Sandbox Reserved 1774

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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Thyroid Stimulating Hormone Receptor (TSHR) Structure and Function

Introduction

Figure 1: TSH binds TSHR on the surface of thyroid cells in the HPT signaling axis pathway, which regulates metabolism and growth.

In humans, the hypothalamic-pituitary-thyroid (HPT) signaling axis regulates functions including metabolism, growth, organ development, and neural differentiation . In this pathway, the thyroid stimulating hormone receptor (TSHR) activates transcription of thyroid hormones thyroxine (T3) and triiodothyronine (T4) in response to ligand binding by thyroid stimulating hormone (TSH) . After a brief introduction to the biological significance of TSHR, this page explores the structure of TSHR and its significance to TSH binding and receptor activation.

Biological Significance of TSHR

The HPT signaling axis involves the brain, thyroid gland, and bloodstream circulation. In the first step of the pathway, thyrotropin releasing hormone (TRH) is secreted by the hypothalamus, which in turn stimulates the anterior pituitary gland to produce TSH . TSH binds to TSHR on the surface of thyroid cells and triggers the production of T3 and T4 through G-protein coupled receptor (GPCR) signaling . T3 and T4 circulate in the bloodstream and enter cells via thyroid hormone transporters to regulate metabolic functions including neural differentiation, metabolism, and growth and development. Additionally, T3 and T4 act in a negative feedback loop to inhibit further TSH production .

Dysregulation of TSHR can lead to disease. In Grave's disease, antibody analogs of TSH cause overactivation of TSHR, leading to clinical symptoms of hyperthyroidism . In contrast, congenital mutations which inactivate TSHR can lead to hypothyroidism, which results in growth retardation and neurologic impairment if left untreated .

Molecular Structure and Function of TSHR

Role of TSHR Domains

To understand how the structure of TSHR contributes to its function, it is helpful to become familiar with the three main domains of TSHR. First is the extracellular domain (ECD), which is concave in shape. It is also called the leucine rich region because it is made primarily of beta sheets which are rich in leucine . The ECD contains lysine residues which play a key role in TSH binding. Second is the transmembrane domain (TMD) , which is composed of seven transmembrane alpha helices which are connected by extracellular loops (ECL). The TMD undergoes a conformation change upon ligand binding that activates the intracellular GPCR signal cascade . The third region of the TSHR is the hinge region, which plays a key role in the movement and stability of the TSHR. The details of the hinge mechanism are discussed in the proceeding section.

Figure 2: (Left) In the downright, inactive state, TSH cannot bind and no signaling activation occurs. (Right) In the upright, active state, binding of TSH leads to GPCR signaling activation and production of T3 and T4 hormones.

Importance of Hinge Region to Signaling Activation

The TSHR hinges between two states: active and inactive. When the extracellular domain is hinged down, the receptor is inactive and no signaling activation occurs. When the extracellular domain raises into the upright position, the hinge region deforms and interacts with the extracellular loops to cause a conformation change in the TMD and corresponding G-protein activation . While transition between the active and inactive states occurs spontaneously, favoring of one state over the other is influence by hinge interactions and ligand binding . When stabilized in the upright conformation, the activated GPCR signaling pathway results in transcription of thyroid hormones T3 and T4 (Fig. 2) .

Modulation of TSHR signaling would not be possible without the hinge region, which accommodates up-and-down rotation of the extracellular domain as a rigid body about an imaginary 55 degree axis . During this transition, the hinge region undergoes slinky-like deformation and is displaced approximately 5 Angstroms upward as it uncoils . The hinge region pulls on the linked transmembrane helices as it stretches, shifting TM helix 7 approximately 4 Angstroms inward and leading to G-protein signaling activation , .

Fig. 3 Animation of TSHR hinging between the active (upright, 7T9I) and inactive (downward, 7T9M) conformations. The ECD rotates 55 degrees as a rigid body.

Stabilizing Interactions in the Hinge

To understand stabilizing interactions which accommodate the hinge motion, the hinge region can be subdivided into the hinge helix, which lies at the intersection of the extracellular and transmembrane domains; helix 1, which sticks up and serves as a binding platform for the TSH ligand; the linker region, which connects helix 1 with the p10 region; and the p10 region, a conserved 10-amino acid sequence which connects to transmembrane helix 7 and undergoes most of the deformation .

Two key disulfide bridges within the hinge region help to maintain its structure and orientation .

1. The first disulfide bridge connects the hinge helix with the linker region
2. The second disulfide bridge connects the hinge helix with the p10 region.

The upright, active conformation of the hinge is stabilized by its respective interactions with the EC and TM domains .

1. A hydrophobic interaction occurs between Y279 in the hinge helix and I486 in EC loop region 1, which protrudes from the TM helices.
2. An ionic interaction occurs between K660 in TM helix 7 and E409 in the p10 region.

If the stabilizing interactions are disrupted, TSHR function is affected. For instance, the mutation I496F has been observed to cause constitutive receptor activation and decreased sensitivity to the TSH ligand, suggesting that the bulkier phenylalanine strengthens the hydrophobic interaction too much, leading to overactivation. Contrastingly, TSHR underactivation results from disrupting the ionic interaction with an E409A mutation, which is associated with diminished receptor activation and TSH potency .

Ligand Binding

Binding of Thyroid Stimulating Hormone to TSHR

The thyroid stimulating hormone binds to the extracellular domain by complementary shape. The ECD is curved and compliments the curvature of TSH similar to how a baseball fits into a glove. There are also several key ionic interactions between the TSH and TSHR. The key ionic interactions occur in the seat belt region of TSH which is highlighted in yellow. The seatbelt region is located in the beta subunit of the TSH. The first ionic interaction is Glu118 from TSH and Lys58 from the ECD.The second interaction is between Asp111 from the TSH and Lys209 from the ECD. These interactions form salt bridges between the ECD and the TSH which allows for specificity of binding for TSH to TSHR ,.

Other key interactions that allow for specificity of binding are polar and nonpolar interactions between TSH and helix 1. Helix 1 contains several polar residues that interact with surrounding nonpolar residues like Leu62 and Phe17. Positively charged Arg54 was also seen to interact with Helix 1. These interactions increase the activation potency and help activate the push and pull mechanism of the hinge region ,.

Ligand Regulation of Signaling Activation

Figure 2 TSHR in active and inactive binding states. Left is TSH bound to TSHR. Middle is M22 bound to TSHR. Right is CS-17 bound to TSHR.

The interactions between the ligand and the receptor have important consequences for disease states. In the image shown to the right are three different states of TSHR. The left-most structure is TSH bound to TSHR in the upright active conformation. In the middle receptor-ligand pair, M22 is bound to TSHR is in the upright state and prevents transition to the down state because of steric clash with the membrane. This conformation causes constitutive activation and the elevated levels of thyroid hormones which are found in a person with Grave's disease. On the right-most side is CS-17 bound to the TSHR. In contrast to TSH and M22 binding, CS-17 binds and locks TSHR in the down, inactive conformation. This prevents the signaling cascade to translation and causes constitutive inactivation .

These different ways to active and inactive TSHR could represent potential therapies for someone with Grave's disease or other thyroid-related diseases with overactive TSH binding. Whereas current therapies target T3/T4 synthesis or destroy the gland using artificial hormones, these diseases could instead be targeted with something like CS-17 which would compete with M22 and TSH to lessen overactivation.