Sandbox Reserved 1779

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This Sandbox is Reserved from February 27 through August 31, 2023 for use in the course CH462 Biochemistry II taught by R. Jeremy Johnson at the Butler University, Indianapolis, USA. This reservation includes Sandbox Reserved 1765 through Sandbox Reserved 1795.

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The thyrotropin receptor with TSH bound. TSHR is shown in dark blue with TSH (light green) bound. The receptor is bound to its G-protein with the various subunits of the G-protein shown in pink, red, turquoise, and yellow. PDB:7UTZ

Drag the structure with the mouse to rotate

1 Introduction
2 Structure
3 TSHR Agonists and Antagonists
- 3.1 M22 Agonist
- 3.2 CS-17 Inverse Agonist

Introduction

Figure 1. TSHR with TSH bound. The extracellular and transmembrane domains of the GPCR are shown in green, the hinge region in cyan, and thyrotropin bound in magenta. PDB:7UTZ

Thyroid hormones exercise essential functions related to activity of thyroid cells as well as metabolic processes and oxygen consumption^[1]. Misregulation of thyroid hormone levels is the cause of many disorders related to hypo- or hyperthyroidism. Thus, understanding the signaling of synthesis and release of these hormones will have applications in treating thyroid hormone disorders^[1]. The initiation of the synthesis and release of these hormones is caused by the glycoprotein, thyroid stimulating hormone (TSH), which is released by the anterior pituitary gland^[1]. The release of TSH from the anterior pituitary is regulated by thyroid-releasing hormone (TRH) which is released by the hypothalamus. When stimulated by TRH, the anterior pituitary releases TSH. When stimulated by TSH, the thyroid gland will produce and release the the thyroid hormones T4 and T3. T3 is the "active form" of the hormone, however it accounts for only 20% of the thyroid hormone that is released after stimulus by TSH. The T4 that predominates in release from the thyroid will be converted to T3 in the bloodstream. High levels of T3 and T4 can negatively regulate the release of TSH from the anterior pituitary, constituting a negative feedback loop ^[2]. The thyrotropin receptor is a G-protein coupled receptor on the surface the thyroid gland cells. TSHR is responsible for binding TSH and transduces signal to initiate synthesis and release of thyroid hormones. In addition to TSH, autoantibodies may also bind to TSHR causing inhibition or activation of its desired function. (Figure 1)^[3]^[4]

Structure

Active and Inactive Form

Figure 2: Inactive form of the thyrotropin receptor shown in blue (PDB: 7T9M). Active form of the thyrotropin receptor shown in green (PDB: 7T9I).

The TSHR protein exists in dynamic equilibrium between two states: active and inactive (Figure 2). will bind and keep the active state in the up position as a result of clashes between bound TSH and the cell membrane.^[5]. cause this clash on the .

Structural Overview

The thyrotropin receptor has an extracellular domain (ECD) that is composed of a as well as a hinge region. The links the ECD to the seven transmembrane helices , which span from the ECD to the intracellular loops ^[6]. Thyrotropin binding causes a conformational change in the ECD that is transduced through the transmembrane helices. In the active state, the ECD is in the "up" position, while in the inactive state, the ECD is in the "down" state, closer to the cell membrane. A "push-pull" mechanism is proposed for the ECD's conformational change between active and inactive states. In the "push" model, TSH binds to the receptor and sterically clashes with the cellular membrane, forcing the ECD up away from the membrane. In the pull model, a short α-helix interacts with TSH to pull the ECD up. The active (up) form of the ECD causes a conformation shift in the TMD which causes differential interactions with a heterotrimeric , initiating intracellular signaling^[3].

Leucine Rich Repeats

The Leucine Rich Repeat Domain (LRRD) is part of the of TSHR and contains . A unique feature of this region is that it is composed entirely of β-pleated sheets. These β-pleated sheets of the LRRD provide a concave binding surface for TSH, including the residues ^[3]. These interact with in the seatbelt region of TSH forming a salt bridge and assisting in binding TSH ^[5]. This interaction is specific to TSH and TSHR. When other agonists or antagonists bind to the receptor, the change in conformation is a result of different residues interacting, as explained later in the page. The LRRD acts as a probe to receive information from the extracellular environment.

Hinge Region and P10 Peptide

The is a scaffold for the attachment of the LRRD to the 7TMD. The hinge region also impacts TSH binding potency and intracellular cyclic adenosine monophosphate (cAMP) levels, mediated by the activation of the GPCR^[7]. The hinge region's interacts with the through disulfides. The p10 peptide is a conserved sequence that spans from the last β sheet of the LRRD to the first transmembrane helix (TM1) and is an intramolecular agonist for conformational shifts in the 7TMD helices^[8]. The disulfides between the LRRD, the hinge helix, and the p10 are critical to TSH signaling as they transduce signal from the ECD through the hinge helix to the p10 peptide. The upward movement of the LRRD, caused by TSH binding, will cause rotation of the hinge helix. The subsequent movement of the p10 peptide leads to movement of the transmembrane helices, which will cause activation of the G-protein. In addition to activation, the hinge region plays an important role in tightly binding TSH. Residues 382-390 of the hinge region adopt a short helix containing two key residues. Y385 from TSHR is buried into a hydrophobic pocket of TSH. D386 from the receptor forms a salt bridge with R386 of the hormone. that assist in the stable binding of TSH to TSHR allow more potent activation of the receptor^[3]. Even with these key functions, the hinge region itself is not absolutely required for receptor activation^[8]. The hinge region functions as a point of attachment to the 7TMD for the LRRD, and its ability to rotate allows for LRRD shifts between up (active state) and down (inactive state) positions. The interactions that the hinge helix makes with the LRRD and p10 act as an important communication medium between the ECD and an intramolecular agonist directly effecting conformational shifts in the 7TMD.

7 Transmembrane Helices

The ECD of TSHR is anchored to the membrane through seven transmembrane helices (7TMD), characteristic of GPCRs. Conformational changes in the 7TMD activate intracellular G-protein signaling^[6]. Once TSH binds, conformational changes to the p10 peptide are transmitted to the 7TMD. Specifically, hinge helix rotation causes the displacement of the p10 peptide that allows the to migrate towards the center of the 7TMD, increasing van Der Waals contacts. Additionally, K660 of TM7 forms a stabilizing with E409 of the p10 region. Hinge helix movement also rearranges Y279 relative to I486 on the neighboring , which links two transmembrane helices and is located extracellularly. Substitution of these residues leads to substantial shifts in the activation of the thyrotropin receptor. Structurally guided mutagenic studies have shown that replacing isoleucine with a more sizeable phenylalanine decreases TSH signaling potency^[8]^[9].The sixth transmembrane helix of TSHR moves outward from the center of the 7TMD to facilitate α-helix 5 of the α-subunit of the G protein (Gα)^[8]^[10]. Gα is activated for intracellular signaling when GDP is exchanged for GTP and dissociates from the γ- and β-subunits of the G-protein (Gγ and Gβ) to bind with other target proteins. Activation of the Gα is caused by conformational shifts in the 7TMD and three intracellular loops which directly interact with the G-protein^[6]. These conformational shifts in transmembrane helices are the mechanism of changing interactions of the G-protein with the receptor.

TSHR Agonists and Antagonists

Chemical agonists are found in many living systems and serve as a way to activate receptors or pathways that are necessary for a wide array of biological processes. Chemical antagonists block or inhibit biological processes. Different types of agonists/antagonists exist within the body including hormones, antibodies, and neurotransmitters. The body naturally produces autoantibodies that can act as agonists and mimic the activating mechanism of the natural hormone leading to disease.^[11].

M22 Agonist

is a monoclonal antibody that is produced by patients with Graves' Disease. In Graves' disease, autoantibodies mimic TSH function and cause thyroid overactivity. ^[11]. Grave's Disease is an autoimmune disease that is a result of hyperthyroidism, where too much TSH is being produced. This disease effects 1 in 100 Americans and especially women or people older than 30 years of age. The M22 autoantibody activates TSHR by causing a membrane clash with the ECD and the cell membrane, keeping the TSHR in the active state by preventing the TSHR from rotating to the inactive state (Figure 3). M22 mimics TSH activation of TSHR, and is a potent activator for intracellular signaling. ^[5] Although M22 binds in a similar manner to TSH, M22 does not interact with the hinge region when bound to TSHR.^[5] These findings show that the hinge region is not necessary for the activation of TSHR, and leads to the discovery of other methods of activation.

Figure 3: Agonist and antagonist drugs for activating or inactivating the TSHR protein. Here the membrane clashes are demonstrated on TSHR with different agonists attached. CS-17 is orange, TSH is purple, and M22 is blue in the figure. The TSHR protein is green and embedded in the protein.

CS-17 Inverse Agonist

is a monoclonal antibody that acts as an inverse agonist for TSHR constitutive activity. ^[12]. An example of a disease caused by inverse agonists is hypothyroidism. The most common cause of hypothyroidism is Hashimoto’s disease. Without enough TSH to bind TSHR, the pathway remains inactive and thus metabolic processes are inhibited in this pathway. CS-17 interacts with the ECD of the TSHR protein on the of the LRRD, suppressing TSHR function by keeping the receptor in the inactive state (Figure 3). Clash of bound CS-17 with the cell membrane locks TSHR in the inactive form. This type of inhibition is uncommon and is a promising mechanism for future drug design and research to combat hypothyroidism.^[12].

References

↑ ^1.0 ^1.1 ^1.2 Yen PM. Physiological and molecular basis of thyroid hormone action. Physiol Rev. 2001 Jul;81(3):1097-142. doi: 10.1152/physrev.2001.81.3.1097. PMID: 11427693.
↑ Pirahanchi Y, Toro F, Jialal I. Physiology, Thyroid Stimulating Hormone. [Updated 2022 May 8]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK499850/
↑ ^3.0 ^3.1 ^3.2 ^3.3 Duan J, Xu P, Luan X, Ji Y, He X, Song N, Yuan Q, Jin Y, Cheng X, Jiang H, Zheng J, Zhang S, Jiang Y, Xu HE. Hormone- and antibody-mediated activation of the thyrotropin receptor. Nature. 2022 Aug 8. pii: 10.1038/s41586-022-05173-3. doi:, 10.1038/s41586-022-05173-3. PMID:35940204 doi:http://dx.doi.org/10.1038/s41586-022-05173-3
↑ Kohn LD, Shimura H, Shimura Y, Hidaka A, Giuliani C, Napolitano G, Ohmori M, Laglia G, Saji M. The thyrotropin receptor. Vitam Horm. 1995;50:287-384. doi: 10.1016/s0083-6729(08)60658-5. PMID: 7709602.
↑ ^5.0 ^5.1 ^5.2 ^5.3 Faust B, Billesbolle CB, Suomivuori CM, Singh I, Zhang K, Hoppe N, Pinto AFM, Diedrich JK, Muftuoglu Y, Szkudlinski MW, Saghatelian A, Dror RO, Cheng Y, Manglik A. Autoantibody mimicry of hormone action at the thyrotropin receptor. Nature. 2022 Aug 8. pii: 10.1038/s41586-022-05159-1. doi:, 10.1038/s41586-022-05159-1. PMID:35940205 doi:http://dx.doi.org/10.1038/s41586-022-05159-1
↑ ^6.0 ^6.1 ^6.2 Kleinau, G., Worth, C. L., Kreuchwig, A., Biebermann, H., Marcinkowski, P., Scheerer, P., & Krause, G. (2017). Structural–functional features of the thyrotropin receptor: A class A G-protein-coupled receptor at work. Frontiers in Endocrinology, 8. https://doi.org/10.3389/fendo.2017.00086
↑ Yumiko Mizutori, Chun-Rong Chen, Sandra M. McLachlan, Basil Rapoport, The Thyrotropin Receptor Hinge Region Is Not Simply a Scaffold for the Leucine-Rich Domain but Contributes to Ligand Binding and Signal Transduction, Molecular Endocrinology, Volume 22, Issue 5, 1 May 2008, Pages 1171–1182, https://doi.org/10.1210/me.2007-0407
↑ ^8.0 ^8.1 ^8.2 ^8.3 Faust, B., Billesbølle, C.B., Suomivuori, CM. et al. Autoantibody mimicry of hormone action at the thyrotropin receptor. Nature 609, 846–853 (2022). https://doi.org/10.1038/s41586-022-
↑ Virginie Vlaeminck-Guillem, Su-Chin Ho, Patrice Rodien, Gilbert Vassart, Sabine Costagliola, Activation of the cAMP Pathway by the TSH Receptor Involves Switching of the Ectodomain from a Tethered Inverse Agonist to an Agonist, Molecular Endocrinology, Volume 16, Issue 4, 1 April 2002, Pages 736–746, https://doi.org/10.1210/mend.16.4.0816
↑ Goricanec, D., Stehle, R., Egloff, P., Grigoriu, S., Plückthun, A., Wagner, G., & Hagn, F. (2016). Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding. Proceedings of the National Academy of Sciences, 113(26). https://doi.org/10.1073/pnas.1604125113
↑ ^11.0 ^11.1 Nunez Miguel R, Sanders J, Chirgadze DY, Furmaniak J, Rees Smith B. Thyroid stimulating autoantibody M22 mimics TSH binding to the TSH receptor leucine rich domain: a comparative structural study of protein-protein interactions. J Mol Endocrinol. 2009 May;42(5):381-95. Epub 2009 Feb 16. PMID:19221175 doi:10.1677/JME-08-0152
↑ ^12.0 ^12.1 Chen, C.-R., McLachlan, S. M., & Rapoport, B. (2007). Suppression of thyrotropin receptor constitutive activity by a monoclonal antibody with inverse agonist activity. Endocrinology, 148(5), 2375–2382. https://doi.org/10.1210/en.2006-1754

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 The thyrotropin receptor has an extracellular domain (ECD) that is composed of a <scene name='95/952709/Lrrd_real/2'>leucine rich repeat domain (LRRD)</scene> as well as a hinge region. The <scene name='95/952709/Hinge_region_real/2'>hinge region</scene> links the ECD to the seven transmembrane helices <scene name='95/952709/7tm_helices/4'>(7TM domain)</scene>, which span from the ECD to the intracellular loops <ref name= "Keinau et al.">Kleinau, G., Worth, C. L., Kreuchwig, A., Biebermann, H., Marcinkowski, P., Scheerer, P., &amp; Krause, G. (2017). Structural–functional features of the thyrotropin receptor: A class A G-protein-coupled receptor at work. Frontiers in Endocrinology, 8. https://doi.org/10.3389/fendo.2017.00086</ref>. Thyrotropin binding causes a conformational change in the ECD that is transduced through the transmembrane helices. In the active state, the ECD is in the "up" position, while in the inactive state, the ECD is in the "down" state, closer to the cell membrane. A "push-pull" mechanism is proposed for the ECD's conformational change between active and inactive states. In the "push" model, TSH binds to the receptor and sterically clashes with the cellular membrane, forcing the ECD up away from the membrane. In the pull model, a short α-helix interacts with TSH to pull the ECD up. The active (up) form of the ECD causes a conformation shift in the TMD which causes differential interactions with a heterotrimeric <scene name='95/952709/G_protein/2'>G-protein</scene>, initiating intracellular signaling<ref name="Duan et al.">PMID:35940204</ref>.
 === Leucine Rich Repeats ===
-The Leucine Rich Repeat Domain (LRRD) is part of the <scene name='95/952708/Tshr_chainr_ecd/1'>ECD</scene> of TSHR and contains <scene name='95/952707/Lrr/5'>10-11 Leucine Rich Repeats</scene>. A unique feature of this region is that it is composed entirely of β-pleated sheets. These β-pleated sheets of the LRRD provide a concave binding surface for TSH, including the residues <scene name='95/952707/Lrr/5'>K209 and K58</scene> <ref name="Duan et al.">PMID: 35940204</ref>. These interact with <scene name='95/952707/Lrr/5'>N91 and E98</scene> in the seatbelt region of TSH forming a salt bridge and assisting in binding TSH <ref name="Faust">PMID: 35940205</ref>. This interaction is specific to TSH and TSHR. When other agonists or antagonists bind to the receptor, the change in conformation is a result of different residues interacting, as explained later in the page. The LRRD acts as a probe to receive information from the extracellular environment.
+The Leucine Rich Repeat Domain (LRRD) is part of the <scene name='95/952708/Tshr_chainr_ecd/1'>ECD</scene> of TSHR and contains <scene name='95/952707/Lrr/5'>10-11 Leucine Rich Repeats</scene>. A unique feature of this region is that it is composed entirely of β-pleated sheets. These β-pleated sheets of the LRRD provide a concave binding surface for TSH, including the residues <scene name='95/952707/Interactions_with_thyrotropin/4'>K209 and K58</scene> <ref name="Duan et al.">PMID: 35940204</ref>. These interact with <scene name='95/952707/Interactions_with_thyrotropin/4'>N91 and E98</scene> in the seatbelt region of TSH forming a salt bridge and assisting in binding TSH <ref name="Faust">PMID: 35940205</ref>. This interaction is specific to TSH and TSHR. When other agonists or antagonists bind to the receptor, the change in conformation is a result of different residues interacting, as explained later in the page. The LRRD acts as a probe to receive information from the extracellular environment.
 ===Hinge Region and P10 Peptide===
 The <scene name='95/952709/Hinge_region_real/5'>hinge region</scene> is a scaffold for the attachment of the LRRD to the 7TMD. The hinge region also impacts TSH binding potency and intracellular cyclic adenosine monophosphate (cAMP) levels, mediated by the activation of the GPCR<ref name="Mizutori et al.">Yumiko Mizutori, Chun-Rong Chen, Sandra M. McLachlan, Basil Rapoport, The Thyrotropin Receptor Hinge Region Is Not Simply a Scaffold for the Leucine-Rich Domain but Contributes to Ligand Binding and Signal Transduction, Molecular Endocrinology, Volume 22, Issue 5, 1 May 2008, Pages 1171–1182, https://doi.org/10.1210/me.2007-0407</ref>. The hinge region's <scene name='95/952709/Hinge_helix_rotation/2'>hinge helix</scene> interacts with the <scene name='95/952709/P10_peptide_region/3'>p10 peptide</scene> through disulfides. The p10 peptide is a conserved sequence that spans from the last β sheet of the LRRD to the first transmembrane helix (TM1) and is an intramolecular agonist for conformational shifts in the 7TMD helices<ref name="Faust et al.">Faust, B., Billesbølle, C.B., Suomivuori, CM. et al. Autoantibody mimicry of hormone action at the thyrotropin receptor. Nature 609, 846–853 (2022). https://doi.org/10.1038/s41586-022-</ref>. The disulfides between the LRRD, the hinge helix, and the p10 are critical to TSH signaling as they transduce signal from the ECD through the hinge helix to the p10 peptide. The upward movement of the LRRD, caused by TSH binding, will cause rotation of the hinge helix. The subsequent movement of the p10 peptide leads to movement of the transmembrane helices, which will cause activation of the G-protein. In addition to activation, the hinge region plays an important role in tightly binding TSH. Residues 382-390 of the hinge region adopt a short helix containing two key residues. Y385 from TSHR is buried into a hydrophobic pocket of TSH. D386 from the receptor forms a salt bridge with R386 of the hormone. <scene name='95/952709/Binding_interactions_hinge/2'>Interactions</scene> that assist in the stable binding of TSH to TSHR allow more potent activation of the receptor<ref name="Duan et al.">PMID:35940204</ref>. Even with these key functions, the hinge region itself is not absolutely required for receptor activation<ref name="Faust et al.">Faust, B., Billesbølle, C.B., Suomivuori, CM. et al. Autoantibody mimicry of hormone action at the thyrotropin receptor. Nature 609, 846–853 (2022). https://doi.org/10.1038/s41586-022-</ref>. The hinge region functions as a point of attachment to the 7TMD for the LRRD, and its ability to rotate allows for LRRD shifts between up (active state) and down (inactive state) positions. The interactions that the hinge helix makes with the LRRD and p10 act as an important communication medium between the ECD and an intramolecular agonist directly effecting conformational shifts in the 7TMD.