Mutations in the mouse TSH receptor equivalent to human constitutively activating TSH receptor mutations also cause constitutive activity.

Constitutively activating mutations in the human thyroid-stimulating hormone (TSH) receptor (TSHr) have been identified as the most prevalent molecular cause of non-autoimmune hyperthyroidism. To investigate the feasibility of an animal model for non-autoimmune hyperthyroidism, we introduced two mutations in the mouse TSHr which had previously been identified in the human TSHr. The two human mutations showed strong differences in TSH binding, basal cAMP and IP accumulation. In the human TSHr, the Ile 486 Phe mutation causes a high increase of basal cAMP accumulation and also basal stimulation of the inositol phosphate pathway, whereas the Val 509 Ala mutation results in a low increase of basal cAMP accumulation without affecting IP signaling. RNA was isolated from mouse thyroid tissue and reverse transcribed. A 2.4 kb PCR product from the mouse TSHr was cloned into the pGEM-T vector system. Ile was substituted with Phe at codon 486 and Val with Ala at codon 509. These mutated mouse TSHrs were subcloned in the pSVL expression vector. After transient expression in COS-7 cells, basal and TSH-stimulated cAMP and IP accumulation, cell surface expression and TSH binding were determined and directly compared to the human TSHr. Whereas constitutively activating mutations of the human parathyroid hormone (PTH)/PTH-related peptide receptor showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor, these two mouse TSHr mutations resulted in constitutive activity similar to the homologous mutations in the human TSHr. Therefore, it should be possible to establish a mouse model for non-autoimmune hyperthyroidism by homologous recombination to study the pathogenetic mechanisms of non-autoimmune hyperthyroidism.     

Delineation of the discontinuous-conformational epitope of a monoclonal antibody displaying full in vitro and in vivo thyrotropin activity

An experimental murine model of Graves' disease was used to produce monoclonal antibodies (mAbs) with thyroid stimulating activity. Two of these, IRI-SAb2 and IRI-SAb3, showed particularly high potency (in the low nanomolar range) and efficacy. IRI-SAb2 behaved as a full agonist of the human TSH receptor (TSHr), even when tested in physiological salt concentrations. Both IRI-SAb2 and IRI-SAb3 were displaced from the TSHr by autoantibodies from patients with Graves' disease or harboring thyroid-blocking antibodies, but not from control subjects or patients with Hashimoto thyroiditis. The epitopes of IRI-SAb2 and IRI-SAb3 were precisely mapped, at the amino acid level, to the amino-terminal portion of the concave portion of the horseshoe structure of TSHr ectodomain. They overlap closely with each other and, surprisingly, with the epitope of a mAb with blocking activity. When injected iv in mice, both mAbs caused biological and histological signs of hyperthyroidism. Unexpectedly, they also triggered an inflammatory response in the thyroid glands. Delineation of the conformational epitopes of these stimulating antibodies opens the way to the identification of the molecular mechanisms implicated in the activation of the TSHr.     

Thyroid disruptor 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) prevents internalization of TSH receptor

The thyroid-stimulating hormone (TSH) receptor (TSHr) was made specifically fluorescent by insertion of a tetracysteine motif (TSHr-FlAsH) into the C-terminal end and transiently transfected into COS-7 and HeLa cells. The observation that TSH administration caused the intracellular level of cAMP to increase in both TSHr-FlAsH-transfected cell types indicated that the FlAsH binding motif did not alter normal TSHr functioning. When transfected into HeLa cells and stimulated with TSH, the TSHr-FlAsH receptor exhibited a pronounced perinuclear labelling pattern, whereas labelling remained on the cell surface following pre-incubation with 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). Chinese hamster ovary (CHO)-TSHr cells probed with anti-TSHr antibodies were fluorescent mainly in the proximity of the plasma membrane, with fluorescence being primarily restricted to a juxta-nuclear position when exposed to 10 mU/ml TSH for 1 or 5 min. However, in the presence of DDT, the anti-TSHr fluorescence maintained a peripheral location along the cell plasma membrane, even if CHO-TSHr cells were stimulated with TSH for 1 and 5 min. To verify that DDT acted specifically on the TSHr, CHO cells transfected with the A₂a receptor were used as controls. Following a 1-min stimulation with 5’-(N-ethyl-carboxamido)-adenosine, A₂a receptors were gradually internalized regardless of the presence of DDT in the culture medium. Finally, immunoelectron microscopy of CHO-TSHr cells showed that a 1-min exposure to TSH sufficed to displace anti-TSHr antibodies tagged with 10-nm gold particles into coated pits and vesicles but that their superficial location was retained along the plasma membrane in the presence of DDT.     

In vitro and in vivo regulation of thyrotropin receptor mRNA levels in dog and human thyroid cells.

Regulation of thyrotropin (TSH) receptor (TSHr) mRNA accumulation as compared with two other thyroid differentiation markers (thyroglobulin and thyroperoxidase (TPO] has been investigated by Northern blot. In dogs in vivo, chronic stimulation of the thyroid TSHr mRNA although it increased the levels of thyroglobulin and TPO mRNA. In dogs treated with thyroxin, the quiescent thyroids expressed normal levels of TSHr and TPO mRNA but depressed levels of thyroglobulin mRNA. In primary cultures of dog thyrocytes, dedifferentiation of the cells by treatment with epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate led to decreased TSHr mRNA levels and nearly abolished thyroglobulin and TPO gene expression. However, TSHr mRNA was always present, compatible with the fact that these cells, when treated by TSH, reexpress differentiation. Treatment of the cells with TSH or forskolin transiently increased the TSHr mRNA level after 20 h, an effect inhibited by cycloheximide. This up-regulation was confirmed at the protein level: forskolin-treated cells showed an enhanced cAMP response to TSH and an increased binding of labeled TSH to their membranes. Long term TSH treatment led to a slight down-regulation of TSHr mRNA in dog thyrocytes, but in human thyroid cells no marked down-regulation was observed.     

Augmentative effect of polyethylene glycol, polyvinyl alcohol and dextran on thyroid-stimulating antibody stimulated cAMP production in FRTL-5 cells and CHO cells expressing human TSH receptor

Previously we reported the augmentative effect of nonionic hydrophilic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA) and dextran on thyroid-stimulating antibody (TSAb) activity in porcine thyroid cell assays. We examined whether a similar phenomenon occurs in FRTL-5 thyroid cells and CHO cells expressing the human (h) TSH receptor (CHO-hTSHR cells). As with porcine thyroid cells, PEG 22.5% precipitated crude IgG from serum of patients with Graves' disease, significantly increased cAMP production as compared with PEG 12.5% precipitated crude IgG in both FRTL-5 cells and CHO-hTSHR cells. PEG 5% augmented purified-TSAb-IgG-stimulated cAMP production in both cell assays. TSAb activities and positivity by the direct assay using whole serum (0.05 ml) in the presence of 5% PEG in untreated Graves' patients were significantly increased as compared with the absence of 5% PEG. The augmentative effects of PVA 10% or dextran T-70 10% on TSAb-IgG-stimulated cAMP production were also observed in both cell assays. PVA 10% did not augment TSH-stimulated cAMP production in spite of weak augmentation by dextran 10% in both cell assays. Lack of the augmentative effects of PEG 5%, PVA 10% and dextran 10% on cAMP produced by GTPgammaS, forskolin and pituitary adenylate cyclase activating polypeptide was observed in both cell assays. The augmentative effects of these polymers in both cell assays similar to porcine thyroid cells suggest that there is no apparent species specificity among human, porcine and rat thyroid cells as far as TSH receptor linked cAMP production in cell membranes existed.     

The thyrotropin (thyroid-stimulating hormone) receptor is expressed on murine dendritic cells and on a subset of CD45RBhigh lymph node T cells: functional role for thyroid-stimulating hormone during immune activation

Thyroid-stimulating hormone (TSH), a central neuroendocrine mediator of the hypothalamus-pituitary-thyroid axis, has been shown to affect various aspects of immunological development and function. To gain a better understanding of TSH involvement within the mammalian immune system, the expression and distribution of the TSH receptor (TSHr) has been studied by immunoprecipitation and by flow cytometric analyses. Using highly enriched populations of B cells, T cells, and dendritic cells, trace amounts of TSHr were precipitated from B cells and T cells, whereas high levels of TSHr were precipitated from the dendritic cell fraction. Flow cytometric analyses of TSHr expression on splenic and lymph node T cells revealed a major difference between those tissues in that only 2-3% of splenic T cells were TSHr+, whereas 10-20% of CD4+8- and CD4-8+ lymph node T cells expressed the TSHr, which was exclusively associated with CD45RB(high) cells and was not expressed during or after activation. The TSHr was not present on cells of the immune system during fetal or neonatal life. However, recombinant TSHbeta was found to significantly enhance the phagocytic activity of dendritic cells from adult animals and to selectively augment IL-1beta and IL-12 cytokine responses of dendritic cells following phagocytic activation. These findings identify a novel immune-endocrine bridge associated with professional APCs and naive T cells.     

Differential regulation of thyrotropin receptor and thyroglobulin mRNA accumulation at the cellular level: an in situ hybridization study.

Regulation of TSH receptor (TSHr) mRNA accumulation has been investigated in canine thyrocytes in primary culture by in situ hybridization experiments; the effects of the mitogenic thyrotropin (TSH), epidermal growth factor (EGF), and phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) have been compared. Apart from their mitogenic action, TSH enhances, while EGF and phorbol ester inhibit, the expression of differentiation. The TSHr gene was transcribed in almost all the cells cultured in control conditions (serum free medium supplemented with insulin). Addition of TSH slightly upregulated (twofold) the expression (mRNA) of the TSHr gene. This positive effect was maintained for 20 and 44 h of treatment. EGF and TPA reduced transiently the TSHr mRNA accumulation but did not suppress it. In these different conditions, the TSHr mRNA was homogeneously distributed within the cell population. This contrasted strongly with the effects of TSH, EGF, and TPA on the expression of the thyroglobulin gene, a prominent marker of thyroid cell differentiation: in this case, the regulation was much tighter (high range of stimulation by TSH, strong inhibition by EGF, and suppression of Tg gene expression by TPA) and displayed a great variability of the level of individual cellular response. The fact that the TSHr gene was little modulated and remained expressed regardless of the treatment may reflect the physiological role of the receptor which is the main connection of the thyrocyte to the regulation network.     

Proper targeting and activity of a nonfunctioning thyroid-stimulating hormone receptor (TSHr) combining an inactivating and activating TSHr mutation in one receptor.

Activating mutations of the thyroid-stimulating hormone receptor (TSHr) have been identified as a cause of toxic adenomas. Germline-inactivating TSHr mutations have been described as a cause of congenital hypothyroidism. The effects of combining activating and inactivating mutations within a single receptor was studied. The double mutant T477I/P639S contained an activating TSHr mutation (P639S) together with an inactivating one (T477I). The other one (I486M/P639S) contained two activating mutations. Constructs were expressed in COS-7 cells and basal and TSH-stimulated cyclic AMP (cAMP) accumulation and inositol phosphate (IP) production were determined. The expression at the cell surface was studied both with binding and fluorescence-activated cell scanning analysis. Our results show that the effect of combining the two activating mutations is an increase in the constitutive activity only for the cAMP pathway and not for the IP pathway suggesting that different mutations result in receptor conformations with different relative abilities to couple to Gs-alpha or Gq-alpha. Surprisingly the double mutant containing the T477I behaves as an activating receptor with constitutive activity both for the cAMP and IP pathways. These data show that an inactive form of the TSHr which is trapped inside a cell after transfection is able to gain the membrane surface when combined with an activated form of the receptor.     

Differential regulation of thyrotropin receptor and thyroglobulin mRNA accumulation at the cellular level: An in situ hybridization study

Regulation of TSH receptor (TSHr) mRNA accumulation has been investigated in canine thyrocytes in primary culture by in situ hybridization experiments; the effects of the mitogenic thyrotropin (TSH), epidermal growth factor (EGF), and phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) have been compared. Apart from their mitogenic action, TSH enhances, while EGF and phorbol ester inhibit, the expression of differentiation. The TSHr gene was transcribed in almost all the cells cultured in control conditions (serum free medium supplemented with insulin). Addition of TSH slightly upregulated (twofold) the expression (mRNA) of the TSHr gene. This positive effect was maintained for 20 and 44 h of treatment. EGF and TPA reduced transiently the TSHr mRNA accumulation but did not suppress it. In these different conditions, the TSHr mRNA was homogeneously distributed within the cell population. This contrasted strongly with the effects of TSH, EGF, and TPA on the expression of the thyroglobulin gene, a prominent marker of thyroid cell differentiation: in this case, the regulation was much tighter (high range of stimulation by TSH, strong inhibition by EGF, and suppression of Tg gene expression by TPA) and displayed a great variability of the level of individual cellular response. The fact that the TSHr gene was little modulated and remained expressed regardless of the treatment may reflect the physiological role of the receptor which is the main connection of the thyrocyte to the regulation network.     

Lysine 183 and glutamic acid 157 of the TSH receptor: two interacting residues with a key role in determining specificity toward TSH and human CG

A naturally occurring mutation in the ectodomain of the TSH receptor (TSHr), K183R, has been described recently in a familial case of gestational hyperthyroidism. Hyperthyroidism was explained by the widening of the specificity of the mutant receptor toward human CG (hCG). In the present study, we attempted to understand in molecular terms the structure-phenotype relationships of this mutant in light of the available structural model of TSHr ectodomain established on the template of the atomic structure of the porcine ribonuclease inhibitor. To this aim, we studied by site-directed mutagenesis and functional assays in transfected COS cells the effects of substituting amino acids with different physicochemical properties for lysine 183. Unexpectedly, all TSHr mutants displayed widening of their specificity toward hCG. Molecular dynamics simulations suggested that the gain of function would be secondary to the release of a nearby glutamate residue (E157) from a salt bridge with K183. This hypothesis was supported by further site-directed mutagenesis experiments showing that the presence of an acidic residue in position 157, or in its vicinity, was required to observe the increase in sensitivity to hCG (an acidic residue in position 183 can partially fulfill the role of a free acidic residue in position 157 when tested on the background of a E157A mutant). Our results suggest also that additional natural mutations (especially K183M, N, or Q) in position 183 of TSHr are expected to be found in gestational hyperthyroidism.     

The Identification of Novel Protein-Protein Interactions in Liver that Affect Glucagon Receptor Activity

Glucagon regulates glucose homeostasis by controlling glycogenolysis and gluconeogenesis in the liver. Exaggerated and dysregulated glucagon secretion can exacerbate hyperglycemia contributing to type 2 diabetes (T2D). Thus, it is important to understand how glucagon receptor (GCGR) activity and signaling is controlled in hepatocytes. To better understand this, we sought to identify proteins that interact with the GCGR to affect ligand-dependent receptor activation. A Flag-tagged human GCGR was recombinantly expressed in Chinese hamster ovary (CHO) cells, and GCGR complexes were isolated by affinity purification (AP). Complexes were then analyzed by mass spectrometry (MS), and protein-GCGR interactions were validated by co-immunoprecipitation (Co-IP) and Western blot. This was followed by studies in primary hepatocytes to assess the effects of each interactor on glucagon-dependent glucose production and intracellular cAMP accumulation, and then in immortalized CHO and liver cell lines to further examine cell signaling. Thirty-three unique interactors were identified from the AP-MS screening of GCGR expressing CHO cells in both glucagon liganded and unliganded states. These studies revealed a particularly robust interaction between GCGR and 5 proteins, further validated by Co-IP, Western blot and qPCR. Overexpression of selected interactors in mouse hepatocytes indicated that two interactors, LDLR and TMED2, significantly enhanced glucagon-stimulated glucose production, while YWHAB inhibited glucose production. This was mirrored with glucagon-stimulated cAMP production, with LDLR and TMED2 enhancing and YWHAB inhibiting cAMP accumulation. To further link these interactors to glucose production, key gluconeogenic genes were assessed. Both LDLR and TMED2 stimulated while YWHAB inhibited PEPCK and G6Pase gene expression. In the present study, we have probed the GCGR interactome and found three novel GCGR interactors that control glucagon-stimulated glucose production by modulating cAMP accumulation and genes that control gluconeogenesis. These interactors may be useful targets to control glucose homeostasis in T2D.     

Stable expression of the human TSH receptor in CHO cells and characterization of differentially expressing clones

The human thyrotropin receptor cDNA was transfected in CHO cells and individual clones were isolated. They were tested for their response to thyrotropin, forskolin and antibodies from a patient with high levels of thyroid stimulating antibodies. Several clones were characterized extensively with respect to membrane binding of labeled thyrotropin, cAMP accumulation in response to thyrotropin and kinetics of cAMP production. Data for three representative clones are presented. Receptor number as assessed by membrane binding of labeled thyrotropin, and cAMP production, measured in a thyrotropin response bioassay, are correlated. The Kd value for the human thyrotropin receptor expressed in CHO was estimated to be 50 pM.     

Biological effect of anti-fucosyl GM1 ganglioside antibody on cyclic adenosine 3',5'-monophosphate production in FRTL5 rat thyroid cells

Addition of specific anti-fucosyl GM1 antibody raised in a rabbit caused dose-dependent inhibition of endogenous and thyrotropin (TSH)- or thyroid stimulating antibody-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in cultured FRTL5 rat thyroid cells. Further, the antibody inhibited the cAMP increase induced by prostaglandin E1 and forskolin. However, anti-fucosyl GM1 antibody did not affect the binding of [125I]bovine TSH to solubilized porcine thyroid TSH receptor or to FRTL5 cells. In conclusion, fucosyl GM1 is one of the specific membrane components of thyrocytes and appears to be involved in adenylate cyclase stimulation or cAMP generation. Further, the biological effects of the ganglioside do not seem to be mediated by the TSH receptor, suggesting a post receptor mechanism.     

A new structural model for the thyrotropin (TSH) receptor, as determined by covalent cross-linking of TSH to the recombinant receptor in intact cells: evidence for a single polypeptide chain.

The most widely held model for the human TSH receptor is of holoreceptor of 80 kDa with two subunits of approximately 50 and 30 kDa linked by disulfide bridges, with the former subunit containing the major hormone-binding site. We reexamined this model by covalently cross-linking radiolabeled TSH to the recombinant human TSH receptor stably expressed in Chinese hamster ovary (CHO) cells. When cross-linking was performed after the preparation of CHO membranes, analysis of hormone-receptor complexes under reducing and nonreducing conditions provided results supporting the two-subunit TSH receptor model. In contrast, however, cross-linking of TSH to the TSH receptor in intact CHO cells before membrane preparation revealed, even under reducing conditions, an approximately 100-kDa receptor as well as an approximately 54-kDa hormone-binding subunit. The approximately 100-kDa holoreceptor size is consistent with the size of the TSH receptor, as predicted from its derived amino acid sequence. The proportions of the approximately 100-kDa TSH receptor and the 54-kDa fragment varied in different experiments, suggesting the occurrence of proteolytic cleavage. Cross-linking of radiolabeled TSH to intact cells expressing a mutant TSH receptor (TSHR-D1) lacking amino acids 317-366 localized the proteolytic cleavage site to just up-stream of amino acid residue 317. In summary, the present data obtained by cross-linking TSH to recombinant human TSH receptors in intact cells provides evidence that the receptor exists in vivo as an approximately 100-kDa glycoprotein with a single polypeptide chain with intramolecular disulfide bridges.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of separate determinants on the thyrotropin receptor reactive with Graves' thyroid-stimulating antibodies and with thyroid-stimulating blocking antibodies in idiopathic myxedema: these determinants have no homologous sequence on gonadotropin receptors.

Deletions, substitutions, or mutations of the rat TSH receptor extracellular domain between residues 20 and 107 (all residue numbers are determined by counting from the methionine start site) have been made by site-directed mutagenesis of receptor cDNA. After transfection in Cos-7 cells, constructs were evaluated for their ability to bind [125I]TSH or respond to TSH and thyroid-stimulating antibodies (TSAbs) from Graves' patients in assays measuring cAMP levels of the transfected cells. Assay results were compared to results from Cos-7 cells transfected with wild-type receptor constructs or vector alone. We identify threonine-40 as a TSAb-specific site whose mutation to asparagine, but not alanine, reduces TSAb activity 10-fold, but only minimally affects TSH-increased cAMP levels. We show that thyroid-stimulating blocking antibodies (TSBAbs), which block TSH or TSAb activity and are found in hypothyroid patients with idiopathic myxedema, continue to inhibit TSH-stimulated cAMP levels when threonine-40 is mutated to asparagine or alanine, suggesting that TSBAbs interact with different TSH receptor epitopes than the TSAb autoantibodies in Graves' patients. This is confirmed by the demonstration that these TSBAbs interact with high affinity TSH-binding sites previously identified at tyrosine-385 or at residues 295-306 of the extracellular domain of the TSH receptor. This is evidenced by a loss in the ability of TSBAbs to inhibit TSAb activity when these residues are mutated or deleted, respectively. Since the TSAb and TSBAb epitopes are in regions of the extracellular domain of the TSH receptor that have no homology in gonadotropin receptors, these data explain at least in part the organ-specific nature of TSH receptor autoantibodies in autoimmune thyroid disease. Data are additionally provided which indicate that residues 30-37 and 42-45, which flank the TSAb epitope at threonine-40, appear to be ligand interaction sites more important for high affinity TSH binding than for the ability of TSH to increase cAMP levels and that cysteine-41 is critical for TSH receptor conformation and expression on the surface of the cell. Thus, despite unchanged maximal values for TSH-increased cAMP levels, substitution of residues 42-45 or deletion of residues 30-37 results in receptors, which, by comparison to wild-type constructs, exhibit significantly worsened Kd values for TSH binding than EC50 values for TSH- or TSAb-increased cAMP activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

A conserved Asn in transmembrane helix 7 is an on/off switch in the activation of the thyrotropin receptor

The thyrotropin (TSH) receptor is an interesting model to study G protein-coupled receptor activation as many point mutations can significantly increase its basal activity. Here, we identified a molecular interaction between Asp(633) in transmembrane helix 6 (TM6) and Asn(674) in TM7 of the TSHr that is crucial to maintain the inactive state through conformational constraint of the Asn. We show that these residues are perfectly conserved in the glycohormone receptor family, except in one case, where they are exchanged, suggesting a direct interaction. Molecular modeling of the TSHr, based on the high resolution structure of rhodopsin, strongly favors this hypothesis. Our approach combining site-directed mutagenesis with molecular modeling shows that mutations disrupting this interaction, like the D633A mutation in TM6, lead to high constitutive activation. The strongly activating N674D (TM7) mutation, which in our modeling breaks the TM6-TM7 link, is reverted to wild type-like behavior by an additional D633N mutation (TM6), which would restore this link. Moreover, we show that the Asn of TM7 (conserved in most G protein-coupled receptors) is mandatory for ligand-induced cAMP accumulation, suggesting an active role of this residue in activation. In the TSHr, the conformation of this Asn residue of TM7 would be constrained, in the inactive state, by its Asp partner in TM6.