An enzymatic basis for Lewis blood types in man.

Milk from women with blood type Le(a+) or Le(b+) contains a specific fucosyltransferase not found in the milk of women with blood type Le(a- b-). The enzyme, a guanosine diphosphate L-fucose: N-acetyl-beta-D-glucosaminylsaccharide alpha-4-L-fucosyltransferase is apparently required for the synthesis of the structural determinants of Le(a) and Le(b) specificity, both of which contain fucose in an alpha-1,4 linkage to N-acetylglucosamine. The same enzyme is also involved in the synthesis of milk oligosaccharides, as two oligosaccharides which contain this linkage are absent from the milk of women with Le(a- b-) blood type.

Relationships of the structure and function of the interferon receptor to hormone receptors and establishment of the antiviral state.

This report describes similarities between the structure and function of the interferon receptor and receptors for glycoprotein hormones and several bacterial toxins. Specifically, it describes several common molecular and mechanistic elements, including: (a) the presence of a glycoprotein as well as a ganglioside component in the receptor; (b) changes in membrane structure as a consequence of interferon action; (c) interferon-induced intracellular cyclic adenosine 3':5'-monophosphate changes; and (d) alterations in the flux of certain ions across the membrane. Since interferon has an antiviral effect, these results define a relationship between hormonal perturbation of cellular events and the ability of an agent to prevent or suppress viral infections of cells. Further definition of these relationships should be important to our understanding of the oncogenic state, of hormonal effects on the oncogenic state, and of other human diseases in which hormonal perturbations of non-target tissues or cross-reactivity of receptors could be pathogenic.

Differences in the electrophysiological response to I- and the inhibitory anions SCN- and ClO-4, studied in FRTL-5 cells.

The electrophysiological properties of the Na+/I- symporter (NIS) were examined in a cloned rat thyroid cell line (FRTL-5) using the whole-cell patch-clamp technique. When the holding potential was between -40 mV and -80 mV, 1 mM NaI and NaSCN induced an immediate inward current which was greater with SCN- than with I-. The reversal potential for I- and SCN- induced membrane currents was +50 mV. This is close to the value of +55 mV calculated by the Nernst equation for Na+. These results are consistent with I- and SCN- translocation via the NIS that is energized by the electrochemical gradient of Na+ and coupled to the transport of two or more Na+. There was no change in the membrane current recording with ClO-4 indicating that ClO-4 was either not transported into the cell, or the translocation was electroneutral. ClO-4 addition, however, did reverse the inward currents induced by I- or SCN-. These effects of I-, SCN- and ClO-4 on membrane currents reflect endogenous NIS activity since the responses duplicated those seen in CHO cells transfected with NIS. There were additional currents elicited by SCN- in FRTL-5 cells under certain conditions. For example at holding potentials of 0 and +30 mV, 1 mM SCN- produced an increasingly greater outward current. This outward current was transient. In addition, when SCN- was washed off the cells a transient inward current was detected. Unlike SCN-, 1-10 mM I- had no observable effect on the membrane current at holding potentials of 0 and +30 mV. The results indicate FRTL-5 cells may have a specific SCN- translocation system in addition to the SCN- translocation by the I- porter. Differences demonstrated in current response may explain some of the complicated influx and efflux properties of I-, SCN- and ClO-4 in thyroid cells.

Sodium/proton exchange in the maintenance of intracellular pH in FRTL-5 thyroid cells.

In the present study we describe a Na+/H+ exchange system in FRTL-5 rat thyroid cells and its role in the regulation of intracellular pH (pHi). The pHi of these cells was measured in acidic medium (pH 6.5) using the equilibrium distribution of 14C-labeled 5,5'-dimethyl-oxazolidine-2,4-dione. The estimated pHi in 10 separate experiments was 7.30 +/- 0.01 (mean +/- SE). The pHi declined to 6.87 +/- 0.02 when equimolar concentrations of choline+ replaced Na+ in the incubation medium, but was restored when cells were placed in a medium containing 50 mM Na+. The necessity for external Na+ to maintain pHi, when cells are exposed to acidic solutions, is consistent with a Na+/H+ antiport system in these cells. This is further supported by the observation that pHi decreased to 6.70 +/- 0.01 when amiloride, an inhibitor of Na+/H+ exchange, was added, and that this agent prevented the recovery of pHi in Na+-depleted cells after addition of Na+ to the medium. This report also provides additional information concerning the relationship between H+ and I- uptake and suggests that there are two I- transport systems: one that is Na+-dependent, 4,4'-diisothiocyano 2,2'-disulfonic acid stilbene-sensitive, and not associated with intracellular acidification, and the other operative at an external pH of 6.5 and associated with intracellular acidification.

Effect of actinomycin D on iodide transport in FRTL-5 thyroid cells.

TSH-induced I- uptake in FRTL-5 thyroid cells requires new protein synthesis. During the induction of I- uptake, which takes nearly 60 h to reach its maximum, two waves of protein synthesis can be identified: one during the first 8 h and another after 24-30 h, each involving different proteins. Cycloheximide (CH) added during the first 10 h of a 48-h incubation with TSH completely inhibits the induction of I- uptake; 58% inhibition is observed with CH added at 24 h; no inhibition is observed when CH is added 36 h after TSH. Like CH, actinomycin D (ActD), added at the beginning of the 48-h period, inhibits TSH induction of I- uptake; partial inhibition (83%, 72%, and 28%) is observed when ActD is added at 1, 5, and 10 h, respectively. Treatment with ActD at 24 h (ActD-treated cells), however, paradoxically increases I- uptake (1.8- to 3.5-fold over the control value). The characteristics of I- uptake in ActD-treated cells are the same as those in untreated cells; both are Na+ dependent and can be inhibited in a comparable manner by anions. Kinetic measurements of I- transport indicate that ActD increases the rate of I- influx (2-fold or greater increase in maximum velocity without a significant change in Km), with only minor changes in I- release. Enhanced I- uptake in ActD-treated cells is inhibited by the simultaneous (24-h) administration of CH, indicating that protein synthesis is required for the late ActD effect. Despite an overall 2-fold decrease in protein synthesis in cells treated with ActD at 24 h, the synthesis of individual proteins maximally induced by TSH during the first 8 h is increased, whereas that of some proteins maximally synthesized after 24-30 h is markedly reduced. The present data indicate that TSH-induced I- uptake in FRTL-5 cells involves a regulatory action of TSH that operates at the mRNA level.

Iodine suppression of iodide uptake in FRTL-5 thyroid cells.

Exposure of FRTL-5 cells to iodide (I-) in excess of 3 microM suppresses the concentrative uptake of I-. The depression of I- uptake measured at the steady state is due to decrease in the rate of I- influx and not to an effect on I- efflux. Exposure to NaI is associated with decreased T4 secretion and also depressed Na+-dependent amino acid accumulation. The depression in I- and amino acid transports increases proportionately with the duration of exposure and concentration of I- used but is not associated with alterations in FRTL-5 cell cAMP levels. The I- suppression effect is blocked, however, when methimazole is present during the incubation with NaI. In agreement with studies in vivo, I- suppression in FRTL-5 cells appears to depend on an intermediate in the organification process and to be independent of a TSH-induced cAMP-mediated action.

Norepinephrine and thyrotropin stimulation of iodide efflux in FRTL-5 thyroid cells involves metabolites of arachidonic acid and is associated with the iodination of thyroglobulin.

Ca2+-dependent and TSH-, norepinephrine (NE)-, and A23187-induced iodide (I-) efflux from FRTL-5 rat thyroid cells is inhibited by quinacrine and trifluoroperazine, agents that inhibit phospholipase A2 activity. Furthermore, I- efflux can be stimulated by an activator of phospholipase A2 activity, melittin. Phospholipase A2 action releases arachidonic acid from phospholipids; arachidonic acid enhances I- efflux in FRTL-5 cells. Inhibitors of arachidonic acid metabolism via the lipoxygenase pathway, 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, and via the cytochrome P450-linked epoxygenase pathway, piperonyl butoxide and 2-diethylaminoethyl-2,2-diphenyl valerate, but not an inhibitor of the cyclooxygenase pathway, indomethacin, can inhibit TSH-, NE-, and A23187-induced I- efflux. TSH, NE, and arachidonic acid stimulation of I- efflux in FRTL-5 cells is associated with increased iodination of thyroglobulin, which is blocked by 10 microM 5,8,11,14-eicosatetraynoic acid and 50 microM piperonyl butoxide. The data thus suggest that TSH- and NE-induced I- efflux from FRTL-5 thyroid cells involves lipoxygenase and/or epoxygenase metabolites of arachidonic acid, released from phospholipids upon Ca2+-dependent activation of phospholipase A2. Since this process is associated with the iodination of thyroglobulin, TSH- and NE-induced I- efflux in FRTL-5 cells may represent the transport of I- from the cell into the follicular lumen in vivo.

Iodide transport in a continuous line of cultured cells from rat thyroid.

The properties of TSH-dependent iodide (I-) uptake are defined for a cloned, continuously growing, functioning rat thyroid cell line (FRTL-5 cells). Since these cells grow without a lumen and are therefore restricted in their ability to iodinate thyroglobulin, the FRTL-5 cells offer the opportunity to directly study transport processes across the membrane into the cell as well as the process whereby I- moves from the cell. FRTL-5 cells concentrate I- approximately 30-fold and exhibit many of the properties of I- uptake seen in thyroid tissue slice and primary cell culture systems. In these cells, accumulation of I- is consistent with a Na+-dependent carrier model for I- uptake, and effects on the influx and efflux processes can be dissociated. Because FRTL-5 cells can be maintained in culture indefinitely and can provide large quantities of a homogeneous functional thyroid cell preparation for study, these cells offer the unique opportunity to further define the mechanism and kinetics of I- transport in a less complex system.

Effect of thyrotropin on iodide efflux in FRTL-5 cells mediated by Ca2+.

A functioning rat thyroid cell line (FRTL-5) responds acutely to the addition of TSH, norepinephrine, and the Ca2+ ionophore A23187 by a depression in iodide (I-) uptake levels. The decrease in I- content measured at the steady state depends on the presence of external Ca2+ and can be accounted for by an effect on stimulated I- efflux. As contrasted to the prolonged time (hours and days) involved in the stimulatory effect of TSH on I- uptake, the acute response to TSH is 1) seen within 5 min and maintained for about 20 min, 2) maximum, at a 1 X 10(-7)M concentration of TSH compared with the concentration of 1 X 10(-9)M necessary for the stimulatory effect, 3) independent of whether the cells are growing in the presence or absence of TSH, and 4) not mimicked by the addition of (Bu)2cAMP. The results suggest that TSH and adrenergic stimulation lead to increased membrane permeability to I- which is mediated by an elevation in the intracellular Ca2+ concentration.

Norepinephrine and thyroid-stimulating hormone induce inositol phosphate accumulation in FRTL-5 cells.

3H-Labeled inositol phosphate accumulation is observed when prelabeled FRTL-5 cells (a rat thyroid cell line) are exposed to norepinephrine (NE) or TSH. The presence of inositol trisphosphate among the products implicates a phosphodiesterase-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate. The response to NE is much greater than that to TSH. This may be explained by the ability of cAMP to inhibit inositol phosphate accumulation in these cells. The stimulation by NE is inhibited by alpha 1-adrenergic receptor antagonists and is markedly potentiated in medium of reduced Ca2+ concentration. After chronic withdrawal of TSH from the growth medium, the magnitude of the response to NE is considerably reduced; however, there is no substantial shift in the dose-response curve. This reflects the dependency of alpha 1-adrenergic receptor expression on TSH in the FRTL-5 cell. In contrast, the characteristics of inositol phosphate accumulation induced by acute treatment with TSH are similar in cells maintained in the presence or absence of a low concentration of this hormone, and correlate well with the iodide efflux and iodination of thyroglobulin observed in response to TSH. These results support the hypothesis that TSH may mediate certain of its physiological effects through cAMP-independent mechanisms, such as phospholipid/Ca2+ and C-kinase pathways.

Thyrotropin-stimulated iodide transport mediated by adenosine 3',5'-monophosphate and dependent on protein synthesis.

Iodide (I-) uptake by FRTL-5 cells, a functioning rat thyroid cell line, is TSH dependent. The effects of TSH withdrawal are not apparent until 1 day; 1 week is required to reduce I- uptake to a minimal level. The readdition of TSH leads to a return of the I--concentrating ability after a latency of 12-24 h. The reappearance of I- uptake induced by TSH is mimicked by (Bu)2cAMP and agents that elevate intracellular cAMP levels in these cells, such as forskolin, cholera toxin, and a Graves' disease serum. The appearance of I- uptake after TSH occurs 12 h after the appearance of TSH-induced [35S]methionine incorporation. Cycloheximide blocks both the TSH- and (Bu)2cAMP-induced increases in methionine incorporation and I- uptake to the same extent and in an identical concentration-dependent manner. TSH-induced [35S]methionine incorporation is associated with increased radiolabeling of several specific proteins, as revealed by gel electrophoresis; none, however, is radiolabeled coincident in time with the appearance of TSH-induced I- uptake. Several proteins whose apparent synthesis is induced by TSH also exhibit TSH-dependent phosphorylation.

Familial goiter in bongo antelope (Tragelaphus eurycerus).

Congenital defects in thyroglobulin (Tg) synthesis in animals have proven to be useful models for the study of Tg synthesis and regulation. Defects in Tg synthesis have been well described in Afrikander cattle, Australia Merino sheep, and goats in The Netherlands. This report describes a study of goiter in a nondomesticated bovine species, bongo antelope (Tragelaphus eurycerus), an African bovid. Three animals housed at the National Zoological Park, Washington, D.C. were studied; two had visible goiters, and a third bongo had microscopic evidence of goiter. Tg extracted from thyroid glands or thyroid colloid from these animals had a high mol wt component that was greater than 220K daltons and differed in apparent mol wt from 19S Tg from domestic cattle. Thyroid extracts also had thyroid albumin; albumin was more than half the total protein in colloid extract. The animals with goiter were euthyroid according to their circulating levels of thyroid hormones.

Repeatedly passed FRTL-5 rat thyroid cells can develop insulin and insulin-like growth factor-I-sensitive cyclooxygenase and prostaglandin E2 isomerase-like activities together with altered basal and thyrotropin-responsive thymidine incorporation into DNA.

Repeatedly passed or aged rat FRTL-5 thyroid cells develop a high level of basal [3H]thymidine incorporation into DNA and a reduced response to TSH in medium containing 5% serum and insulin (5H medium). The basal [3H]thymidine incorporation into DNA of aged cells can exceed the TSH-induced increase in earlier passages of the same cell line (fresh cells) and the TSH response decreases from more than 10-fold above basal in fresh cells to less than 2-fold in aged cells. This change is not associated with a loss of the diploid karyotype, a change in basal cAMP levels, or a change in dependence on TSH for cell growth. Attenuation of the TSH response in the [3H]thymidine incorporation assay is more evident than the reduced effect of TSH on cAMP levels or iodide transport; moreover, the TSH effect on cAMP levels does not correlate with that on [3H] thymidine incorporation as a function of hormone concentration. The high basal activity in [3H]thymidine incorporation into DNA in aged cells is due to an increased responsiveness to insulin, insulin-like growth factor-I (IGF-I), or serum. Thus, removal of serum and insulin from the medium eliminates the high basal [3H]thymidine incorporation into DNA, and this activity is restored by insulin or IGF-I in a concentration-dependent manner. The increased responsiveness of aged cells to insulin or IGF-I is inhibited by indomethacin or hydrocortisone and is associated with insulin or IGF-I, but not TSH, stimulation of cyclooxygenase and prostaglandin E2 (PGE2) isomerase-like activity. Fresh cells, in contrast, require TSH plus insulin or IGF-I to increase these activities. Increased responsiveness of cyclooxygenase activity to insulin or IGF-I in aged cells reflects at least in part an increase in cyclooxygenase mRNA levels. We suggest that insulin/IGF-I stimulation of PGE2 production leads to the high basal thymidine incorporation into DNA in aged cells maintained in TSH-depleted (5H) medium; the reduced stimulation by TSH of cAMP content or iodide uptake may reflect PG inhibition (negative feedback regulation) of cAMP production.

Tetanus toxin interactions with the thyroid: decreased toxin binding to membranes from a thyroid tumor with a thyrotropin receptor defect and in vivo stimulation of thyroid function.

Normal rat thyroid membranes adsorb neurotoxicity when incubated with purified tetanus toxin. Membranes from a rat thyroid tumor with a thyrotropin receptor defect adsorb very little neurotoxicity when similarly evaluated. This inability of the tumor membranes to adsorb neurotoxicity is correlated with a defect in their ability to bind both 125I-labeled tetanus toxin and [125I]iodothyrotropin. The effect of tetanus toxin on the release of radioiodine from the thyroids of appropriately prepared mice has been measured by adapting methods used for the bioassay of thyrotropin. One minimum lethal dose of tetanus toxin given sc caused a significant release of radioiodine into the blood of mice 48 h after injection. In mice subjected to the stress of prior bleedings or anesthesia, the release of radioiodine from the thyroid by tetanus toxin was accelerated, i.e., the increase in blood radioiodine could be measured 24 h after injection. These results again suggest that tetanus toxin may interact with thyrotropin receptors on thyroid plasma membranes. The "sympathetic overactivity syndrome" seen in some patients with tetanus and the syndrome characterized as "thyroid storm" in patients with Graves' disease are discussed as they may relate to these observations.

Hyposialylated thyroglobulin in a patient with congenital goiter and hypothyroidism.

A large family (14 children) with congenital goiter whose parents are first cousins was studied. Thyroid tissue was obtained, after 125I in vivo labeling, from one of the siblings (JBM). Gel filtration of thyroid proteins indicated that thyroglobulin (Tg) eluted as a single symmetrical peak in the same position as authentic 19S Tg. Gel electrophoresis in a 7.5% sodium dodecyl sulfate-polyacrylamide gel revealed a major band with the same mobility and immunoreactivity as normal 19S Tg. Hydrolysis of the patient's Tg indicated that most of the radioactivity was mono- and diiodotyrosines. The yield of T4 from JBM Tg (26 pmol/mg protein) was 5-fold less than normal thyroid tissue (140 pmol/mg protein) and approximately half of that in thyroid tissue from endemic goiter (51 pmol/mg). Total T3 released from JBM Tg was similar to the other two tissues. When the carbohydrate content of normal and patient Tg was analyzed, there was no differences in glucosamine, galactose or mannose content. However, unlike normal and endemic-goiter Tg, that had a mean sialic acid content of 7.3 and 5.6 micrograms/mg protein, respectively, the sialic acid concentration of the patients Tg was only 0.3 microgram/mg. Sialyltransferase activity was readily demonstrated in homogenate from normal thyroid or endemic goiter, but no sialyltransferase activity was detectable in a homogenate of JBM-thyroid tissue. We conclude that the finding of severely hyposialylated Tg is linked to a defect in iodotyrosine coupling seen in this patient with a possibly abnormal migration of Tg into the follicular lumen.

Characterization of the optimal stimulatory effects of graves' monoclonal and serum immunoglobulin G on adenosine 3',5'-monophosphate production in fRTL-5 thyroid cells: a potential clinical assay.

Immunoglobulin G (IgG) preparations derived from the sera of patients with hyperthyroidism due to Graves' disease (TSAb) as well as a monoclonal IgG derived from heterohybridoma fusions of Graves' lymphocytes augmented cAMP levels in a continuous strain of functioning rat thyroid cells (clone FRTL-5) in culture. Optimal stimulation was the same for both types of IgG preparations when measured after 2 h of incubation with 5 X 10(4) cells/well and using cells maintained in a nongrowth, TSH-deficient medium for 7 days. At low IgG concentrations, the stimulatory activities of both preparations exhibited a linear dependence on concentration and similar Ka values (approximately 4 X 10(-8) M) despite the fact that 65% of the Graves' serum IgG preparations had a significantly better ability to inhibit TSH binding to membrane preparations. The Ka value for TSH in the same assay was about 5 X 10(-12) M. Using this cell assay, 90% of a series of hyperthyroid Graves' IgG preparations exhibited stimulating activity, a value comparable to the frequency of positive results found by ourselves and others using human thyroid cell and slice systems. In contrast, only 10% of patients who were euthyroid 1 yr after antithyroid drug withdrawal (n = 21) exhibited stimulating activity, and no stimulating activity was detected in patients with nontoxic nodular goiter (n = 11), toxic adenoma (n = 5), or thyroid carcinoma (n = 6). The studies suggest that an optimized rat FRTL-5 thyroid cell system is a clinically useful and convenient alternative to human thyroid cell and slice systems for detecting TSAbs.

Thyrotropin and norepinephrine stimulate the metabolism of phosphoinositides in FRTL-5 thyroid cells.

Hormone-induced changes in phospholipid metabolism were examined in a functioning rat thyroid cell line (FRTL-5). Stimulation of FRTL-5 cells, prelabeled with 32P, with TSH or NE resulted in a rapid decrease in the radioactivity of both phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-monophosphate (PIP). The effects of TSH on phospholipid metabolism and calcium mobilization are independent of those on adenylate cyclase. This suggests that the TSH receptor may be unique in that it activates enzyme cascades involved in cAMP production and Ca2+ mobilization.

Inhibition of iodide transport in rat thyroid cells using N-substituted anthranilic acid derivatives.

The purpose of this study was to test the effects of chloride channel blockers on iodide uptake in thyroid cells, in the hope of eventually using these blockers to identify and isolate a putative iodide transporter. The chloride channel blockers used in this report are derivatives of N-substituted anthranilic acid and were synthesized using published procedures. For these studies FRTL-5 cells, a line of continuous-growing rat thyroid cells, were used as a model system to study effects on iodide transport. In these cells, there are at least two ways for transmembrane iodide movements, a sodium-dependent influx step and a proposed channel that normally mediates iodide efflux. Two derivatives studied decreased iodide accumulation in FRTL-5 cells, but were found also to lower intracellular pH and ATP levels. To simplify interpretation of the effect of the drugs on iodide transport, we extended the studies using plasma membrane vesicles made from pig thyroid. Iodide entry in these vesicles depended on a sodium gradient and was independent of ATP levels. Iodide transport in plasma membrane vesicles and FRTL-5 cells was measured at 30 sec when the uptake was nearly linear and therefore likely to reflect iodide entry. The uptake was measured using three concentrations of iodide and three of drug. Kinetic analysis of the data described a competitive inhibition by the drugs with a Ki of approximately 250 microM. In summary, N-substituted anthranilic acid derivatives reversibly inhibit iodide entry in FRTL-5 cells and pig plasma membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulated iodide uptake in FRTL-5 cells preincubated with Graves' immunoglobulins in NaCl-free medium: a sensitive assay for thyroid-stimulating antibodies.

The present study was designed to increase the sensitivity of iodide uptake assay for detecting thyroid-stimulating antibodies (TSAb). Based on the previous observations that TSAb are more active to increase cAMP levels in the NaCl-free condition, we improved the assay procedure and defined the optimum conditions: FRTL-5 cells were incubated with immunoglobulin (IgG) in NaCl-free medium for 3 days, and then 125I uptake in the cells was determined after 60 min. The sensitivity of iodide uptake assay for TSAb increased 3-fold by the modification, when assessed by the IgG concentration required to elicit the same response. The described assay is as sensitive as that using cAMP measurement in NaCl-free buffer. Thus, it could detect TSAb in all 21 patients with active Graves' disease and in 7 of 8 with ophthalmic Graves' disease but not in 29 of 30 control subjects. Thyroid stimulating activities assessed by these two assays correlated with each other (n = 29, r = 0.707, p < 0.001). False positive results obtained in 4 hypothyroid patients with Hashimoto's thyroiditis (serum TSH concentrations, 11-171 mU/L) could be prevented using anti-TSH antibodies. In summary, the described assay allows evaluation of stimulated thyroid function directly without affecting the detection of TSAb.

Multicomponent structure of the thyrotropin receptor: relationship to Graves' disease.

The thyrotropin receptor is proposed to contain both a glycoprotein and a ganglioside component. Monoclonal antibodies have been developed against soluble thyroid TSH receptor preparations and using Graves' lymphocytes. These show that initial recognition of thyrotropin requires the glycoprotein component, but that monoclonal antibodies to this component block thyrotropin function (blocking antibodies) rather than mimic thyrotropin. Monoclonal antibodies which stimulate thyroid activity in cultured cell systems (cAMP increase) or mouse bioassays, all interact with gangliosides. Using monoclonal antibodies to the glycoprotein component of the thyrotropin receptor, we show that two protein bands, molecular weights 18,000-23,000 and 50,000-55,000, are precipitated from detergent-solubilized preparations. Using a crosslinking procedure with 125I-labeled thyrotropin, we show that thyrotropin binding is related to the disappearance of the 18,000-23,000 molecular weight band on sodium dodecylsulfate gels and the appearance of a 30,000-33,000 molecular weight thyrotropin-membrane component complex. Higher molecular weight thyrotropin-membrane complexes of 75,000-80,000 and 250,000 are visualized when binding studies are performed at pH 7.4 in physiologic medium; larger amounts of the 30,000-33,000 complex are evident at pH 6.0 in a low salt medium. It is thus proposed that the glycoprotein component of the thyrotropin receptor is composed of two subunits with apparent molecular weights of 18,000-23,000 and 50,000-55,000; that the 18,000-23,000 subunit interacts with thyrotropin; and that different receptor subunits can exist depending on in vitro binding conditions. Using monoclonal-stimulating antibodies or natural autoimmune IgG preparations from patients' sera, we show that stimulating antibodies exhibit species-specific binding to human thyroid ganglioside preparations. Individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.