Hormone- and antibody-mediated activation of the thyrotropin receptor.

Thyroid-stimulating hormone (TSH), through activation of its G-protein-coupled thyrotropin receptor (TSHR), controls the synthesis of thyroid hormone-an essential metabolic hormone1-3. Aberrant signalling of TSHR by autoantibodies causes Graves' disease (hyperthyroidism) and hypothyroidism, both of which affect millions of patients worldwide4. Here we report the active structures of TSHR with TSH and the activating autoantibody M225, both bound to the allosteric agonist ML-1096, as well as an inactivated TSHR structure with the inhibitory antibody K1-707. Both TSH and M22 push the extracellular domain (ECD) of TSHR into an upright active conformation. By contrast, K1-70 blocks TSH binding and cannot push the ECD into the upright conformation. Comparisons of the active and inactivated structures of TSHR with those of the luteinizing hormone/choriogonadotropin receptor (LHCGR) reveal a universal activation mechanism of glycoprotein hormone receptors, in which a conserved ten-residue fragment (P10) from the hinge C-terminal loop mediates ECD interactions with the TSHR transmembrane domain8. One notable feature is that there are more than 15 cholesterols surrounding TSHR, supporting its preferential location in lipid rafts9. These structures also highlight a similar ECD-push mechanism for TSH and autoantibody M22 to activate TSHR, therefore providing the molecular basis for Graves' disease.

Autoantibody mimicry of hormone action at the thyrotropin receptor.

Thyroid hormones are vital in metabolism, growth and development1. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR)2. In patients with Graves' disease, autoantibodies that activate the TSHR pathologically increase thyroid hormone activity3. How autoantibodies mimic thyrotropin function remains unclear. Here we determined cryo-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. Thyrotropin selects an upright orientation of the extracellular domain owing to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody from a patient with Graves' disease selects a similar upright orientation of the extracellular domain. Reorientation of the extracellular domain transduces a conformational change in the seven-transmembrane-segment domain via a conserved hinge domain, a tethered peptide agonist and a phospholipid that binds within the seven-transmembrane-segment domain. Rotation of the TSHR extracellular domain relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to other G-protein-coupled receptors with large extracellular domains.

De novo triiodothyronine formation from thyrocytes activated by thyroid-stimulating hormone.

The thyroid gland secretes primarily tetraiodothyronine (T4), and some triiodothyronine (T3). Under normal physiological circumstances, only one-fifth of circulating T3 is directly released by the thyroid, but in states of hyperactivation of thyroid-stimulating hormone receptors (TSHRs), patients develop a syndrome of relative T3 toxicosis. Thyroidal T4 production results from iodination of thyroglobulin (TG) at residues Tyr5 and Tyr130, whereas thyroidal T3 production may originate in several different ways. In this study, the data demonstrate that within the carboxyl-terminal portion of mouse TG, T3 is formed de novo independently of deiodination from T4 We found that upon iodination in vitro, de novo T3 formation in TG was decreased in mice lacking TSHRs. Conversely, de novo T3 that can be formed upon iodination of TG secreted from PCCL3 (rat thyrocyte) cells was augmented from cells previously exposed to increased TSH, a TSHR agonist, a cAMP analog, or a TSHR-stimulating antibody. We present data suggesting that TSH-stimulated TG phosphorylation contributes to enhanced de novo T3 formation. These effects were reversed within a few days after removal of the hyperstimulating conditions. Indeed, direct exposure of PCCL3 cells to human serum from two patients with Graves' disease, but not control sera, led to secretion of TG with an increased intrinsic ability to form T3 upon in vitro iodination. Furthermore, TG secreted from human thyrocyte cultures hyperstimulated with TSH also showed an increased intrinsic ability to form T3 Our data support the hypothesis that TG processing in the secretory pathway of TSHR-hyperstimulated thyrocytes alters the structure of the iodination substrate in a way that enhances de novo T3 formation, contributing to the relative T3 toxicosis of Graves' disease.

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.

Fourth ventricular thyrotropin induces satiety and increases body temperature in rats.

Besides its well-known action to stimulate thyroid hormone release, thyrotropin mRNA is expressed within the brain, and thyrotropin and its receptor have been shown to be present in brain areas that control feeding and gastrointestinal function. Here, the hypothesis that thyrotropin acts on receptors in the hindbrain to alter food intake and/or gastric function was tested. Fourth ventricular injections of thyrotropin (0.06, 0.60, and 6.00 µg) were given to rats with chronic intracerebroventricular cannulas aimed at the fourth ventricle. Thyrotropin produced an acute reduction of sucrose intake (30 min). The highest dose of thyrotropin caused inhibition of overnight solid food intake (22 h). In contrast, subcutaneous administration of corresponding thyrotropin doses had no effect on nutrient intake. The highest effective dose of fourth ventricular thyrotropin (6 µg) did not produce a conditioned flavor avoidance in a standardized two-bottle test, nor did it affect water intake or gastric emptying of glucose. Thyrotropin injected in the fourth ventricle produced a small but significant increase in rectal temperature and lowered plasma levels of tri-iodothyronin but did not affect plasma levels of thyroxine. In addition, there was a tendency toward a reduction in blood glucose 2 h after fourth ventricular thyrotropin injection ( P = 0.056). In conclusion, fourth ventricular thyrotropin specifically inhibits food intake, increases core temperature, and lowers plasma levels of tri-iodothyronin but does not affect gastromotor function.

Thyroid-stimulating hormone stimulation downregulates autophagy and promotes apoptosis in chondrocytes.

Subclinical hypothyroidism (SCH) patients have normal thyroid hormone levels but increased thyroid stimulating hormone (TSH) level in serum. It has been reported that high TSH is related to abnormal skeletal development in mice with hypothyroidism. However, the cellular mechanism is not fully understood. In the present study, we aim to investigate the direct effects of TSH stimulation on chondrocytes, and the putative role of autophagy in this process. By using EdU incorporation assay and flow cytometry for mitochondrial membrane potential assay, we demonstrated deceased proliferation and promoted apoptosis in TSH stimulated primary mouse chondrocytes. And the balance of Bcl-2 and BAX expression on protein level was broken. More interestingly, the expression of autophagic markers Beclin-1 and LC3II was reduced in TSH stimulated chondrocytes, accompanied by less autophagosomes and accumulated p62 protein, indicating an impaired autophagic flux. More interestingly, mTOR was upregulated and AMPK activity was decreased in TSH stimulated PMCs, suggesting that mTOR/AMPK pathway is get involved in the regulation of TSH on autophagy in PMCs. Collectively, we found an increased apoptosis and suppressed autophagy in TSH stimulated primary chondrocytes, which is meaningful in understanding the effects of increased TSH level on articular cartilage and the role of autophagy in this process, and thus provide a potential novel therapeutic target in related cartilage damages.

Mechanisms of Action of TSHR Autoantibodies.

The availability of human monoclonal antibodies (MAbs) to the TSHR has enabled major advances in our understanding of how TSHR autoantibodies interact with the receptor. These advances include determination of the crystal structures of the TSHR LRD in complex with a stimulating autoantibody (M22) and with a blocking type autoantibody (K1-70). The high affinity of MAbs for the TSHR makes them particularly suitable for use as ligands in assays for patient serum TSHR autoantibodies. Also, M22 and K1-70 are effective at low concentrations in vivo as TSHR agonists and antagonists respectively. K1-70 has important potential in the treatment of the hyperthyroidism of Graves' disease and Graves' ophthalmopathy. Small molecule TSHR antagonists described to date do not appear to have the potency and/or specificity shown by K1-70. New models of the TSHR ECD in complex with various ligands have been built. These models suggest that initial binding of TSH to the TSHR causes a conformational change in the hormone. This opens a positively charged pocket in receptor-bound TSH which attracts the negatively charged sulphated tyrosine 385 on the hinge region of the receptor. The ensuing movement of the receptor's hinge region may then cause activation. Similar activation mechanisms seem to take place in the case of FSH and the FSHR and LH and the LHR. However, stimulating TSHR autoantibodies do not appear to activate the TSHR in the same way as TSH.

[New achievements in the development and study of the mechanisms of action of the low molecular weight agonists of receptors of the thyroid-stimulating and the luteinizing hormones].

Pituitary glycoprotein hormones, luteinizing (LH) and thyroid-stimulating (TSH), exert their regulatory effects on cells through the G protein-coupled receptors, specifically binding to their extracellular domain. There is an alternative way of activation of LH and TSH receptors, when low molecular weight organic molecules bind to an allosteric site of the receptors which is localized within their transmembrane channel. Low molecular weight agonists have many advantages over glycoprotein hormones, among them a high efficiency not only in the case of the parenteral but also in the oral administration, low immunogenicity, chemical stability, and a low cost. Unlike pituitary glycoprotein hormones with the agonistic activity, low molecular weight compounds may be either agonists or inverse agonists and neutral antagonists. Recently it was shown that low molecular weight agonists of LH receptor are able to stimulate its mutant forms by restoring the processing of receptor in a cell, and by increasing its sensitivity to LH, which is important for the treatment of reproductive dysfunctions caused by mutations in the LH receptor. This review summarizes the recent achievements that are linked with the development of low molecular weight regulators of TSH and LH receptors and the study of their mechanisms of action. It also presents the author' data concerning the creation of new low molecular weight agonists of LH receptor based on the thienopyrimidine structure, which are effective both in vitro, and in vivo in different ways of administration.

[Peptide 612-627 of thyrotropin receptor and its modified derivatives as the regulators of adenylyl cyclase in the rat thyroid gland].

The regulation of the specific activity of the thyroid gland is carried by thyroid-stimulating hormone (TSH) through TSH receptor (TSHR). This receptor is coupled to different types of G-proteins, including the G(s)-proteins, through which TSH stimulates the enzyme adenylyl cyclase (AC). As the application of TSH in medicine is limited, the development of selective regulators of TSHR with agonistic and antagonistic activity is carried out. One of the approaches to their creation is to develop the peptides corresponding to functionally important regions of TSHR which are located in its intracellular loops (ICL) and are involved in the binding and activation of G-proteins. We have synthesized peptide corresponding to the C-terminal region 612-627 of the third ICL of TSHR and its derivatives modified by palmitic acid residue (at the N- or the C-terminus) or by polylysine dendrimer (at the N-terminus), and studied their effect on the basal and TSH-stimulated AC activity in the membrane fraction isolated from the rat thyroid. The most active was peptide 612-627-K(Pal)A modified by palmitate at the C-terminus, where in TSHR the hydrophobic transmembrane region is located. At the micromolar concentrations the peptide increased AC activity and reduced the AC stimulating effect of TSH. The action of the 612-627-K(Pal)A has been directed onto TSHR homologous to it, as indicated by the following facts: 1) the inhibition of G(s)-protein, the downstream component of AC system, by treating the membranes with cholera toxin led to the blocking of peptide AC effect, 2) this effect was not detected in the tissues where no TSHR, 3) the peptide did not significantly affect the AC stimulating effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer are far behind the 612-627-K(Pal)A in their ability to activate AC in the thyroid, while the peptide modified by palmitate at the N-terminus was inactive. At the same time, the peptide modified by dendrimer was comparable to the 612-627-K(Pal)A in the ability to inhibit the AC effect of TSH, but, although to a lesser extent that it decreased the AC effects of other hormones, demonstrating the low receptor specificity. Thus, these data point to the high efficiency of peptide 612-627-K(Pal)A, as a regulator of TSHR, and the prospects of creating the drugs based on it to control the thyroid functions in pathology.

A novel bioassay for anti-thyrotrophin receptor autoantibodies detects both thyroid-blocking and stimulating activity.

Autoantibodies to the thyrotrophin (TSH) receptor (anti-TSHR) are unique, in that they are involved directly in the pathophysiology of certain autoimmune thyroid diseases (AITD). Thyroid-stimulating antibodies (TSAb) act as agonists that activate the thyroid gland and cause Graves' disease. Other anti-TSHR antibodies block TSH and can cause hypothyroidism. Thyroid-blocking antibodies (TBAb) have not been studied as extensively as TSAb. We developed a TBAb bioassay based on a cell line that expresses a chimeric TSHR. The 50% inhibitory concentration of the chimeric Chinese hamster ovary (CHO)-Luc cells was more than five-fold lower compared with the wild-type CHO-Luc cells. We tested the performance of this bioassay using a thyroid-blocking monoclonal antibody K1-70, established an assay cut-off and detected TBAb in 15 of 50 (30%) patients with AITD. Interestingly, the assay detects both TSAb and TBAb and measures the net activity of a mixture of both types of antibodies. There was a high correlation (R(2) 0·9, P < 0·0001) between the results of the TSAb assay and the negative percentage inhibition of the TBAb assay. The TBAb bioassay was approximately 20-fold more sensitive than a commercially available TSHR binding assay (TRAb). In contrast to TRAb, sera with high levels of TBAb activity were able to be diluted several hundred-fold and still exhibit blocking activity above the cut-off level. Thus, this TBAb bioassay provides a useful tool for measuring the activity of anti-TSHR antibodies and may help clinicians to characterize the diverse clinical presentations of patients with AITD.

How one TSH receptor antibody induces thyrocyte proliferation while another induces apoptosis.

Thyroid stimulating hormone (TSH) activates two major G-protein arms, Gsα and Gq leading to initiation of down-stream signaling cascades for survival, proliferation and production of thyroid hormones. Antibodies to the TSH receptor (TSHR-Abs), found in patients with Graves' disease, may have stimulating, blocking, or neutral actions on the thyroid cell. We have shown previously that such TSHR-Abs are distinct signaling imprints after binding to the TSHR and that such events can have variable functional consequences for the cell. In particular, there is a great contrast between stimulating (S) TSHR-Abs, which induce thyroid hormone synthesis and secretion as well as thyroid cell proliferation, compared to so called "neutral" (N) TSHR-Abs which may induce thyroid cell apoptosis via reactive oxygen species (ROS) generation. In the present study, using a rat thyrocyte (FRTL-5) ex vivo model system, our hypothesis was that while N-TSHR-Abs can induce apoptosis via activation of mitochondrial ROS (mROS), the S-TSHR-Abs are able to stimulate cell survival and avoid apoptosis by actively suppressing mROS. Using fluorescent microscopy, fluorometry, live cell imaging, immunohistochemistry and immunoblot assays, we have observed that S-TSHR-Abs do indeed suppress mROS and cellular stress and this suppression is exerted via activation of the PKA/CREB and AKT/mTOR/S6K signaling cascades. Activation of these signaling cascades, with the suppression of mROS, initiated cell proliferation. In sharp contrast, a failure to activate these signaling cascades with increased activation of mROS induced by N-TSHR-Abs resulted in thyroid cell apoptosis. Our current findings indicated that signaling diversity induced by different TSHR-Abs regulated thyroid cell fate. While S-TSHR-Abs may rescue cells from apoptosis and induce thyrocyte proliferation, N-TSHR-Abs aggravate the local inflammatory infiltrate within the thyroid gland, or in the retro-orbit, by inducing cellular apoptosis; a phenomenon known to activate innate and by-stander immune-reactivity via DNA release from the apoptotic cells.

Small-molecule agonists for the thyrotropin receptor stimulate thyroid function in human thyrocytes and mice.

Seven-transmembrane-spanning receptors (7TMRs) are prominent drug targets. However, small-molecule ligands for 7-transmembrane-spanning receptors for which the natural ligands are large, heterodimeric glycoprotein hormones, like thyroid-stimulating hormone (TSH; thyrotropin), have only recently been reported, and none are approved for human use. We have used quantitative high-throughput screening to identify a small-molecule TSH receptor (TSHR) agonist that was modified to produce a second agonist with increased potency. We show that these agonists are highly selective for human TSHR versus other glycoprotein hormone receptors and interact with the receptor's serpentine domain. A binding pocket within the transmembrane domain was defined by docking into a TSHR homology model and was supported by site-directed mutagenesis. In primary cultures of human thyrocytes, both TSH and the agonists increase mRNA levels for thyroglobulin, thyroperoxidase, sodium iodide symporter, and deiodinase type 2, and deiodinase type 2 enzyme activity. Moreover, oral administration of the agonist stimulated thyroid function in mice, resulting in increased serum thyroxine and thyroidal radioiodide uptake. Thus, we discovered a small molecule that activates human TSHR in vitro, is orally active in mice, and could be a lead for development of drugs to use in place of recombinant human TSH in patients with thyroid cancer.

Thyrotropin receptor stimulates internalization-independent persistent phosphoinositide signaling.

The thyrotropin [thyroid-stimulating hormone (TSH)] receptor (TSHR) is known to acutely and persistently stimulate cAMP signaling and at higher TSH concentrations to acutely stimulate phosphoinositide signaling. We measured persistent signaling by stimulating TSHR-expressing human embryonic kidney-EM293 cells with TSH and measuring cAMP or inositol monophosphate (IP1) production, a measure of phosphoinositide signaling, 60 min or longer after TSH removal. In contrast to persistent cAMP production, persistent IP1 production increased progressively when TSH exposure was increased from 1 to 30 min, whereas the rates of decay of persistent signaling were similar. A small-molecule agonist and a thyroid-stimulating antibody also caused persistent IP1 and cAMP signaling. A small-molecule inverse agonist and a neutral antagonist inhibited TSH-stimulated persistent IP1 production, whereas the inverse agonist but not the neutral antagonist inhibited persistent cAMP production. As with persistent cAMP production, persistent IP1 production was not affected when TSHR internalization was inhibited or enhanced. Moreover, Alexa546-TSH-activated TSHR internalization was not accompanied by Gα(q) coupling protein internalization. Thus, transient exposure to high concentrations of TSH causes persistent phosphoinositide and cAMP signaling that is not dependent on internalization. To our knowledge, this is the first demonstration of persistent activation by any G protein-coupled receptor (GPCR) via the Gα(q) pathway and of two G protein-mediated pathways by any GPCR.

Identification of key amino acid residues in a thyrotropin receptor monoclonal antibody epitope provides insight into its inverse agonist and antagonist properties.

CS-17 is a murine monoclonal antibody to the human TSH receptor (TSHR) with both inverse agonist and antagonist properties. Thus, in the absence of ligand, CS-17 reduces constitutive TSHR cAMP generation and also competes for TSH binding to the receptor. The present data indicate that for both of these functions, the monovalent CS-17 Fab (50 kDa) behaves identically to the intact, divalent IgG molecule (150 kDa). The surprising observation that CS-17 competes for TSH binding to the human but not porcine TSHR enabled identification of a number of amino acids in its epitope. Replacement of only three human TSHR residues (Y195, Q235, and S243) with the homologous porcine TSHR residues totally abolishes CS-17 binding as detected by flow cytometry. TSH binding is unaffected. Of these residues, Y195 is most important, with Q235 and S243 contributing to CS-17 binding to a much lesser degree. The functional effects of CS-17 IgG and Fab on constitutive cAMP generation by porcinized human TSHR confirm the CS-17 binding data. The location of TSHR amino acid residues Y195, Q235, and S243 deduced from the crystal structure of the FSH receptor leucine-rich domain provides valuable insight into the CS-17 and TSH binding sites. Whereas hormone ligands bind primarily to the concave surface of the leucine-rich domains, a major portion of the CS-17 epitope lies on the opposite convex surface with a minor component in close proximity to known TSH binding residues.

Quantitative high-throughput screening using a live-cell cAMP assay identifies small-molecule agonists of the TSH receptor.

The thyroid-stimulating hormone (TSH; thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of 7-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates the growth and function of thyroid follicular cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small-molecule agonists of the TSHR are available. To screen for novel TSHR agonists, the authors miniaturized a commercially available cell-based cyclic adenosine 3',5' monophosphate (cAMP) assay into a 1536-well plate format. This assay uses an HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal homogeneous time-resolved fluorescence cAMP-based assay. Forty-nine compounds in several structural classes have been confirmed as the small-molecule TSHR agonists that will serve as a starting point for chemical optimization and studies of thyroid physiology in health and disease.

Suppression of thyrotropin receptor constitutive activity by a monoclonal antibody with inverse agonist activity.

TSH binding to the TSH receptor (TSHR) induces thyrocyte growth and proliferation primarily by activating the adenylyl cyclase signaling pathway. Relative to the other glycoprotein hormone receptors, the TSHR has considerable ligand-independent (constitutive) activity. We describe a TSHR monoclonal antibody (CS-17) with the previously unrecognized property of being an inverse agonist for TSHR constitutive activity. This property is retained, even when constitutive activity is extremely high consequent to diverse TSHR extracellular region mutations. A similar effect on an activating mutation at the base of the sixth transmembrane helix (not accessible to direct CS-17 contact) indicates that CS-17 is acting allosterically. Administered to mice in vivo, CS-17 reduces serum T(4) levels. The CS-17 epitope is conformational and a significant portion lies in the C-terminal region of the TSHR leucine-rich domain (residues 260-289). By interacting with the large TSHR extracellular domain, CS-17 is, to our knowledge, the first antibody reported to be an inverse agonist for a member of the G protein receptor superfamily. After humanization of its murine constant region, CS-17 has the potential to be an adjunctive therapeutic agent in athyreotic patients with residual well-differentiated thyroid carcinoma as well as pending definitive treatment in some selected hyperthyroidism states.

Structural determinants for G-protein activation and specificity in the third intracellular loop of the thyroid-stimulating hormone receptor.

The selectivity of G-protein recognition is determined by the intracellular loops (ICLs) of seven-transmembrane-spanning receptors. In a previous study, we have shown that the N-terminal and central portions of ICL2 from F525 to D530 participate in dual Galphas-/Galphaq-protein activation by the thyroid-stimulating hormone receptor (TSHR). ICL3 is another major determinant for G-protein activation. Therefore, the aim of our study was to identify important amino acids within ICL3 of the TSHR to gain insight in more detail about its specific function for Galphas- and Galphaq-protein activation and selectivity. Single-alanine substitutions of residues in the N-terminal, middle, and C-terminal region of ICL3 were generated. N-terminal residues Y605 and V608 and C-terminal positions K618, K621, and I622 were identified as selectively important for Galphaq activation, whereas mutations in the center of ICL3 had no effect on TSHR signaling. Our findings provide evidence for an amino acid pattern in the N- and C-terminal part of ICL3, which is involved in Galphaq-mediated signaling. Furthermore, molecular modeling of interaction of TSHR ICL2 and 3 with Galphaq suggests three potential contact sites: TSHR C-terminal ICL3 with beta5-6 loop of Galphaq, TSHR ICL2 residues I523-R531 with beta2-3 loop and N-terminal helix of Galphaq, and TSHR ICL2/transmembrane helix (TMH) 3+ICL3/TMH6 with C-terminal tail of Galphaq.

Iodinated TG in Thyroid Follicles Regulate TSH/TSHR Signaling for NIS Expression.

Our previous research has suggested that high degree of iodinated thyroglobulin (TG) may inhibit the expression and function of sodium iodide symporter (NIS), but the underlying mechanism remains unclear. In present study, we discuss a newly constructed follicle model in vitro, which was used to simulate the follicular structure of the thyroid and explore the regulatory roles of iodinated TG in the follicular lumen on NIS expression. The results showed that both NIS expression and PKA activity were increased in lowly iodinated TG group, while decreased NIS expression with increased PKC activity was found in highly iodinated TG group. Also, NIS expression was increased in PKA agonist-treated group, while decreased NIS was found in PKC agonist-treated group. Moreover, when the PLC-PKC pathway was blocked by PKC-specific inhibitor, highly iodinated TG significantly promoted the expression of NIS. However, when the cAMP-PKA pathway was blocked by a PKA-specific blocker, highly iodinated TG slightly suppressed NIS expression. TG with a low degree of iodination had the reverse effect on NIS. When the PLC-PKC pathway was blocked, TG with a low degree of iodination slightly promoted NIS expression. However, when the cAMP-PKA pathway was blocked, TG with a low degree of iodination greatly inhibited NIS expression. All these suggested that iodinated TG inhibited the expression of NIS by PLC-PKC pathway and promoted NIS expression via the cAMP-PKA pathway. When highly iodinated TG was present, the PLC-PKC pathway became dominant. In the presence of lowly iodinated TG, the cAMP-PKA became the major pathway.

Evaluation of small-molecule modulators of the luteinizing hormone/choriogonadotropin and thyroid stimulating hormone receptors: structure-activity relationships and selective binding patterns.

The substituted thieno[2,3-d]pyrimidine 3 (Org 41841), a partial agonist for the luteinizing hormone/choriogonadotropin receptor (LHCGR) and the closely related thyroid-stimulating hormone receptor (TSHR), was fundamentally altered, and the resulting analogues were analyzed for their potencies, efficacies, and specificities at LHCGR and TSHR. Chemical modification of the parent compound combined with prior mutagenesis of TSHR provided compelling experimental evidence in support of computational models of 3 binding to TSHR and LHCGR within their transmembrane cores. Biochemical analysis of a specific modification to the chemical structure of 3 provides additional evidence of a H-bond between the ligand and a glutamate residue in transmembrane helix 3, which is conserved in both receptors. Several key interactions were surveyed to determine their respective biochemical roles in terms of both van der Waals dimensions and hydrogen bond capacity and the respective relationship to biological activity.

Emerging strategies for managing differentiated thyroid cancers refractory to radioiodine.

Efficient treatment of radio refractory thyroid cancer is still a major challenge. The recent identification of genetic and epigenetic alterations present in almost all differentiated tumors has revealed novel molecular targets, which can hopefully be exploited to create new treatments for these tumors. This review looks briefly at some of the innovative strategies currently being investigated for the treatment the radioiodine-resistant thyroid cancers.