Discovery of a Positive Allosteric Modulator of the Thyrotropin Receptor: Potentiation of Thyrotropin-Mediated Preosteoblast Differentiation In Vitro.

Recently, we showed that TSH-enhanced differentiation of a human preosteoblast-like cell model involved a ß-arrestin 1 (ß-Arr 1)-mediated pathway. To study this pathway in more detail, we sought to discover a small molecule ligand that was functionally selective toward human TSH receptor (TSHR) activation of ß-Arr 1. High-throughput screening using a cell line stably expressing mutated TSHRs and mutated ß-Arr 1 (DiscoverX1 cells) led to the discovery of agonists that stimulated translocation of ß-Arr 1 to the TSHR, but did not activate Gs-mediated signaling pathways, i.e., cAMP production. D3-ßArr (NCGC00379308) was selected. In DiscoverX1 cells, D3-ßArr stimulated ß-Arr 1 translocation with a 5.1-fold greater efficacy than TSH and therefore potentiated the effect of TSH in stimulating ß-Arr 1 translocation. In human U2OS-TSHR cells expressing wild-type TSHRs, which is a model of human preosteoblast-like cells, TSH upregulated the osteoblast-specific genes osteopontin (OPN) and alkaline phosphatase (ALPL). D3-ßArr alone had only a weak effect to upregulate these bone markers, but D3-ßArr potentiated TSH-induced upregulation of ALPL and OPN mRNA levels 1.6-fold and 5.5-fold, respectively, at the maximum dose of ligands. Furthermore, the positive allosteric modulator effect of D3-ßArr resulted in an increase of TSH-induced secretion of OPN protein. In summary, we have discovered the first small molecule positive allosteric modulator of TSHR. As D3-ßArr potentiates the effect of TSH to enhance differentiation of a human preosteoblast in an in vitro model, it will allow a novel experimental approach for probing the role of TSH-induced ß-Arr 1 signaling in osteoblast differentiation.

(99m)Tc-labeled-rhTSH analogue (TR1401) for imaging poorly differentiated metastatic thyroid cancer.

BACKGROUND: Differentiated thyroid carcinomas originating from thyroid follicular cells are frequent tumors of the thyroid with relatively good prognosis due to improved surgical techniques and follow-up procedures. Poorly differentiated thyroid cancers, which lose iodine uptake ability, in most cases still express thyrotropin (TSH) receptors (TSHR). Therefore, the aim of this study was to radiolabel a superagonist recombinant human TSH (rhTSH) analogue for imaging poorly differentiated thyroid cancer. METHODS: The TSHR superagonist, TR1401, was labeled with (99m)Tc using an indirect method via succinimidyl-6-hydrazinonicotinate hydrochloride conjugation. In vitro quality controls included SDS-PAGE, cysteine challenge, and cell-binding assay on TSHR positive cell lines (JP09 and ML-1). In vivo studies included tumor targeting experiments in athymic nude CD-1 mice xenografted with several different TSHR positive cells (JP09, K1, and ML-1) and TSHR negative cells (JP02) as control. RESULTS: The superagonist rhTSH analogue TR1401 was labeled with high labeling efficiency (>95%) and high specific activity (9250 MBq/mg). The labeled molecule retained its biologic activity and structural integrity. In tumor targeting experiments, a focal uptake of radiolabeled TR1401 was observed in TSHR positive cells but not in TSHR negative cells. The same observation was made in a dog with spontaneous intraglandular thyroid cancer. CONCLUSIONS: We were able to radiolabel the rhTSH superagonist analogue TR1401 with (99m)Tc efficiently with retention of in vitro and in vivo binding capacity to TSHR. The relative role of such novel radiopharmaceutical versus (131)I scanning of thyroid cancer will require future histopathologic and clinical studies, but it may open new perspectives for presurgical staging of thyroid cancer, and diagnosis of radioiodine negative local relapses and/or distant metastases.

Carbohydrate-mediated polyethylene glycol conjugation of TSH improves its pharmacological properties.

Thyrogen (thyrotropin alfa for injection), recombinant human TSH (rhTSH), has been successfully used to enhance diagnostic radioiodine scanning and thyroglobulin testing in the follow-up of patients with thyroid cancer and as an adjunctive treatment for radioiodine thyroid remnant ablation. However, the short half-life of rhTSH in the circulation requires a multidose regimen. We developed novel sialic acid-mediated and galactose-mediated conjugation chemistries for targeting polyethylene glycol (PEG) to the three N-linked glycosylation sites on the protein, to prolong plasma half-life by eliminating kidney filtration and potential carbohydrate-mediated clearance. Conjugates of different PEG sizes and copy numbers were screened for reaction yield, TSH receptor binding, and murine phamacokinetics/pharmacodynamics studies. The best performing of these products, a 40-kDa mono-PEGylated sialic acid-mediated conjugate, exhibited a 3.5-fold longer duration of action than rhTSH in rats, as a 5-fold lower affinity was more than compensated by a 23-fold extension of circulation half-life. Biochemical characterization confirmed conjugation through the sialic acids. Correlation of PEG distribution on the three N-linked glycosylation sites and the PEG effect on receptor binding supported the previously reported structure-function relationship of rhTSH glycosylation. This long-acting rhTSH has the potential to significantly improve patient convenience and provider flexibility while reducing potential side effects associated with a sudden elevation of serum TSH.

Effects of recombinant human thyroid-stimulating hormone superagonists on thyroidal uptake of 18F-fluorodeoxyglucose and radioiodide.

BACKGROUND: Superagonist analogs of human thyroid-stimulating hormone (hTSH) may stimulate the uptake of (131)I-iodide and (18)F-fluorodeoxyglucose ((18)F-FDG) in thyroid carcinomas to a greater degree than hTSH. We herein report the potency and efficacy of two hTSH analogs, TR1401 and TR1402, to stimulate radioiodide and (18)F-FDG uptake in FRTL-5 cells and compared the effects of hTSH and TR1401 on radioiodide uptake in the thyroid in vivo in mice. METHODS: The effects of hTSH analogs on intracellular levels of cAMP, uptake of (131)I-iodide, and (18)F-FDG were studied in FRTL-5 cells to determine the stimulatory potency and efficacy of the compounds by calculating half-maximum effective concentration (EC(50)) values and maximal stimulatory effects (E(max)). Biodistribution studies (n = 96) and positron emission tomography/computed tomography imaging studies (single animals) on thyroid (125)I/(124)I-iodide uptake were performed with T3-suppressed CD-1 mice in a dose-dependent manner (3, 10, and 30 µg/animal). RESULTS: The EC(50) values of TR1401 and TR1402 demonstrated a 90-fold or 800-fold higher potency for their capacity to increase intracellular cAMP levels in comparison with hTSH (p < 0.05). Similar results were demonstrated for the stimulation of (18)F-FDG uptake. Bovine TSH, TR1401, and TR1402 were 85%-490% more potent to increase iodide uptake than hTSH (p < 0.05). TR1402 was 30% more efficacious to stimulate iodide uptake than hTSH. The agonist-induced increase in radiotracer uptake was paralleled by increases in NIS and GLUT-1 expression. Ex vivo biodistribution studies showed an increased iodide uptake in the thyroid of TR1401-treated mice at the low dose of 3 µg/animal in comparison with hTSH-treated mice (n = 16, p < 0.05). Positron emission tomography/computed tomography imaging studies confirmed the increased thyroidal iodide uptake in TR1401-treated mice in vivo. CONCLUSIONS: TR1401 and TR1402 have considerably higher potency than hTSH to stimulate thyroidal iodide and (18)F-FDG uptake in vitro. Moreover, in vivo studies indicated that at low but not higher doses, TR1401 induced an enhanced ability for the thyroid to concentrate iodide compared with hTSH. These properties makes TR1401 and TR1402 interesting candidates for use in humans to enhance uptake of radioiodine and (18)F-FDG by metastases and recurrences of thyroid carcinoma.

The superagonistic activity of bovine thyroid-stimulating hormone (TSH) and the human TR1401 TSH analog is determined by specific amino acids in the hinge region of the human TSH receptor.

Bovine TSH (bTSH) has a higher affinity to the human TSHR (hTSHR) and a higher signaling activity than human TSH (hTSH). The molecular reasons for these phenomena are unknown. Distinct negatively charged residues (Glu297, Glu303, and Asp382) in the hinge region of the hTSHR are known to be important for bTSH binding and signaling. To investigate the potential relevance of these positions for differences between bTSH and hTSH in the interaction to the hTSHR, we determined bTSH- and hTSH-mediated cAMP production of several substitutions at these three hinge residues. To examine specific variations of hTSH, we also investigated the superagonistic hTSH analog TR1401 (TR1401), whose sequence differs from hTSH by four additional positively charged amino acids that are also present in bTSH. To characterize possible interactions between the acidic hTSHR positions Glu297, Glu303, or Asp382 and the additional basic residues of TR1401, we investigated TR1401 binding and signaling properties. Our data reveal increased cAMP signaling of the hTSHR using TR1401 and bTSH compared with hTSH. Whereas Asp382 seems to be important for bTSH- and TR1401-mediated but not for hTSH-mediated signaling, the substitution E297K exhibits a decreased signaling for all three TSH variants. Interestingly, bTSH and TR1401 showed only a slightly different binding pattern. These observations imply that specific residues of the hinge region are mediators of the superagonistic activity of bTSH and TR1401 in contrast to hTSH. Moreover, the simultaneous localization of binding components in the glycoprotein hormone molecule and the receptor hinge region permits important reevaluation of interacting hormone receptor domains.

The synthetic TRH analogue taltirelin exerts modality-specific antinociceptive effects via distinct descending monoaminergic systems.

BACKGROUND AND PURPOSE: Exogenously administered thyrotropin-releasing hormone (TRH) is known to exert potent but short-acting centrally-mediated antinociceptive effects. We sought to investigate the mechanisms underlying these effects using the synthetic TRH analogue taltirelin, focusing on the descending monoaminergic systems in mice. EXPERIMENTAL APPROACH: The mice received systemic or local injections of taltirelin combined with either central noradrenaline (NA) or 5-hydroxytryptamine (5-HT) depletion by 6-hydroxydopamine (6-OHDA) or DL-p-chlorophenylalanine (PCPA), respectively, or blockade of their receptors. The degree of antinociception was determined using the tail flick and tail pressure tests. KEY RESULTS: Subcutaneously (s.c.) administered taltirelin exhibited dose-dependent antinociceptive effects in the tail flick and tail pressure tests. These effects appeared to be primarily supraspinally mediated, since intracerebroventricularly (i.c.v.) but not intrathecally (i.t.) injected taltirelin generated similar effects. Depletion of central NA abolished only the analgesic effect of taltirelin (s.c. and i.c.v.) on mechanical nociception. By contrast, depletion of central 5-HT abolished only its analgesic effect on thermal nociception. Intraperitoneal (i.p.) and i.t. injection of the alpha2-adrenoceptor antagonist yohimbine respectively reduced the analgesic effect of taltirelin (s.c. and i.c.v.) on mechanical nociception. By contrast, the 5-HT1A receptor antagonist WAY-100635 (i.p. and i.t.) reduced the effect of taltirelin (s.c. and i.c.v.) on thermal nociception. Neither the 5-HT2 receptor antagonist ketanserin nor the opioid receptor antagonist naloxone altered the antinociceptive effect of taltirelin. CONCLUSIONS AND IMPLICATIONS: These findings suggest that taltirelin activates the descending noradrenergic and serotonergic pain inhibitory systems, respectively, to exert its analgesic effects on mechanical and thermal nociception.

Bioengineering of human thyrotropin superactive analogs by site-directed "lysine-scanning" mutagenesis. Cooperative effects between peripheral loops.

We have previously engineered the first superactive analogs of human thyrotropin (hTSH) by using a novel design strategy. In this study, we have applied homology comparisons focusing on the alphaL3 loop of the common alpha-subunit of human glycoprotein hormones. Seven highly variable amino acid residues were identified, and charge-scanning mutagenesis revealed three previously unrecognized modification permissive domains and four gain-of-function lysine substitutions. Such gain-of-function mutations were hormone- and receptor-specific and dependent on location and basic charge. Cooperativity of individual substitutions was established in double and triple lysine mutants. In combinations of the most potent alphaL3 loop analog with two previously characterized loop analogs, a higher degree of cooperativity for the alphaL3 loop analog compared with both the alphaL1 loop analog and the hTSH-betaL3 loop analog was observed. We demonstrated that spatially distinct regions of the common alpha-subunit contribute differentially to the interaction of hTSH with its receptor and that combinations of two modified loops on the same and on opposite sides of the hTSH molecule display similar increases in in vitro biopotency. In addition, combination of all three superactive loops showed cooperativity in receptor binding and activation resulting in the most potent hTSH superactive analog described to date.

Engineering human glycoprotein hormone superactive analogues.

We report the generation of superactive analogues of human glycoprotein hormones, with potential applications in thyroid and reproductive disorders. Current biological and structural data were used to rationalize mutagenesis. The 11-20 region in the alpha-subunit with a cluster of lysine residues forms a previously unrecognized domain critical for receptor binding and signal transduction, as well as an important motif in the evolution of glycoprotein hormone activities. The gradual elimination of basic residues in the alpha-subunit coincided with the evolutionary divergence of the hominids from the Old World monkeys. By selective reconstitution of certain critical residues present in homologous nonhuman hormones we have developed human thyroid stimulating hormone and chorionic gonadotropin analogues with substantial increases in receptor binding affinity and bioactivity, thus providing a paradigm for the design of novel therapeutic protein analogues.

Synthetic analogs of the carboxyl-terminus of beta-thyrotropin: the importance of basic amino acids in receptor binding activity.

Previously, using a synthetic peptide strategy, we determined that four distinct regions of human beta-thyrotropin (beta TSH) were responsible for interaction of TSH with the TSH receptor. The most potent of these four regions was the carboxyl-terminus of the subunit, represented by the peptide sequence beta 101-112, which inhibited binding of radiolabeled beta TSH to receptor in radioreceptor assay with an IC50 of approximately 100 microM. In the current studies, we systematically substituted the native amino acids in region beta 101-112 with alanine, and we have determined which residues within this span are important to the binding activity of TSH to its receptor. Substitution of Lys101, Asn103, Tyr104, Cys105, Lys107, and Lys110 with alanine each caused a significant fall in activity as compared to the native sequence, whereas substitution at the remaining positions had little or no effect. Because three of these residues are positively charged at physiologic pH, we hypothesized that this charge may be important to the binding activity of the sequence. We modified the charge characteristics of the region by synthesizing two series of analogs in which the residues identified in the alanine substitution studies were substituted with Arg, D-Lys, and D-Arg at each position. In addition, a series of analogs containing basic residues, either added to or substituted for nonbasic residues in the sequence beta 101-112, was synthesized. Substitution of Arg, D-Lys, and D-Arg for Lys101, Lys107, and Lys110 had little effect on activity; however, inclusion of additional basic residues in the beta 101-112 sequence significantly enhanced the inhibitory activity of the region.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of carbohydrate in thyrotropin action assessed by a novel approach using enzymatic deglycosylation.

Deglycosylation of thyrotropin (TSH) and gonadotropins by chemical methods virtually abolishes their biological activity without impairing receptor binding activity. Recent reports have suggested that enzymatic deglycosylation, using endoglycosidases caused a much smaller decrease, if any, in the potency of the glycoprotein hormones without altering the Vmax. However, in these studies complete removal of the carbohydrate chains from the hormones was not unequivocally documented. We have prepared completely deglycosylated bovine TSH by endoglycosidase F digestion of its subunits, which were more readily deglycosylated than the intact hormone. The deglycosylated subunits were separated from any incompletely digested subunits by concanavalin A affinity chromatography. Carbohydrate compositional analysis, using a highly sensitive pulsed amperometric detection method coupled to ion-exchange high performance liquid chromatography, was performed to ascertain the complete removal of the glycan moieties from the subunits. The deglycosylated subunits thus prepared were recombined to obtain deglycosylated TSH dimer. Receptor binding activity of bTSH was minimally affected by the carbohydrate removal. In an in vitro bioassay using stimulation of cyclic AMP production in FRTL-5 cells, deglycosylated bTSH showed reduced activity with a potency 5-10-fold lower than that of control, although the Vmax remained unaltered. In contrast, the deglycosylated bTSH showed a reduction in Vmax, when assayed for its adenylyl cyclase stimulating activity in bovine thyroid membranes. Previous reports using chemical methods have apparently overestimated the effects of deglycosylation, probably because of altered protein conformation, while those using endoglycosidases have apparently underestimated these effects, probably because of incomplete deglycosylation.

Inhibition of thyrotropin binding to receptor by synthetic human thyrotropin beta peptides.

In order to study the structure and function relationships of the thyrotropin (TSH)-specific beta-subunit, we produced 11 synthetic overlapping peptides containing the entire 112-amino acid sequence of human beta TSH and tested them for activity in TSH radioreceptor assay using both human and porcine thyroid membranes. Synthetic peptides representing four regions of the beta-subunit demonstrated the ability to inhibit binding of 125I-bovine TSH to crude thyroid membranes. The peptide representing the -COOH terminus of the subunit (beta 101-112) possessed highest binding activity, inhibiting binding of labeled TSH with an EC50 of 80 microM. The remaining active peptides were: beta 71-85 (104 microM), beta 31-45 (186 microM), beta 41-55 (242 microM), and beta 1-15 (331 microM). Specificity of the binding activity was shown by the inability of the peptides representing the remainder of the subunit to inhibit binding of label and by the inability of any of the peptides to inhibit binding of 125I-epidermal growth factor to the same thyroid membranes. The low affinity of the peptides as compared with native hormone is in agreement with previous studies of synthetic alpha-subunit peptides and, further, suggests that the interaction of beta TSH with receptor is multifaceted, requiring cooperative binding of these sites for the observed high affinity of the whole hormone. These studies are in agreement with previous predictions of active regions by chemical modification but add two regions to the list, showing the utility of the synthetic peptide strategy in the study of peptide hormone structure-activity relationships.

A sensitive thyrotropin (TSH) bioassay based on iodide uptake in rat FRTL-5 thyroid cells: comparison with the adenosine 3',5'-monophosphate response to human serum TSH and enzymatically deglycosylated bovine and human TSH.

A sensitive in vitro assay based on the uptake of 125I by the FRTL-5 rat thyroid cell line has been applied to the measurement of TSH bioactivity from different sources. In this bioassay various human pituitary reference preparations showed similar potencies; the limit of detection and the half-maximal response were 1.60 +/- 0.1 (+/- SE) and 9.70 +/- 0.40 microU/0.5 ml, respectively; however, when compared to pituitary TSH from other species, human TSH was 29- and 10-fold less biopotent than bovine and rat TSH, respectively. The iodide uptake response to TSH was inhibited by the presence of human serum in a dose-dependent fashion, but pretreatment of serum with 10% polyethylene glycol restored TSH activity. The iodide uptake response was compared to the stimulation of cAMP production in the measurement of serum TSH bioactivity from human samples after immunoaffinity purification. In the cAMP production bioassay, immunoaffinity-purified serum TSH showed increased bioactivity in patients with primary hypothyroidism and TSH-secreting pituitary tumor compared to that in normal subjects, while in the iodide uptake bioassay minimal differences were detected among the different groups. To investigate further structure-function relationships of TSH in FRTL-5 cells we studied the effects of deglycosylated purified pituitary bovine and human TSH on both bioassays. Using two new enzymes, peptide-N-glycosidase and endo-beta-N-acetylglucosaminidase F, we removed one carbohydrate chain from TSH alpha and all three chains from TSH, respectively. In the iodide uptake bioassay both enzymes induced a 2-fold decrease in TSH biopotency, while in the cAMP production bioassay this decrease was only present with peptide-N-glycosidase-treated TSH. In summary, 1) the iodide uptake bioassay system in FRTL-5 cells represents a valid and sensitive method for the measurement of TSH bioactivity from different sources and can be applied to serum samples with elevated TSH concentrations by simple pretreatment with polyethylene glycol without immunoaffinity purification; 2) the enzymatic removal of one carbohydrate chain from both bovine and human TSH significantly decrease their biological activity, assessed as cAMP production and iodide uptake response in FRTL-5 cells, while the removal of three carbohydrate chains induces a significant decrease only in the iodide uptake bioassay: and 3) the disparate results for cAMP and iodide uptake in both human samples and deglycosylated pituitary TSH suggest that in addition to cAMP, other second messengers may play a role in TSH action.

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

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

The role of the carbohydrate moiety in thyrotropin action.

The relative binding affinity of deglycosylated human TSH was 6-fold higher than that of native TSH. Although deglycosylated human TSH significantly stimulated adenylate cyclase, it was less effective than the native hormone. When deglycosylated human TSH was added with bovine TSH, however, a dose-dependent antagonism was observed. In particular, submaximal and maximal concentrations of bovine TSH and deglycosylated human TSH resulted in cAMP values much lower than the sum of activities of the individual hormones. The data suggest that although the effects of TSH deglycosylation are not as dramatic as with the gonadotropins, the carbohydrates of TSH appear to be required for maximal activation of adenylate cyclase by the hormone.

The effects of thyrotropin-releasing hormone, metabolites and analogues on locomotor activity in rats.

Thyrotropin-releasing hormone (TRH) has generally been reported to increase locomotor activity in rats; however there are also some negative reports. In order to identify the possible causes for this discrepancy, the effects of intra-cerebroventricular injection of TRH, its metabolites 'acid TRH' (TRH-OH) and His-Pro-diketopiperazine (DKP), and two analogues 3-methyl-His-TRH and RX 77368 (3,3-dimethyl-Pro-TRH), were assessed using photocell activity cages. All compounds were tested in groups of eight rats in the afternoon (1300-1700 h), but in addition TRH and DKP were tested in two further groups of rats during the morning (0900-1230 h). TRH and DKP failed to induce a significant rise in activity during the morning test period, but TRH did have a significant effect when tested in the afternoon. Both TRH and TRH-OH caused dose dependent increases in locomotor activity, whereas DKP and the two analogues had no effect. This stimulation of activity was shown to be at least partly mediated by dopamine since locomotor enhancement was blocked in a second experiment using the dopamine antagonist alpha-Flupenthixol. The results are discussed in terms of actions on the mesolimbic dopamine system, and the importance of circadian variations within this system to the expression of peptide effects in general.

Thyrotropin-daunomycin conjugate shows receptors-mediated cytotoxicity in cultured thyroid cells.

Daunomycin (DM), a potent chemotherapeutic agent, was linked to bovine thyrotropin (TSH) directly (TSH-DM) and indirectly (TSH-alpha-lactalbumin-DM) by covalent cross-linking methods. The activity of these hormone-daunomycin conjugates was tested in vitro on the rabbit thyroid cells in primary culture. Both TSH-DM and TSH-alpha-lactalbumin-DM conjugates inhibited [3H]leucine incorporation and cell proliferation, while free TSH did not. Their inhibitory action was more potent than that of free daunomycin. The inhibition appeared to be specific to the thyroid cells and diminished in the presence of a large amount of free TSH, suggesting that the cytotoxic action of these TSH-DM conjugates was a receptor-mediated phenomenon.

Dansylated thyrotropin as a probe of hormone-receptor interactions.

A strongly fluorescent 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of bovine thyrotropin has been prepared. The dye-conjugated hormone is bioactive and shares, essentially unchanged, the membrane binding and adenylate cyclase stimulatory activities of the native hormone. Binding of 125I-labeled dansyl-thyrotropin to thyroid plasma membranes is sensitive to inhibition by gangliosides and, as is the case for the binding of 125I-thyrotropin, galactosyl-N-acetylgalactosaminyl[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GDIb) is the most potent binding inhibitor. Gangliosides interact with dansyl-thyrotropin, causing a large increase of the quantum yield and a 5- to 10-nm blue shift of the emission maximum of the hormone-bound naphthalene chromophore; gangliosides cause no change in the fluorescent properties of the free dye. The fluorescence enhancement caused by gangliosides can be specifically reversed by unlabeled thyrotropin. The effect of gangliosides on dansyl-thyrotropin fluorescence is strongly salt-dependent; salts cannot, however, reverse the formation of the dansyl-thyrotropin.ganglioside complex once it has formed. The salt data suggest that the association of the ganglioside with dansyl-thyrotropin is dominated by electrostatic interactions, but that salt-independent, short range interactions, most likely hydrophobic, dominate the dissociation of the dansyl-thyrotropin-ganglioside adduct. Sucrose gradient centrifugation, ultracentrifugation, and fluorescence polarization data indicate that the gangliosides are micellar in nature under the conditions of these experiments. Acid titration of dansyl-thyrotropin causes a marked quenching of dansyl fluorescence which in part reflects dissociation of the hormone into its constituent alpha and beta subunits. In the presence of GDIb, but not N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GDIa), pH-dependent quenching and subunit dissociation are essentially eliminated. Circular dichroism results and fluorescence polarization studies support the interpretation that the ganglioside interaction causes a conformational change in the thyrotropin molecule. The acid titration data together with differences in the ability of gangliosides to influence the tyrosine fluorescence of the thyrotropin molecule indicate that different gangliosides induce different conformational perturbations in the thyrotropin molecule.