Activation of the cAMP Pathway by the TSH Receptor Involves Switching of the Ectodomain from a Tethered Inverse Agonist to an Agonist

Vlaeminck-Guillem, Virginie; Ho, Su-Chin; Rodien, Patrice; Vassart, Gilbert; Costagliola, Sabine

doi:10.1210/mend.16.4.0816

Cited by 161 publications

(140 citation statements)

References 54 publications

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“…We used 2 approaches to show that compound 2 binds to the serpentine domain of TSHR in contrast to the natural ligand TSH, which binds primarily to the large N-terminal ectodomain (31). We used a TSHR mutant that does not contain the N-terminal ectodomain and that was shown previously not to be activated by TSH (24) and showed that it was activated by compound 2. We also used a 3D homology model of TSHR (14,23), which was based on the x-ray crystallographic structure of opsin (25), to predict the binding pocket for the small-molecule agonist and predict the amino acid residues within the pocket that interact with compound 2.…”

Section: Discussionmentioning

confidence: 99%

“…To show that compound 2 binds to the serpentine domain, we tested the activation of a TSHR mutant in which the large amino-terminal ectodomain responsible for TSH binding was deleted. This truncated TSHR (KFLR) was shown by Vlaeminck-Guillem et al (24) not to bind or be activated by TSH. Compound 2 exhibited 12% lower efficacy and lower potency (EC 50 of 1.7 M) at KFLR than TSHR (Fig.…”

Section: Compound 2 Binds To the Transmembrane Helical Bundle Of Tshrmentioning

confidence: 96%

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

Neumann¹,

Huang

Titus

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

113

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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.7TMR ͉ G protein-coupled receptor ͉ low-molecular-weight ligands ͉ radioiodide uptake ͉ TSH receptor S mall-molecule agonists and antagonists for 7-transmembrane-spanning receptors (7TMRs; G protein-coupled receptors) make up approximately 40% of the drugs in clinical use (1). The natural ligands for the 7TMRs for which drugs have been discovered are themselves primarily small molecules, such as norepinephrine, adenosine, and acetylcholine. Smallmolecule ligands for the subclass of 7TMRs for which the natural ligands are 30-kDa heterodimeric glycoprotein hormones have recently begun to be described, but none have been approved for use in humans. For example, small-molecule agonists (2-5) and an antagonist (6) for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR), and small-molecule agonists (7-9) and antagonists (10-12) for the FSH receptor (FSHR) have been reported. The thyrotropin (thyroid-stimulating hormone, TSH) receptor (TSHR) is the third member of the glycoprotein hormone receptor subfamily. We recently reported smallmolecule agonists (13) and an antagonist (14) for human TSHR; however, the agonists were of low affinity and were shown to activate TSHRs only in a model cell system in which human TSHRs were ectopically over-expressed.The physiologic role of TSHRs in the hypothalamic-pituitarythyroid axis is well known. TSH, which is produced in the thyrotrophs of the anterior pituitary gland, is secreted into the circulation in response to TSH-releasing hormone stimulation and in turn stimulates the function of the thyroid follicular cells (i.e., thyrocytes) leading, in particular, to increases in size ...

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Section: Discussionmentioning

confidence: 99%

Section: Compound 2 Binds To the Transmembrane Helical Bundle Of Tshrmentioning

confidence: 96%

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

Neumann¹,

Huang

Titus

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

113

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“…Mutations were introduced in the hTSHr by site-directed mutagenesis as described previously (32). The primers are available upon request.…”

Section: Construction Of Tshr Mutantsmentioning

confidence: 99%

An Activation Switch in the Rhodopsin Family of G Protein-coupled Receptors

Urizar

Claeysen

Deupí

et al. 2005

Journal of Biological Chemistry

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106

100

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We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche؉ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of ϳ14 and ϳ10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of ϳ13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.Genome sequencing projects have identified the G proteincoupled receptor (GPCR) 1 family as one of the largest class of proteins with more than 800 human sequences. These sequences have been classified into five main families, glutamate, rhodopsin, adhesion, frizzled/taste2, and secretin (1). GPCRs are involved in passing chemical signals across the cell membrane. The incoming signal may arrive in the form of neurotransmitters, peptides, divalent cations, proteases, hormones, and sensory stimuli such as photons and gustatory or odorant molecules (2, 3). Atomic level details of a three-dimensional structure of a GPCR are only known for the inactive form of rhodopsin, the light photoreceptor protein of rod cells (4, 5). Rhodopsin is formed by an extracellular N terminus, seven ␣-helices, which cross the cellular membrane (TM 1-TM 7) connected by hydrophilic loops, and a cytoplasmic C terminus containing an ␣-helix (HX 8) parallel to the cell membrane. The overall structure of the helical bundle seems common in the rhodopsin-like GPCR family because of the large number of conserved sequence patterns in these 7 TM segments (6): GN in TM 1, (N/S)LxxxD in TM 2, (D/E)RY in TM 3, CWxP in TM 6, and NPxxY(x) 5,6 F in TM 7 and HX 8, among others. Activation of GPCRs is commonly discussed in terms of the extended ternary complex model, which proposes an equilibrium between inactive and active states (7). Recent advances in the ␤ 2 -adrenergic receptor have shown that ag...

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“…Whereas direct interaction between the agonist and different regions of the TMD takes place for the majority of GPCRs, a favoured current model holds that GpHr activation involves switching of the ECD from an inverse agonist into a tethered agonist of the TMD, on binding of the hormone 3,15 . This model provides a rational explanation to the observation that the TSHr can be activated by binding of ligands with little, if any sequence identity (TSH, thyrostimulin, autoantibodies) as well as by point mutation in a specific residue of the ECD 3,16,17 .…”

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confidence: 99%

Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers

et al. 2012

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Glycoprotein hormone receptors show strong negative cooperativity. As a consequence, at physiological hormone concentrations, a single agonist binds to a receptor dimer. Here we present evidence that constitutively active receptors lose cooperative allosteric regulation in direct relation with their basal activity. The most constitutive mutants lost nearly all cooperativity and showed an increase of initial tracer binding, reflecting the ability of each protomer to bind with equal affinity. Allosteric interaction between the protomers takes place at the transmembrane domain. The allosteric message resulting from hormone binding to the ectodomain of one protomer travels 'downward' to its transmembrane domain, before affecting the transmembrane domain of the other protomer. This results in transmission of an 'upward' message lowering the binding affinity of the ectodomain of the second protomer. our results demonstrate a direct relation between the conformational changes associated with activation of the transmembrane domain and the allosteric behaviour of glycoprotein hormone receptors dimers.

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Activation of the cAMP Pathway by the TSH Receptor Involves Switching of the Ectodomain from a Tethered Inverse Agonist to an Agonist

Cited by 161 publications

References 54 publications

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

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

An Activation Switch in the Rhodopsin Family of G Protein-coupled Receptors

Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers

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