Glycoprotein Hormone Receptors Research Articles

Cryo-electron microscopy for GPCR research and drug discovery in endocrinology and metabolism.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, with many GPCRs having crucial roles in endocrinology and metabolism. Cryogenic electron microscopy (cryo-EM) has revolutionized the field of structural biology, particularly regarding GPCRs, over the past decade. Since the first pair of GPCR structures resolved by cryo-EM were published in 2017, the number of GPCR structuresresolved by cryo-EM has surpassed the number resolved by X-ray crystallography by 30%, reaching >650, and the number has doubled every ~0.63 years for the past 6 years. At this pace, it is predicted that the structure of 90% of all human GPCRs will be completed within the next 5-7 years. This Review highlights the general structural features and principles that guide GPCR ligand recognition, receptor activation, Gprotein coupling, arrestin recruitment and regulation by GPCR kinases. The Review also highlights the diversity of GPCR allosteric binding sites and how allosteric ligands could dictate biased signalling that is selective for a G protein pathwayor an arrestin pathway. Finally, the authors use the examples of glycoprotein hormone receptors and glucagon-like peptide 1 receptor to illustrate the effect of cryo-EM on understanding GPCR biology in endocrinology and metabolism, as well as on GPCR-related endocrine diseases and drug discovery.

Tracking conformational transitions of the gonadotropin hormone receptors in a bilayer of (SDPC) poly-unsaturated lipids from all-atom molecular dynamics simulations.

Glycoprotein hormone receptors [thyrotropin (TSHR), luteinizing hormone/chorionic gonadotropin (LHCGR), and follicle stimulating hormone (FSHR) receptors] are rhodopsin-like G protein-coupled receptors. These receptors display common structural features including a prominent extracellular domain with leucine-rich repeats (LRR) stabilized by β-sheets and a long and flexible loop known as the hinge region (HR), and a transmembrane (TM) domain with seven α-helices interconnected by intra- and extracellular loops. Binding of the ligand to the LRR resembles a hand coupling transversally to the α- and β-subunits of the hormone, with the thumb being the HR. The structure of the FSH-FSHR complex suggests an activation mechanism in which Y335 at the HR binds into a pocket between the α- and β-chains of the hormone, leading to an adjustment of the extracellular loops. In this study, we performed molecular dynamics (MD) simulations to identify the conformational changes of the FSHR and LHCGR. We set up a FSHR structure as predicted by AlphaFold (AF-P23945); for the LHCGR structure we took the cryo-electron microscopy structure for the active state (PDB:7FII) as initial coordinates. Specifically, the flexibility of the HR domain and the correlated motions of the LRR and TM domain were analyzed. From the conformational changes of the LRR, TM domain, and HR we explored the conformational landscape by means of MD trajectories in all-atom approximation, including a membrane of polyunsaturated phospholipids. The distances and procedures here defined may be useful to propose reaction coordinates to describe diverse processes, such as the active-to-inactive transition, and to identify intermediaries suited for allosteric regulation and biased binding to cellular transducers in a selective activation strategy.

Clinical Case of Mccune-Albright-Braitsev Syndrome (MAS)

The presented clinical case describes orphan disease known as MAS with manifested symptoms of precocious puberty, recurrent ovarian cysts, fibrous dysplasia, café-au-lait skin pigmentation and abnormal cardiac conduction. The pathogenesis is based on the GNAS gene mutation that cause hyperactivation of glycoprotein hormone receptors and hypersecretion. There are genetic tests that confirm the diagnosis, however, given the high percentage of false negative results, in most cases the disease is diagnosed based on a combination of clinical and laboratory-instrumental data. Given the high clinical variability and absence of management algorithms for patient with this syndrome, the article pinpoints the necessity of thorough examination of patients to select further management tactics. Multidisciplinary approach and collegiate case management will improve diagnosis of the disease and prevent the development of severe complications.

Ancestral glycoprotein hormone-receptor pathway controls growth in C. elegans.

In vertebrates, thyrostimulin is a highly conserved glycoprotein hormone that, besides thyroid stimulating hormone (TSH), is a potent ligand of the TSH receptor. Thyrostimulin is considered the most ancestral glycoprotein hormone and orthologs of its subunits, GPA2 and GPB5, are widely conserved across vertebrate and invertebrate animals. Unlike TSH, however, the functions of the thyrostimulin neuroendocrine system remain largely unexplored. Here, we identify a functional thyrostimulin-like signaling system in Caenorhabditis elegans. We show that orthologs of GPA2 and GPB5, together with thyrotropin-releasing hormone (TRH) related neuropeptides, constitute a neuroendocrine pathway that promotes growth in C. elegans. GPA2/GPB5 signaling is required for normal body size and acts through activation of the glycoprotein hormone receptor ortholog FSHR-1. C. elegans GPA2 and GPB5 increase cAMP signaling by FSHR-1 in vitro. Both subunits are expressed in enteric neurons and promote growth by signaling to their receptor in glial cells and the intestine. Impaired GPA2/GPB5 signaling causes bloating of the intestinal lumen. In addition, mutants lacking thyrostimulin-like signaling show an increased defecation cycle period. Our study suggests that the thyrostimulin GPA2/GPB5 pathway is an ancient enteric neuroendocrine system that regulates intestinal function in ecdysozoans, and may ancestrally have been involved in the control of organismal growth.

Glycoprotein hormone receptors in GtoPdb v.2023.1

Glycoprotein hormone receptors (provisional nomenclature [47]) are activated by a non-covalent heterodimeric glycoprotein made up of a common α chain (glycoprotein hormone common alpha subunit CGA, P01215), with a unique β chain that confers the biological specificity to FSH, LH, hCG or TSH. There is binding cross-reactivity across the endogenous agonists for each of the glycoprotein hormone receptors. The deglycosylated hormones appear to exhibit reduced efficacy at these receptors [122, 31].

Mechanism of hormone and allosteric agonist mediated activation of follicle stimulating hormone receptor

Follicle stimulating hormone (FSH) is an essential glycoprotein hormone for human reproduction, which functions are mediated by a G protein-coupled receptor, FSHR. Aberrant FSH-FSHR signaling causes infertility and ovarian hyperstimulation syndrome. Here we report cryo-EM structures of FSHR in both inactive and active states, with the active structure bound to FSH and an allosteric agonist compound 21 f. The structures of FSHR are similar to other glycoprotein hormone receptors, highlighting a conserved activation mechanism of hormone-induced receptor activation. Compound 21 f formed extensive interactions with the TMD to directly activate FSHR. Importantly, the unique residue H6157.42 in FSHR plays an essential role in determining FSHR selectivity for various allosteric agonists. Together, our structures provide a molecular basis of FSH and small allosteric agonist-mediated FSHR activation, which could inspire the design of FSHR-targeted drugs for the treatment of infertility and controlled ovarian stimulation for in vitro fertilization.

FSHR activation through small molecule modulators: Mechanistic insights from MD simulations

Follicle-stimulating hormone receptor (FSHR) is a glycoprotein hormone receptor that plays a vital role in reproduction, cancer progression and osteoporosis. Owing to its therapeutic importance, several small molecule modulators have been identified by researchers through high throughput studies that usually include virtual screening of chemical libraries followed by in vitro validation through radio-ligand binding assays, cAMP accumulation and luciferase-based luminescence assays. The binding site of these modulators and structural changes that accompany modulator binding remains elusive. Here, we address these aspects through molecular docking and MD simulations on well-studied FSHR modulators and comparing the domain motions between agonist/FSH bound and antagonist bound FSHR structures. It was observed that agonist and antagonist modulators bind to the same site, but interact with distinct residues in transmembrane domain(TMD). FSHR(TMD) residues Ile522, Ala595, Ile602 and Val604 were found to interact only with agonist. Notably, these residues are conserved in the close homolog luteinizing hormone/choriogonadotropin receptor (LHCGR) and participate in interaction with its agonist Org43553. We observed distinctly prominent domain motions and conformational changes in TM helices 3, 4 and 6 for agonist bound FSHR structure. These structural changes have also been reported for LHCGR, and few GPCR members suggesting an important and well conserved mechanism of GPHR activation that could be exploited for design of novel modulators.

Brain atlas for glycoprotein hormone receptors at single-transcript level.

There is increasing evidence that anterior pituitary hormones, traditionally thought to have unitary functions in regulating single endocrine targets, act on multiple somatic tissues, such as bone, fat, and liver. There is also emerging evidence for anterior pituitary hormone action on brain receptors in mediating central neural and peripheral somatic functions. Here, we have created the most comprehensive neuroanatomical atlas on the expression of TSHR, LHCGR, and FSHR. We have used RNAscope, a technology that allows the detection of mRNA at single-transcript level, together with protein level validation, to document Tshr expression in 173 and Fshr expression in 353 brain regions, nuclei and subnuclei identified using the Atlas for the Mouse Brain in Stereotaxic Coordinates. We also identified Lhcgr transcripts in 401 brain regions, nuclei and subnuclei. Complementarily, we used ViewRNA, another single-transcript detection technology, to establish the expression of FSHR in human brain samples, where transcripts were co-localized in MALAT1-positive neurons. In addition, we show high expression for all three receptors in the ventricular region-with yet unknown functions. Intriguingly, Tshr and Fshr expression in the ependymal layer of the third ventricle was similar to that of the thyroid follicular cells and testicular Sertoli cells, respectively. In contrast, Fshr was localized to NeuN-positive neurons in the granular layer of the dentate gyrus in murine and human brain-both are Alzheimer's disease-vulnerable regions. Our atlas thus provides a vital resource for scientists to explore the link between the stimulation or inactivation of brain glycoprotein hormone receptors on somatic function. New actionable pathways for human disease may be unmasked through further studies.

Integrative model of the FSH receptor reveals the structural role of the flexible hinge region

The follicle-stimulating hormone receptor (FSHR) belongs to the glycoprotein hormone receptors, a subfamily of G-protein-coupled receptors (GPCRs). FSHR is involved in reproductive processes such as gonadal development and maturation. Structurally, the extensive extracellular domain, which contains the hormone-binding site and is linked to the transmembrane domain by the hinge region (HR), is characteristic for these receptors. How this HR is involved in hormone binding and signal transduction is still an open question. We combined invitro and in situ chemical crosslinking, disulfide pattern analysis, and mutation data with molecular modeling to generate experimentally driven full-length models. These models provide insights into the interface, important side-chain interactions, and activation mechanism. The interface indicates a strong involvement of the connecting loop. A major rearrangement of the HR seems implausible due to the tight arrangement and fixation by disulfide bonds. The models are expected to allow for testable hypotheses about signal transduction and drug development for GPHRs.

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.

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.

Signal Transduction of C-Terminal Phosphorylation Regions for Equine Luteinizing Hormone/Chorionic Gonadotropin Receptor (eLH/CGR).

This study aimed to investigate the signal transduction of phosphorylation sites at the carboxyl (C)-terminal region of equine luteinizing hormone/chorionic gonadotropin receptor (eLH/ CGR). The eLH/CGR has a large extracellular domain of glycoprotein hormone receptors within the G protein-coupled receptors. We constructed a mutant (eLH/CGR-t656) of eLH/ CGR, in which the C-terminal cytoplasmic tail was truncated at the Phe656 residue, through polymerase chain reaction. The eLH/CGR-t656 removed 14 potential phosphorylation sites in the intracellular C-terminal region. The plasmids were transfected into Chinese hamster ovary (CHO)-K1 and PathHunter Parental cells expressing β-arrestin, and agonist-induced cAMP responsiveness was analyzed. In CHO-K1 cells, those expressing eLH/CGR-t656 were lower than those expressing eLH/CGR wild-type (eLH/CGR-wt). The EC50 of the eLH/ CGR-t656 mutant was approximately 72.2% of the expression observed in eLH/CGR-wt. The maximal response in eLH/CGR-t656 also decreased to approximately 43% of that observed in eLH/CGR-wt. However, in PathHunter Parental cells, cAMP activity and maximal response of the eLH/CGR-t656 mutant were approximately 173.5% and 100.8%, respectively, of that of eLH/CGR-wt. These results provide evidence that the signal transduction of C-terminal phosphorylation in eLH/CGR plays a pivotal role in CHO-K1 cells. The cAMP level was recovered in PathHunter Parental cells expressing β-arrestin. We suggest that the signal transduction of the C-terminal region phosphorylation sites is remarkably different depending on the cells expressing β-arrestin in CHO-K1 cells.

Screen of small fragment mutations within the sheep thyroid stimulating hormone receptor gene associated with litter size

The thyroid stimulating hormone receptor (TSHR), a glycoprotein hormone receptor, plays an important role in metabolic regulation and photoperiod control in the time of reproduction in birds and mammals. Previous genome-wide association studies revealed that the TSHR gene was related to reproduction and its function was identified in female reproduction, but rare studies reported the polymorphism of TSHR gene. However, the molecular mutations of the TSHR gene in sheep have not been reported so far. Herein, we explored potential polymorphisms of the sheep TSHR gene, and a 29 bp nucleotide sequence variant (rs1089565492) was identified in the AUW sheep. There were three genotypes of the 29 bp variant locus detected which named ‘II’ ‘DD’ and ‘ID’ been identified. Association analysis results showed the 29 bp variant was significantly associated with the litter size of the AUW sheep (p < 0.05). This finding suggests that the 29 bp nucleotide sequence variant within TSHR gene could be a candidate marker of reproduction traits for sheep breeding improving through the marker-assisted selection (MAS).

Cell-Surface Loss of Constitutive Activating and Inactivating Mutants of Eel Luteinizing Hormone Receptors

The present study aimed to investigate the mechanism of cell surface receptor loss by two constitutively activating mutants (designated L469R, and D590Y) and two inactivating mutants (D417N and Y558F) of the luteinizing hormone receptor (LHR) in the Japanese eel Anguilla japonica, known to naturally occur in human LHR transmembrane domains. We investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in HEK 293 cells. The expression level of wild-type eel LHR was considered to be 100%, and the expression levels of L469R and D417N were 97% and 101%, respectively, whereas the expression levels of D590Y and Y558F slightly increased to approximately 110% and 106%, respectively. The constitutively activating mutants L469R and D590Y exhibited a decrease in cell surface loss in a manner similar to that of wild-type eel LHR. The rates of loss of cell surface agonist-receptor complexes were observed to be very rapid (2.6-6.2 min) in both the wild-type eel LHR and activating mutants. However, cell surface receptor loss in the cells expressing inactivating mutants D417N and Y558F was slightly observed in the cells expressing inactivating mutants D417N and Y558F, despite treatment with a high concentration of agonist. These results provide important information on LHR function in fish and the regulation of mutations of highly conserved amino acids in glycoprotein hormone receptors.

Thyroid Disorders.

Thyroid Disorders.

Structures of full-length glycoprotein hormone receptor signalling complexes.

Luteinizing hormone and chorionic gonadotropin are glycoprotein hormones that are related to follicle-stimulating hormone and thyroid-stimulating hormone1,2. Luteinizing hormone and chorionic gonadotropin are essential to human reproduction and are important therapeutic drugs3-6. They activate the same G-protein-coupled receptor, luteinizing hormone-choriogonadotropin receptor (LHCGR), by binding to the large extracellular domain3. Here we report four cryo-electron microscopy structures of LHCGR: two structures of the wild-type receptor in the inactive and active states; and two structures of the constitutively active mutated receptor. The active structures are bound to chorionic gonadotropin and the stimulatory G protein (Gs), and one of the structures also contains Org43553, an allosteric agonist7. The structures reveal a distinct 'push-and-pull' mechanism of receptor activation, in which the extracellular domain is pushed by the bound hormone and pulled by the extended hinge loop next to the transmembrane domain. A highly conserved 10-residue fragment (P10) from the hinge C-terminal loop at the interface between the extracellular domain and the transmembrane domain functions as a tethered agonist to induce conformational changes in the transmembrane domain and G-protein coupling. Org43553 binds to a pocket of the transmembrane domain and interacts directly with P10, which further stabilizes the active conformation. Together, these structures provide a common model for understanding the signalling of glycoprotein hormone receptors and a basis for drug discovery for endocrine diseases.

Constitutive Activating Eel Luteinizing Hormone Receptors Induce Constitutively Signal Transduction and Inactivating Mutants Impair Biological Activity.

In contrast to the human lutropin receptor (hLHR) and rat LHR (rLHR), very few naturally occurring mutants in other mammalian species have been identified. The present study aimed to delineate the mechanism of signal transduction by three constitutively activating mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of the eel LHR, known to be naturally occurring in human LHR transmembrane domains. The mutants were constructed and measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO)-K1 cells. The activating mutant cells expressing eel LHR-M410T, L469R, and D590Y exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response without agonist treatment, respectively. However, inactivating mutant cells expressing D417N and Y558F did not completely impaired signal transduction. Specifically, signal transduction in the cells expressing activating mutant L469R was not occurred with a further ligand stimulation, showing that the maximal response exhibited approximately 53% of those of wild type receptor. Our results suggested that the constitutively activating mutants of the eel LHR consistently occurred without agonist treatment. These results provide important information of LHR function in fish and regulation with regard to mutations of highly conserved amino acids in glycoprotein hormone receptors.

From cell surface to signalling and back: the life of the mammalian FSH receptor.

Follicle-stimulating hormone receptor (FSHR) is a class A G protein-coupled receptor that belongs to the subfamily of glycoprotein hormone receptors (GPHRs). The interaction of FSH with FSHR triggers downstream signaling pathways that play a central role in mammalian reproduction, such as folliculogenesis in females and the maintenance of spermatogenesis in males. This warrants a detailed investigation into FSHR, from its genesis, to the post-translational modifications that enable it to become functionally competent, followed by its trafficking to the cell membrane. Subsequently, FSH-stimulated Gs uncoupling and transduction of G protein-mediated signaling pathways takes place, after which the receptor undergoes β-arrestin-mediated internalization and may trigger other noncanonical signaling pathways. The majority of the FSH-FSHR complexes are recycled back to the cell surface and only a small proportion are routed to lysosomal degradation pathways, thus completing the lifecycle of the FSH receptor. Information about important epitopes and aspects of FSH receptor function has been gleaned from a number of sources, including structure-function studies on both naturally occurring and induced mutations, single nucleotide polymorphisms, peptides and antipeptide antibodies corresponding to predicted functional residues, X-ray crystallography analysis and high resolution imaging studies, in addition to the information available for the other GPHRs. In this review, we have traversed through the life cycle of the FSH receptor and discuss the reproductive pathophysiologies that could result from an impairment in receptor function, as may arise from defects during its journey from its birth to its degradation. Moreover, the unresolved questions and challenges that require exploration have been highlighted.

Glycoprotein hormone receptors (version 2020.4) in the IUPHAR/BPS Guide to Pharmacology Database

Glycoprotein hormone receptors (provisional nomenclature [45]) are activated by a non-covalent heterodimeric glycoprotein made up of a common α chain (glycoprotein hormone common alpha subunit CGA, P01215), with a unique β chain that confers the biological specificity to FSH, LH, hCG or TSH. There is binding cross-reactivity across the endogenous agonists for each of the glycoprotein hormone receptors. The deglycosylated hormones appear to exhibit reduced efficacy at these receptors [120].

Glycoprotein G-protein Coupled Receptors in Disease: Luteinizing Hormone Receptors and Follicle Stimulating Hormone Receptors.

Signal transduction by luteinizing hormone receptors (LHRs) and follicle-stimulating hormone receptors (FSHRs) is essential for the successful reproduction of human beings. Both receptors and the thyroid-stimulating hormone receptor are members of a subset of G-protein coupled receptors (GPCRs) described as the glycoprotein hormone receptors. Their ligands, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and a structurally related hormone produced in pregnancy, human chorionic gonadotropin (hCG), are large protein hormones that are extensively glycosylated. Although the primary physiologic functions of these receptors are in ovarian function and maintenance of pregnancy in human females and spermatogenesis in males, there are reports of LHRs or FSHRs involvement in disease processes both in the reproductive system and elsewhere. In this review, we evaluate the aggregation state of the structure of actively signaling LHRs or FSHRs, their functions in reproduction as well as summarizing disease processes related to receptor mutations affecting receptor function or expression in reproductive and non-reproductive tissues. We will also present novel strategies for either increasing or reducing the activity of LHRs signaling. Such approaches to modify signaling by glycoprotein receptors may prove advantageous in treating diseases relating to LHRs or FSHRs function in addition to furthering the identification of new strategies for modulating GPCR signaling.