thyroid tissues. We found: neoplastic thyroid tissues bind more labeled EGF than nonneoplastic thyroid tissues; follicular adenomas and carcinomas have higher EGF binding than other thyroid tissues; a weak but significant correlation between specific EGF binding and specific TSH binding, and between specific EGF binding and TSH-stimulated adenylate cyclase activity of the thyroid membrane preparations. These findings are consistent with the hypothesis that TSH stimulates an increase in thyroid EGF receptors by increasing intracellular cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of cytosolic tyrosine kinase activity by propylthiouracil-induced hyperplasia in the rat thyroid.

Hyperplasia of the thyroid gland induced by propylthiouracil (PTU) is a well established model of rapid cell proliferation in vivo. Recent evidence indicates that tyrosine kinase activity is associated with growth factor receptors and oncogene protein products and may have an important regulatory action in the control of cell growth. Thus, we examined tyrosine kinase activity in rat thyroid membrane and cytosol preparations at rest and during PTU-induced hyperplasia. Although kinase activity was present in a crude microsomal membrane preparation, no change was observed during thyroid growth. In contrast, tyrosine kinase activity assayed with the artificial substrate poly(Glu,Na:Tyr) 4:1 was present in normal rat thyroid cytosol and increased 2- to 6-fold during the rapid phase of hyperplasia in the first 5-10 days of PTU treatment. It declined to control values by day 15, when the size and DNA content of the thyroid reached a plateau. Preincubation of the cytosolic preparations with several peptides known to bind to and activate growth factor receptor tyrosine kinases failed to enhance the activity, suggesting, along with the cytosolic localization, that the activity was distinct from these receptors. By gel filtration chromatography and polyacrylamide gel electrophoresis, tyrosine kinase activity was associated with a 55 kDa protein. Partial purification over a poly(Glu,Na:Tyr)4:1-Sepharose column, yielded a protein that appeared capable of autophosphorylation. It is suggested that this tyrosine kinase plays a role in mediating the growth-promoting effects of this model of thyroid cell hyperplasia.

Evidence that partially desialylated variants of human chorionic gonadotropin (hCG) are the factors in crude hCG that inhibit the response to thyrotropin in human thyroid membranes.

Previous studies have revealed that pure unmodified hCG (hCGp) has little potency to inhibit the binding of bovine TSH (bTSH) to human thyroid membranes and to either stimulate adenylate cyclase or inhibit TSH-stimulated adenylate cyclase therein. On the other hand, preparations of crude hCG (hCGc) as well as enzymatically desialylated hCGp (asialo-hCGp) are relatively potent inhibitors of bTSH binding (TBI activity) and bTSH-stimulated adenylate cyclase activity in human thyroid membrane preparations. In the present studies we have sought to identify and characterize the inhibitory moieties in crude hCG that are responsible for these inhibitory activities and to elucidate the properties of their interaction with the TSH receptor in human thyroid membranes. Preparations of hCGc were processed by DEAE-52 chromatography; this separated components of interest, which were not adsorbed, from intact hCGp, which was adsorbed to the column. The former were then subjected to gel filtration on Sephadex G-100, and the earliest eluting fractions, which proved to have the greatest TBI activity, were pooled, rechromatographed, and designated as variant hCG (hCGv). Three different preparations of hCGv were studied. All displayed a lower sialic acid content, by about half, than that in hCGp. Though their potencies varied somewhat, all had significant TBI activity, which was less than that of asialo-hCGp, but more than that of hCGc. Saturation studies revealed that the TBI activities of hCGv and asialo-hCGp were due to a competitive inhibition of bTSH binding at both the high and low affinity bTSH-binding sites, whereas the inhibitory activity of hCGc was exerted primarily at the low affinity binding site. Preparations of hCGv were also capable of inhibiting the adenylate cyclase response to TSH in human thyroid membranes, and Lineweaver-Burk analysis revealed this inhibition to be competitive in nature. As with its TBI activity, the potency of hCGv to inhibit the adenylate cyclase response was intermediate between that of asialo-hCGp and hCGc. Among the three batches of hCGv, their inhibitory effects on bTSH binding and adenylate cyclase activation appeared to vary inversely with with their sialic acid content. Enzymatic desialylation of hCGv increased its potency to that of asialo-hCGp. Several lines of evidence, as follows, indicate that the moieties that comprise hCGv are modified forms of hCGp itself. 1) On a unit weight basis, hCGv was at least as potent as hCGp in its ability to inhibit the binding of [125I]hCG to rat testicular membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of synthetic alpha-subunit peptides on thyrotropin interaction with its receptor.

Synthetic peptides of the alpha-subunit of human glycoprotein hormones have been shown previously to inhibit binding of [125I]iodo-hCG to ovarian membranes, thus indicating the importance of the alpha-subunit in the structure-function relationships of the gonadotropic hormone. These same synthetic alpha-subunit peptides, the sequences of which are common to all human glycoprotein hormones, were found to inhibit the binding of [125I]iodo-TSH to human thyroid membrane preparations and FRTL-5 rat thyroid cells. The active portions of the subunit were represented in synthetic peptides alpha 21-35, alpha 31-45, alpha 26-46, and alpha 81-92, indicating that 2 separate sites within the alpha-subunit have binding activity for TSH. Peptides alpha 26-46 and alpha 31-45 were also found to potently inhibit the stimulation of adenylate cyclase activity by bovine TSH in TSH bioassay using FRTL-5 cells. Seven other synthetic peptides, including the remainder of the 92-amino acid sequence of the alpha-subunit, demonstrated little or no ability to inhibit binding of the tracer or inhibit the bioactivity of intact TSH. The findings were very similar to those of previous studies involving hCG binding, except that the two active sites appeared to be somewhat shifted towards the COOH-terminal end of the subunit. These studies support the concept of the importance of the alpha-subunit in receptor binding of all glycoprotein hormones and demonstrate the utility of the overlapping synthetic peptide strategy in investigations of protein structure-function relationships.

Characterization of a Bioeffective Monoclonal Antibody against Thyrotropin β-Subunit*

To describe a region of the TSH molecule participating in binding to receptor, a monoclonal antibody specific for a TSH epitope shared by beta-subunits of bovine (b), ovine (o), and human (h) TSH was obtained by immunization with mixtures of purified bTSH and hTSH. RIAs showed that the antibody also bound the beta-subunits of bLH, oLH, hLH, and hCG, but not the beta-subunits of porcine LH and TSH. Preincubation of [125I]iodo-bTSH with the antibody completely inhibited binding of the hormone to the TSH receptor of bovine thyroid membrane preparations at pH 7.4 in 50 mM NaCl (ED50 = 10 nM). The antibody also inhibited TSH-induced mitogenesis of FRTL-5 cells (ED50 = 50 nM). We conclude that the antibody binds to a site on the bTSH molecule that participates in high affinity binding of hormone to physiological TSH receptor. The target epitope includes a conserved structural determinant in beta-subunits of the glycoprotein hormones as well as a feature that allows discrimination of porcine hormones from those of bovine, ovine, and human origin.

The active principles of plant extracts with antithyrotropic activity: oxidation products of derivatives of 3,4-dihydroxycinnamic acid.

We have recently reported that freeze-dried extracts (FDE) of certain plants form high molecular weight adducts with bovine TSH (bTSH), preventing it from binding to and stimulating adenylate cyclase in human thyroid membranes. We have now studied 34 pure compounds identical or structurally related to compounds present in FDE from Lycopus or Lithospermum, 2 of the 3 species of active plants studied previously. In studies conducted at 4 C in 20 mM Tris-HCl-0.5% BSA buffer, pH 7.45, eight 3,4-dihydroxylated compounds, all structurally related to cinnamic acid, inhibited the binding of [125I] bTSH to human thyroid membranes. Of these, 4 (caffeic, rosmarinic, chlorogenic, and ellagic acids) are present in the plants, and 4 (3,4-dihydroxyphenylacetic acid, deoxyepinephrine, adenochrome, and nordihydroguaretic acid) are structurally related thereto. These compounds were inactive when tested directly but became active when allowed to undergo auto-oxidation. With all 8 compounds, half-maximum inhibition of [125I]bTSH binding required quantities of oxidized product equivalent to 20-80 micrograms/ml (60-195 microM) of the original compound. Half-maximum inhibitory concentrations of oxidized caffeic and ellagic acids were increased 2- to 4-fold when experiments were performed at 37 C in medium containing 50 mM NaCl. Preincubation of membranes with active oxidation products in concentrations up to 100 micrograms/ml, followed by washing, had no effect on the subsequent binding of [125I]bTSH. As has been shown in the case of FDE, when [125I]bTSH was preincubated with oxidation products of caffeic and ellagic acids and was then chromatographed on Sephadex G-100, its elution pattern was advanced from an apparent mol wt of 30,000 to the void volume, and [125I]bTSH in the early eluting fractions displayed greatly reduced binding to thyroid membrane preparations. Addition of a large excess of unlabeled bTSH during preincubation prevented the shift in the elution pattern of [125I]bTSH produced by these oxidation products. To ascertain whether FDE and active compounds interact with the protein or carbohydrate moieties of bTSH, studies of their effects on the binding and chromatographic behavior of 125I-deglycosylated-bTSH (dg-bTSH) were also performed. Effects were similar to those observed for intact bTSH, suggesting that they do not interact with the carbohydrate moiety of TSH. Preincubation of both bTSH and dg-bTSH with either active FDE or oxidation products of caffeic or rosmarinic acid also greatly decreased their activity in the McKenzie mouse assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Methodological aspects and clinical results of an assay for thyroid-stimulating antibodies: correlation with thyrotropin binding-inhibiting antibodies.

Several methodological aspects concerning the measurement of thyroid-stimulating antibodies (TSAb) were examined using a 5000-g particulate fraction from human thyroid tissue. TSH displayed maximal stimulation at 32 C. An ATP-regenerating system had no influence at ATP concentrations greater than 0.67 mM. Theophylline did not influence cAMP concentrations in the incubation medium after stimulation with increasing dosages of TSH, suggesting the absence of active phosphodiesterases in the membrane preparations. Sera dialyzed against Tris-HCl were as suitable as immunoglobulin G preparations for determination of TSAb. One hundred and nineteen patients with ophthalmic Graves' disease, 42 of whom were hyperthyroid at the time of study, were investigated for TSH binding-inhibiting antibodies ( TBIAb ) and TSAb. In 76% of these patients, concordance between the presence and absence of both activities was found independent of whether they were untreated, treated, or in remission. When gradations of potency in the two systems were compared, linear regression revealed a correlation coefficient of 0.59. The coefficient correlation increased when TBIAb and TSAb were each determined in a single assay using identical thyroid membrane preparations. In four other patients, both TSAb and TBIAb were measured repeatedly during the course of the disease. In three patients, both activities were parallel, whereas in one patient, dissociation of the two activities was found. The data suggest the presence of antibodies with varying stimulating and binding-inhibiting properties. However, in hyperthyroidism of Graves' disease, both activities usually were present in similar quantities.

Monoclonal antibodies to the thyrotropin receptor: the identification of blocking and stimulating antibodies

Monoclonal antibodies to the thyrotropin (TSH) receptor have been obtained from fusions of mouse myeloma cells with spleen cells immunized with solubilized thyroid membrane preparations. Two monoclonal antibodies which inhibit 125I-TSH binding and are reactive with the glycoprotein component of the bovine TSH receptor (11E8 and 13D11), are shown to inhibit basal and TSH stimulated adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. Both antibodies also inhibit 125I-TSH binding in vitro, whether binding is measured at pH 6.0 in low salts and at 0-4 C or at pH 7.4 in 50 mM NaCl and at 37 C. The glycoprotein component is thus a portion of the physiologic TSH receptor in vivo and 125I-TSH binding studies apparently measure the high affinity glycoprotein component under nonphysiologic conditions and conditions more representative of the physiologic milieu. A third monoclonal antibody whose interaction with thyroid membranes is prevented by TSH is shown to stimulate adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. This stimulating antibody only weakly inhibits 125I-TSH binding to thyroid membranes or to the glycoprotein component of the TSH receptor. The 22A6 antibody does, however, immunoprecipitate mixed brain gangliosides, in distinct contrast to the monoclonal antibodies to the glycoprotein receptor component, i.e., 11E8 and 13D11. The results support the speculation that autoimmune antibodies which inhibit TSH binding to thyroid membranes are not necessarily identical to antibodies which stimulate function; that antibodies directed at the high affinity initial site of TSH interaction with a cell can behave as blocking rather than stimulating antibodies and that a possible relationship exists between stimulating antibodies and the low affinity TSH binding sites (gangliosides) on thyroid membranes.

Graves' IgG stimulation of continuously cultured rat thyroid cells: a sensitive and potentially useful clinical assay.

A continuously cultured line of normal rat thyroid (FRTL) cells can be stimulated by immunoglobulin preparations from patients with Graves' disease as measured by increases in intracellular cAMP levels. Responsiveness is concentration-dependent but is delayed in time relative to thyrotropin. Additionally, the cells respond to Graves' immunoglobulins which have no long-acting thyroid stimulator (LATS) activity and are negative when adenylate cyclase stimulation in human thyroid membrane preparations is assayed. No correlation exists between the stimulation activity and the ability of a Graves' immunoglobulin preparation to inhibit thyrotropin binding; cells are responsive even in the presence of such inhibitor activity.

Thyrotropin stimulation of the ADP-ribosyltransferase activity of bovine thyroid membranes.

Thyrotropin increases the ADP-ribosylation activity of bovine thyroid membranes. Rapid ADP-ribosylation of membrane components is followed by increasing ADP-ribosylation of components in the supernatant of the reaction mixture. One of the major membrane proteins ADP-ribosylated in the thyrotropin-stimulated reaction has an approximate molecular weight of 40,000; this same protein is also a major ADP-ribosylated product of the A promoter of cholera toxin and appears to be related to the G regulatory subunit of the adenylate cyclase complex. The ADP-ribosylated products appearing in the supernatant solution comigrate with thyrotropin and preparations of 125I-labeled alpha subunit of thyrotropin; the alpha subunit, but not the beta subunit, of thyrotropin can be ADP-ribosylated by the membrane ADP-ribosyltransferase activity. NAD can be shown to enhance the ability of thyrotropin to stimulate the adenylate cyclase activity of bovine thyroid membrane preparations and of membrane preparations of a rat thyroid tumor whose adenylate cyclase activity is otherwise unresponsive to thyrotropin. The beta subunit of thyrotropin inhibits thyrotropin stimulation of both the ADP-ribosylation and adenylate cyclase activities of the thyroid membrane.

Monoclonal antibodies to the thyrotropin receptor: implications for receptor structure and the action of autoantibodies in Graves disease.

Hybridoma cells have been obtained by fusing P3-NS1/1-Ag4-1 mouse myeloma cells with spleen cells from mice immunized with solubilized preparations of the thyrotropin receptor. Five clones were produced that secrete a monoclonal antibody whose binding to thyroid membranes is specifically inhibited by unlabeled thyrotropin. The antibody interacts with functioning thyroid cells in culture but not with nonfunctioning cells; this interaction is prevented by thyrotropin. The antibodies are capable of competitively blocking thyrotropin binding to bovine thyroid membrane preparations; they prevent 125I-labeled thyrotropin binding to a solubilized preparation of the glycoprotein component of the bovine thyrotropin receptor but are unable to inhibit 125I-labeled thyrotropin binding to liposomes containing gangliosides at comparable concentrations. They prevent 125I-labeled thyrotropin binding to rat, bovine, or human (Graves disease) thyroid membrane preparations. They do not stimulate adenylate cyclase activity in thyroid membrane preparations but can inhibit thyrotropin-stimulated iodide uptake by functioning thyroid cells in culture.

Cyclic AMP release from normal human thyroid slices in response to thyrotrophin.

Slice preparations of normal human thyroid tissue were incubated in vitro with TSH. The cyclic AMP contents of slices were determined at intervals up to 120 min, and cyclic AMP in the incubation medium was also estimated for each incubation period. Slice cyclic AMP levels were related both to incubation time and TSH dose. In response to 10 mU TSH/ml, slice cyclic AMP levels were maximal within 60 min, and were not significantly changed at 120 min. Cyclic AMP was detectable in the medium within 10 min of slice exposure to TSH, and increased throughout the initial 60 min of incubation. Cyclic AMP release during this period was dependent on both TSH dose and incubation time. Between 60-120 min, however, cyclic AMP release partially lost its TSH dose-dependency, and 0.5-5.0 mU TSH/ml were equipotent with respect to the final medium cyclic AMP level attained. Slices incubated without TSH released only small amounts of cyclic AMP, and maximal levels were attained within 20 min. In contrast to the adenylate cyclase response of thyroid membrane preparations, which was stimulated by NaF, suggesting that cyclic AMP release was not a result of the stimulation of damaged cells. These findings demonstrate the importance of cyclic AMP release from human thyroid slices, following in vitro exposure to TSH, and suggest that, after incubation periods such as are used for the functional biodetection of thyroid stimulators, the magnitude of cyclic AMP release may be of quantitative significance.

Membrane-bound tubulin in brain and thyroid tissue.

Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein.