The Activation Mechanism of Glycoprotein Hormone Receptors with Implications in the Cause and Therapy of Endocrine Diseases

Abstract

Glycoprotein hormones (GPHs) are the main regulators of the pituitary-thyroid and pituitary-gonadal axes. Selective interaction between GPHs and their cognate G protein-coupled receptors ensure specificity in GPH signaling. The mechanisms of how these hormones activate glycoprotein hormone receptors (GPHRs) or how mutations and autoantibodies can alter receptor function were unclear. Based on the hypothesis that GPHRs contain an internal agonist, we systematically screened peptide libraries derived from the ectodomain for agonistic activity on the receptors. We show that a peptide (p10) derived from a conserved sequence in the C-terminal part of the extracellular N terminus can activate all GPHRs in vitro and in GPHR-expressing tissues. Inactivating mutations in this conserved region or in p10 can inhibit activation of the thyroid-stimulating hormone receptor by autoantibodies. Our data suggest an activation mechanism where, upon extracellular ligand binding, this intramolecular agonist isomerizes and induces structural changes in the 7-transmembrane helix domain, triggering G protein activation. This mechanism can explain the pathophysiology of activating autoantibodies and several mutations causing endocrine dysfunctions such as Graves disease and hypo-and hyperthyroidism. Our findings highlight an evolutionarily conserved activation mechanism of GPHRs and will further promote the development of specific ligands useful to treat Graves disease and other dysfunctions of GPHRs. Glycoprotein hormone receptors (GPHRs)2 were cloned about 25 years ago (1-3) and, since then, the glycoprotein hormones (GPHs) TSH, LH/hCG, and FSH are considered as the agonists for their respective receptors. In contrast to other rhodopsin-like G protein-coupled receptors (GPCRs), they possess a large extracellular leucine-rich repeat (LRR) hormone-binding domain, which is linked via a hinge region (HR) to the 7-transmembrane helix domain (7TM) (see Fig. 1A). Based on the crystal structure of human FSH bound to the receptor's extracellular domain (ECD), it has been suggested that the FSHR grabs the FSH in a "hand-clasp" mode, orienting the hormone to interact with the extracellular loops and juxtamembrane regions of the 7TM to induce signaling (4). However, to date, all attempts to identify agonistic parts of GPHs have failed, and even extreme high concentrations of hCG did not activate the 7TM of LHR lacking the ECD (5).In addition to TSH and thyrostimulin (6), the TSHR can be activated by mutations in the ECD and by ECD-directed autoantibodies, which cause hyperthyroidism and Graves disease, respectively (7). The detailed mechanism of how the ECD integrates the activating actions of TSH, thyrostimulin, mutations, and autoantibodies to trigger GPHR signal transduction is unsolved, although there are numerous mutagenesis studies (5,8) and crystallographic data of the liganded GPHR ECD (4, 9 -11).Based on findings that proteolytic cleavage of the ECD by trypsin, or artificially generated ECD deletions and truncations, can increase TSHR ac...

Structure-Function Relationships of the Follicle-Stimulating Hormone Receptor

“…The FSHR ECD structure reported by Jiang and colleagues ( 7 ), identified the hinge region as an integral part of the ECD (Figure 1B ), and confirmed previously reported biochemical data on the FSHR and TSHR ( 85 , 90 – 93 ), underlying the role of this region in ligand-stimulated receptor activation. These and other studies ( 94 ) have also suggested that the ECD of the glycoprotein hormone receptors acts as a tethered inverse agonist. In this scenario, the ECD acts as an agonist upon ligand binding and activates the sequence 353FNPCEDIMGY362 located in the junction of the carboxyl-terminal end of the hinge region and the 7TMD helix 1, which function as an internal agonist unit (Figure 1B ).…”

FSH for the Treatment of Male Infertility

“…FSH binding to its cognate receptor, the FSHR, leads to the rewiring of a complex intracellular signaling network that profoundly alters gene regulation at several levels. Hormone binding to the Leucine-rich repeat domain provokes conformational changes in the receptor, including the interaction of a sulfated tyrosine within the receptor hinge region with the interface of FSH α and β chains [4,5], and tethering of a decapeptide that protrudes out of the plasma membrane from the first intracellular transmembrane helix [6]. Work is still ongoing to decipher the crystal structure of the entire FSHR, including transmembrane domains, in active conformation.…”

Intervention Strategies into Glycoprotein Hormone Receptors for Modulating (Mal–)function, with Special Emphasis on the TSH Receptor

“…After having already been shown for the LHR in 1999 that a short extracellular sequence near transmembrane helix (TMH) 1 is important for hormone mediated signaling [24], later it was suggested that the intramolecular agonistic unit in TSHR comprises specific amino acids localized immediately prior TMH1 [25] and further experiments refined these insights [26,27]. This was greatly confirmed by a recent peptide screening study that revealed a decapeptide representing a highly conserved sequence shortly prior TMH1 in GPHR as a tethered "internal agonist" [28]. Homology models suggest that this internal agonistic sequence in the hinge region's C-terminus (assumed for all three GPHR subtypes) is i) covalently connected to a short helix (converging helix) in the N-terminus of the hinge region via a disulfide bridge (Cys408-Cys284 in TSHR), ii) located at the transition to TMH1 and is iii) con-sequently embedded in between the extracellular loops of the seven TMHs (reviewed in [29]).…”

“…The internal tethered agonist in the GPHR can obviously also be targeted, which has been shown by adding the sequence of the internal agonist to the receptor as peptide. The peptide FNPCEDIMGY activates non-specifically TSHR and also LHR and FSHR, whereas its mutant peptide FNPCKDIMGY acts as unspecific inhibitor [28]. Both suggest that these peptides directly affect the tethered agonist.…”

“…The hormone (or activating antibodies) bind at the large ectodomain in between the leucine rich repeat domain (LRRD) and the hinge region interactions and probably triggers conformational changes (arrow pale green) at a convergent center of the LRRD and hinge region. LRRD and hinge region are via disulfide bridges (yellow) covalently connected to the short converging helix and to a following internal agonistic sequence identified by Brüser et al [28] (red). This intramolecular agonist located shortly prior transmembrane helix 1 is tightly embedded by the extracellular loops [29] of the heptahelical transmembrane domain (TMD, grey, [49]).…”