Characteristics of a solubilized thyrotropin receptor from bovine thyroid plasma membranes.

The thyrotropin receptor from bovine thyroid plasma membranes has been solubilized using lithium diiodosalicylate, and an assay to measure thyrotropin binding to the solubilized receptor has been developed. Both the solubilized thyrotropin receptor and the thyrotropin receptor on thyroid plasma membranes have effectively identical nonlinear Scatchard plots and negatively sloped Hill plots, i.e. both preparations have receptors which appear to exhibit a similar negatively cooperative relationship. Although the pH optimum of thyrotropin binding to the solubilized receptor is the same as that of the thyroid plasma membrane receptor, pH 6.0, the pH dependency curve of the solubilized receptor is slightly different in its outline. Thyrotropin binding to the solubilized receptor is less sensitive to salt inhibition than is binding to the thyroid plasma membrane receptor; however, optimal binding remains at 0 degrees. The relative affinities of thyrotropin and two glycoprotein hormones which can be considered structural analogs, luteinizing hormone and human chorionic gonadotropin, are 100:10:5, respectively, toward plasma membrane receptors, but 100:25:40 toward the solubilized receptors. The solubilized receptor preparation is heterogeneous in size in that it has binding components with molecular weights of 286,000, 160,000, 75,000, and 15,000 to 30,000. Tryptic digestion converts all three higher molecular weight components to the 15,000 to 30,000 molecular weight species, and the 15,000 to 30,000 molecular weight receptor component has all of the binding properties of the solubilized receptor preparation before tryptic digestion including an identical nonlinear Scatchard plot. It has the same size as and coelutes from Sephadex G-100 with a 15,000 to 30,000 molecular weight receptor released by tryptic digestion of bovine thyroid plasma membranes or tryptic digestion of bovine or dog thyroid cells in culture. The tryptic fragment of the solubilized receptor or preparations has been purified almost 250-fold by affinity chromatography on thyrotropin-Sepharose columns. The binding activity is lost when the solubilized thyrotropin receptor preparation is exposed to beads of neuraminidase-Sepharose or conconavalin A-Sepharose.     

Suspension culture reveals a morphogenetic property of a thyroid epithelial cell line

It is known that freshly dissociated thyroid cell clusters form follicles in suspension culture. Thyroid epithelial cell lines, grown for many generations in vitro, fail to show colloid-containing lumina when cultured as monolayers. Several thyroid cell lines, some transformed, have been tested with respect to their ability to form extracellular lumina when transferred from monolayer to suspension culture. One cell line in particular, the T78 cell line, showed this property when cultured in suspension. Lumina formed within 3 days even in the absence of added thyrotropin (TSH). The ultrastructure of lumina within cell aggregates resembled that of the thyroid follicle in vivo. The ability to undergo morphogenesis may therefore be an intrinsic property of thyroid epithelial cells which is retained for a large number of generations in vitro and is revealed by proper culture conditions. The shift from monolayer to suspension culture may thus lead to the expression of a thyroid differentiated function such as the formation of follicle-like structures.     

Experimental exophthalmos. Binding of thyrotropin and an exophthalmogenic factor derived from thyrotropin to retro-orbital tissue plasma membranes.

Biologically active preparations of 125I-thyrotropin, [3H]thyrotropin, and the [3H]exophthalmogenic factor derived from thyrotropin by partial pepsin digestion have been used to study the binding properties of the thyrotropin receptor on guinea pig retro-orbital tissue plasma membranes. In regard to the optimal conditions of binding, pH, buffer, salt concentrations, and temperature, these properties are the same as those described in any accompanying report concerning thyrotropin binding to bovine thyroid plasma membranes (Tate, R.L., Schwartz, H.I., Holmes, J.M., Kohn, L.D., and Winand, R.J. (1975) J. Biol. Chem. 250, 6509-6515). In addition, thyrotropin receptors on the retro-orbital tissue plasma membranes are similar to thyrotropin receptors on bovine thyroid plasma membranes in their apparent negative cooperativity and in their relative affinities for luteinizing hormone, the beta subunit of thyrotropin, and the alpha subunit of thyrotropin. In contrast, gamma-globulin from patients with malignant exophthalmos enhances binding when added to incubation mixtures containing the retro-orbital tissue plasma membranes but not when added to those containing thyroid plasma membranes. Normal gamma-globulin and gamma-globulin from Graves' disease patients without exophthalmos do not have this property. The gamma-globulin itself does not bind to the membrane except in the presence of thyrotropin or its exophthalmogenic factor derivative. Tryptic digestion of the retro-orbital tissue membranes releases specific thyrotropin and exophthalmogenic factor binding activity into the supernatant phase. Chromatography on Sephadex G-100 indicates that this trypsin-released receptor activity has a molecular weight of 75,000 or greater, rather than 15,000 to 30,000 for the trypsin-released receptor activity from bovine thyroid membranes (Tate, R.L., Schwartz, H.I., Holmes, J.M., Kohn, L.D., and Winand, R.J. (1975) J. Biol. Chem. 250, 6509-6515).     

The differential effect of thyrotropin on the electrical responses of thyroid cells in monolayer cultures of varying duration

The acute effects of thyrotropin on the membrane potential of thyroid cells maintained in the presence or absence of thyrotropin (0.2 U/ml) in the culture medium was determined. Monolayer cultures were prepared from porcine thyroid glands and cultured for 4–17 days after which the culture medium was exchanged for a buffered salt solution for intracellular measurements of the membrane potential. Cells were serially impaled with a microelectrode, first in the absence and then in the presence of 10 mU/ml thyrotropin. Cells cultured for 4–9 days depolarized from ?29.6 ± 1.7 (mean ± S.E.) to ?19.3 ± 1.3 mV within 10 min after acute addition of 10 mU/ml thyrotropin. From 11 to 17 days of culture, basal membrane potentials were lower and, in most instances, cell hyperpolarization occurred within 30 min in response to thyrotropin. There was no difference in electrical response of cells maintained in culture with or without thyrotropin. However, cells cultured with thyrotropin formed follicle-like structures in contrast to the monolayer formation of cells cultured without thyrotropin. The changes in the basal and stimulated electrical responses occur within a time frame similar to that reported for changes in the biosynthetic capacity of thyroid cells in culture. The data further emphasize the possible regulatory role of the cell membrane in stimulus-secretion coupling in the thyroid.     

The differential effect of thyrotropin on the electrical responses of thyroid cells in monolayer cultures of varying duration.

The acute effects of thyrotropin on the membrane potential of thyroid cells maintained in the presence or absence of thyrotropin (0.2 U/ml) in the culture medium was determined. Monolayer cultures were prepared from porcine thyroid glands and cultured for 4--17 days after which the culture medium was exchanged for a buffered salt solution for intracellular measurements of the membrane potential. Cells were serially impaled with a microelectrode, first in the absence and then in the presence of 10 mU/ml thyrotropin. Cells cultured for 4--9 days depolarized from --29.6 +/- 1.7 (mean +/- S.E.) to --19.3 +/- 1.3 mV within 10 min after acute addition of 10 mU/ml thyrotropin. From 11 to 17 days of culture, basal membrane potentials were lower and, in most instances, cell hyperpolarization occurred within 30 min in response to thyrotropin. There was no difference in the electrical response of cells maintained in culture with or without thyrotropin. However, cells cultured with thyrotropin formed follicle-like structures in contrast to the monolayer formation of cells cultured without thyrotropin. The changes in the basal and stimulated electrical responses occur within a time frame similar to that reported for changes in the biosynthetic capacity of thyroid cells in culture. The data further emphasize the possible regulatory role of the cell membrane in stimulus-secretion coupling in the thyroid.     

Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into follicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 μU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 μU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.     

Follicle-like structure and polarized monolayer: Role of the extracellular matrix on thyroid cell organization in primary culture

The thyroid follicle, the morphofunctional unit of thyroid gland, is a spheroidal structure formed by a monolayer of polarized cells surrounding a closed cavity in which thyroglobulin accumulates. Newly isolated porcine thyroid cells reorganize into two types of structures which differ by the orientation of cell polarity: in follicle-like structures, obtained in the presence of TSH, the apical pole delineates a closed cavity and cells express most parameters characteristic of thyroid function; in inside-out follicles the apical pole is oriented towards the culture medium and cells do not express properly the thyroid function. The organization of newly formed follicles can be modified by stimulation of cell migration or by interaction of their apical poles with a new cell environment. Seeded on a hard surface (glass, plastic), cells of follicle-like structures or inside-out follicles formed in suspension migrate giving a monolayer. On the contrary, cells organized into a monolayer treated with hexamethylene bisacetamide, reorganize into follicle-like structures. Inside-out structures reoganize upon interaction of their apical poles with collagen I gel, a coherent matrix, or with a reconstituted basement membrane (RBM), a soft matrix. Overlaid with collagen I, monolayers reorganize into follicles. Embedded in collagen I or in RBM, inside-out follicles reorient their polarity giving functional follicles. On the RBM surface, cells pull on the gel and embed themselves in the soft matrix gel, finally reorienting their polarity to inside-in polarity. When comparing thyroid cells with other epithelial cell types (mammary cells, Sertoli cells), it appears that the obtention in culture of follicle-like structures, ie closed inside-in polarized cell organization, is the best way to express in culture both morphology and function of any specific epithelial tissue, the polarized monolayer in porous bottom culture chamber coming just behind.     

Separation of the glycoprotein and ganglioside components of thyrotropin receptor activity in plasma membranes.

The two components of thyroid plasma membranes known to interact with thyrotropin, i.e., a glycoprotein with specific thyrotropin binding activity and the gangliosides of the thyroid membranes, are shown to segregate differently when membranes are solubilized with lithium diiodosalicylate. Individually examined, the interaction of each component with thyrotropin exhibits a different sensitivity to salts. The data suggest that the thyrotropin receptor on the thyroid membrane is a complex which is composed of both glycoprotein and ganglioside components and that its properties are derived from each component.     

Thyrotropin receptors in thyroid plasma membranes. Characteristics of thyrotropin binding and solubilization of thyrotropin receptor activity by tryptic digestion.

Biologically active bovine 125I-thyrotropin preparations have been prepared, characterized, and used to evaluate the optimal conditions for thyrotropin binding to bovine thyroid plasma membranes in vitro. Binding of 125I-TSH has a pH optimum around 6.0 and is sensitive to the choice and concentration of buffer. Binding is inhibited by salts, especially those containing magnesium and calcium ions; magnesium concentrations optimal for adenylate cyclase assays (2 to 5 mM) result in 85 to 98% inhibition of binding. Binding is temperature sensitive. At 37 degrees binding has its highest initial level; however, instability of the membrane at this temperature causes a rapid loss of binding activity. Binding at 0 degrees is optimal in 30 min and at the same level as initial binding at 37 degrees; since there is no decrease in binding activity, it has been chosen as the optimal temperature. Thyrotropin, luteinizing hormone, the beta subunit of thyrotropin, and the alpha subunit of thyrotropin have relative binding affinities for the thyrotropin receptors of 100, 10, 2, and less than 0.5, respectively. In all of these characteristics, 125I-thyrotropin at 1.5 x 10(-5) M concentrations has the same properties of binding to bovine plasma membranes as do [3H]thyrotropin preparations which have been previously characterized (Amir, S.M., Carraway, T.F., Jr., Kohn, L.D., and Winand, R.V. (1973) J. Biol. Chem. 248, 4092-4100) and used to study binding at 5 x 10(-6) M concentrations. 125I-TSH binding as a function of hormone concentration results in curved Scatchard plots; however, Hill plots of these same binding data are linear and have a slope of 0.65. Taken together, these data suggest that the heterogeneity in thyrotropin binding constants which is evident in the Scatchard plot reflects a negatively cooperative relationship among the thyrotropin receptor sites, i.e. decreased hormonal affinity as hormone concentrations increase. Adenylate cyclase studies yield kinetic plots which also exhibit negative cooperativity; corrections for thyrotropin bound under the adverse binding conditions of the adenylate cyclase assays suggest that Km values for thyrotropin in this enzymatic assay are compatible with binding constants measured by the 125I-thyrotropin preparations. Tryptic digestion destroys binding activity on the thyroid plasma membrane but releases specific thyrotropin receptor activity into the supernatant phase. Chromatography on Sephadex G-100 indicates that this solubilized receptor fragment has a molecular weight between 15,000 and 30,000.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP.

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclic AMP which both promote the aggregation of isolated cells into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cyclic AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: reexpression of specific functions

Isolated porcine thyroid cells cultured in suspension in Eagle Minimum Essential Medium supplemented with calf serum (5-20%) reorganize to form vesicles, i.e. closed structures in which all cells have an inverted polarity as compared to that found in follicles: the apical membranes are bathed by the culture medium. Under these conditions, cells neither concentrate iodide nor respond to acute thyrotropin (TSH) stimulation. When embedded in collagen gel, these vesicles undergo polarity reversal to form follicles. We describe here the change in the orientation of cell polarity and the subsequent reappearance of specific thyroid functions. Six hr after embedding, membrane areas in contact with collagen fibers show basal characteristics. At this time, cells begin to concentrate iodide and to respond to acute TSH stimulation (iodide efflux and increased cAMP levels). Most cells form follicles 24 hr after embedding, but 48 hr are required for the transformation of all vesicles into follicles. This occurs without opening of the tight junctions. Iodide organification is detected 24 hr after embedding, when periodic acid-Schiff positive material, identified as thyroglobulin by immunofluorescence, accumulates in the lumen. Iodide concentration and organification, as well as response to TSH stimulation reach maximal levels after 3 days in the collagen matrix. After a 5-day culture in the collagen matrix in the absence of TSH, cell activity can be stimulated by chronic treatment with low hormone concentrations (10-100 microU/ml). As shown with thyroid cells grown in monolayer on permeable substrates (Chambard M., et al., 1983, J. Cell Biol. 96, 1172-1177), iodide uptake and cAMP-mediated TSH responses are expressed when the halogen and the hormone have direct access to the basal membrane. Organification, on the contrary, requires a closed apical compartment.     

Role of phospholipids in the structure and function of the thyrotropin receptor.

Phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine interact with 125I-thyrotropin and inhibit its binding to thyroid plasma membranes; phosphatidylcholine is not similarly effective. The interaction has been monitored by column chromatography on Sephadex G-100 which shows, for example, that 125I-labeled thyrotropin forms an adduct with phosphatidylinositol but not with phosphatidylcholine. Formation of the 125I-labeled thyrotropin-phosphatidylinositol adduct is dependent on the phosphatidylinositol concentration but can be reversed by both unlabeled thyrotropin and excess membranes. The efficacy of the phospholipid interaction and the phospholipid inhibition of thyrotropin binding to thyroid membranes is paralleled by changes in fluorescence and fluorescence polarization imposed on the 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of thyrotropin. These changes are reversed by unlabeled thyrotropin but not by prolactin, placental lactogen, or growth hormone; similar changes are not observed when phospholipids are incubated with dansylated growth hormone, prolactin, and placental lactogen. Monovalent potassium, sodium, and lithium salts neither prevent nor reverse the formation of the phospholipid-dansyl-thyrotropin adduct; these results contrast with the effects of the same salts on the formation of ganglioside adducts with dansyl-thyrotropin. Despite their ability to interact witw 125I-thyrotropin in solution, neither phosphatidylinositol, phosphatidylserine, nor phosphatidylethanolamine, when incorporated in a liposome, binds the 125I-labeled ligand. These same phospholipids have no effect on ganglioside binding of 125I-labeled thyrotropin when gangliosides are incorporated in a liposome. These phospholipids do, however, modulate the expression of the glycoprotein component of the thyrotropin receptor when it is imbedded in a liposome. The phosphatidylinositol in this case serves as a negative modulator, both by decreasing the incorporation of the glycoprotein component of the receptor into the liposome and by inhibiting the binding activity of the glycoprotein component which is incorporated. Speculation is offered as to a possible role of the phospholipids in the message transmission process which would be consistent with current studies demonstrating a direct interaction of acidic phospholipids with thyrotropin. The effect of phospholipids on liposomes containing the glycoprotein component of the thyrotropin receptor raises the possibility that phospholipids and, in particular, phosphatidylinositol, may also play a role in regulating the insertion and expression of this receptor component in thyroid plasma membranes.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclin AMP which both promote the aggregation of isolated cell into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells to the acute effect of thyroid-stimulating hormone: only cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cycli AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Hexamethylene bisacetamide (HMBA) increases thyroglobulin levels in porcine thyroid cells without increasing cyclic-AMP.

The polar planar compound hexamethylene bisacetamide (HMBA) is an inducer of terminal differentiation which has been extensively studied in the murine erythroleukemia cells (MELC). We have tested this compound in normal porcine thyroid cells in primary culture where it either activates or inhibits the major tissue specific functions of these cells: it induces the reorganization of cells into follicles, prevents the loss of thyrotropin sensitivity in monolayer cells, activates cell growth but inhibits their iodide metabolism. In this paper, we demonstrate that HMBA acts on the total thyroglobulin levels measured in cell layers plus media. This specific marker of thyroid tissue is increased by HMBA both in kinetics and in concentration-response experiments. HMBA per se does not increase the total cyclic AMP measured either during the first hours after stimulation or in the following days when compared to controls. As expected, cyclic AMP in the same experiment increased rapidly within minutes after the cells were challenged by TSH (positive control). Altogether, the results show that the drug HMBA mimics thyrotropin effects on thyroglobulin levels measured in porcine thyroid cells in culture. This modulation cannot be explained by an increase in cyclic AMP, indicating that despite similarities between TSH and HMBA effects, the mechanism of the mode of action of these two molecules is very different.     

Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation.

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into folicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 microU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 microU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.     

Noninactivation of thyrotropin by cultured porcine thyroid cells.

Freshly isolated porcine thyroid cells were cultured in the presence of highly purified porcine thyrotropin. Cells associate into follicles between the second and tenth day of culture and later form a monolayer. The biological and immunological activity of thyrotropin was measured daily in the media. Thyrotropin concentration and biological activity remained unchanged from the onset of the culture up to day 14. Limiting factors influencing thyroglobulin biosynthesis do not appear before day 13. The loss of follicular organization at day 10 cannot be explained by thyrotropin degradation in the medium. Considering the number of receptors per cell and the half life of the thyrotropin . receptor complex in the two dissociation compartments previously demonstrated, it appears in terms of both biological activity and affinity for the receptors that the thyrotropin molecules released from the first compartment do not differ from native molecules. It can be calculated that at least 31% of the molecules released from the second compartment are not inactivated. Thus, it is probable that the catabolism of thyrotropin on the receptor, or near the receptor site, does not play an important role in the regulation of thyroid cell function in vitro.     

Thyrotropin binding to and adenylate cyclase activity of porcine thyroid plasma membranes.

Plasma membranes have been purified from porcine thyroid gland homogenate by discontinuous sucrose gradient centrifugation. The preparations contained specific binding sites for thyrotropin but not for luteinizing hormone or the beta subunits of thyrotropin and luteinizing hormone. Optimum conditions of 125I-labeled thyrotropin binding were pH 6.0-6.5 and 37 degrees C. Thyrotropin binding was reduced by divalent (Ca2+, Mg2+) and monovalent cations (Na+, K+, Li+), 50% inhibition being obtained at 10 mM and 50 mM respectively. Displacement curves of 125I-labeled bovine or porcine thyrotropin by the unlabeled hormone from three species was in the order of increasing concentrations (bovine greater than porcine greater than human) which is the order of decreasing biological activity of these hormone preparations in the assay in vivo in the mouse. The validity of the results was established by controlling that porcine membranes bound the native and the 125I-labeled hormones with equal affinity. A single type of high-affinity (Kd = 0.28 nM) binding sites was detected for bovine and porcine thyrotropins. In contrast, porcine plasma membranes bound human thyrotropin with a lower affinity (Kd = 70 nM). A good correlation was found at equilibrium and in the conditions of the cyclase assay, between receptor occupancy and adenylate cyclase activation for the three hormones.     

Effects of tunicamycin and N-linked oligosaccharide-processing inhibitors on the morphology of cultured porcine thyroid cells

The effects of tunicamycin and of N-linked oligosaccharide-processing inhibitors on the ability of cultured porcine thyroid cells to adhere to a plastic support and to form organized structures were examined. The culture conditions used allowed the epithelial cells to adhere to the support and to form either a monolayer (no thyrotropin) or follicles (thyrotropin 4 mU/ml). The follicles thus obtained tend to disappear after 8 to 9 days, giving rise to a monolayer. Tunicamycin prevented both cell adhesion to the support and formation of organized structures. Swainsonine, an inhibitor of mannosidase II, had no obvious effect. Deoxymannojirimycin, an inhibitor of mannosidase I, did not prevent cell adhesion to the support and formation of monolayers or follicles, but it favored the maintenance of follicles at a time when they were no longer present in controls. It also led to the appearance of some follicles in cultures without thyrotropin. Castanospermine, an inhibitor of glucosidase I, did not prevent cell adhesion but slowed cell spreading, thus delaying monolayer formation. Pronase glycopeptides prepared from cell-surface glycoproteins were examined with respect to their behavior on concanavalin A-Sepharose. The glycopeptides from control cells displayed complex and high-mannose glycans. The content in complex glycans was decreased in inhibitor-treated cells, while that in hybrid or high-mannose glycans was increased, indicating that the inhibitors modify the N-glycan structures. In conclusion, N-glycosylation of glycoproteins is necessary for cellular adhesion to the support. Complex structures do not seem necessary for cell adhesion monolayer or follicle formation. High-mannose structures favor follicular organization, while glucoses on the high mannose structures hinder cell spreading.     

Interaction of the thyrotropin receptor on rat FRTL-5 thyroid cells with thyrotropin and a thyrotropin-stimulating autoantibody from Graves' patients

FRTL-5 rat thyroid cells were either surface-labeled with 125I or biosynthetically labeled with [3H]N-acetylglucosamine, solubilized by lithium diiodosalicylate and immunoprecipitated after sequential exposure to bovine thyrotropin and anti-bovine thyrotropin. Autoradiography of polyacrylamide gels run under denaturing conditions and in the presence of a reducing agent revealed two prominent bands with approximate molecular weights of 66-70 kDa and 47 kDa. Immunoprecipitation of the same radiolabeled and solubilized membrane preparations with a Graves' disease IgG having thyroid stimulating but no thyrotropin-binding inhibiting activity revealed only one major band, migrating near the 47 kDa component reactive with thyrotropin. No bands were immunoprecipitated in control incubations using normal human IgG or substituting radiolabeled, solubilized membranes from a rat thyroid cell line with no thyrotropin receptor activity. Thin layer chromatography of Folch extracts of the [3H]-N-acetylglucosamine-labeled immunoprecipitates obtained by either procedure indicated that a specific thyroid ganglioside was coprecipitated with the immunoprecipitated proteins in both cases.     

Biologic activity of anti-thyrotropin anti-idiotypic antibody

We raised an antihuman thyrotropin anti-idiotypic antibody and showed that it was active at the thyrotropin receptor. Thus this antibody inhibited ¹²⁵I b-TSH binding to thyroid plasma membranes, stimulated adenylate cyclase activity through a guanyl nucleotide-dependent mechanism, increased radioiodide entry rate into isolated porcine thyroid follicular cells, and induced such cultured cells to organize into follicles. All these parameters are typical of thyrotropin action. This work raises the possibility that thyroid stimulating antibodies that cause the hyperthyroidism of Graves disease may be, at least in some patients, anti-thyrotropin anti-idiotypic antibodies. It also offers a novel method whereby antireceptor antibodies used in the isolation and characterization of the receptor may be raised from ligands.