A conformational contribution of the luteinizing hormone–receptor ectodomain to receptor activation

The follicle-stimulating hormone receptor (FSHR) is a G-protein-coupled receptor with a large hormone-specific extracellular domain (amino acids (aa) 1-366) and a characteristic seventransmembrane domain (TMD; aa 367-695). The extracellular domain is composed of leucine-rich repeats (LRRs; aa 18 -259) connected to TMD by a hinge region (HinR; aa 260 -366), whose role in the hormone action is not clearly understood. We generated a novel polyclonal HinR antibody that specifically stimulates cAMP production by HEK 293 cells expressing FSHR in a hormone-independent manner. The monovalent antibody retained the stimulatory potential. The segment of aa 296 -331 in HinR was identified as the binding site for the stimulatory antibody. Deletions of this entire segment or any 10 amino acids within this segment from FSHR led to complete loss of antibody response and, surprisingly, response to the hormone as well despite all mutants exhibiting cell surface receptor density and affinity comparable with the wild type receptor. Interestingly, these mutants exhibited higher basal cAMP production. The mutant lacking LRRs with the intact HinR (⌬18 -259) showed suppressed basal activity, which increased significantly with deletions extending to aa 331. These data suggest that the segment 296 -331 acts as a tethered inverse agonist of the TMD and plays a very critical role in the hormonal activation of FSHR. The mutants lacking LRRs failed to bind FSH, whereas deletions in HinR had no effect on hormone binding, indicating that the LRRs constitute the primary high affinity binding site, whereas HinR may not play a significant role in FSH binding.The human glycoprotein hormones, follicle-stimulating hormone (FSH), 3 thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and the chorionic gonadotropin, are heterodimeric proteins with an identical ␣ subunit annealed noncovalently with the hormone specific ␤ subunit (1). The receptors for these hormones belong to the family of G-protein-coupled receptors and have a hormone-specific large extracellular domain (ECD) believed to be involved in specific interactions with the hormone and a characteristic seven-transmembrane domain (TMD) involved in transducing the signal. The ECD of these receptors can be further divided into two distinct regions, the N-terminal leucine-rich repeat domain (LRRs), with 9 -10 such LRRs being present in each receptor and the hinge region (HinR) that connects the LRRs to the TMD (2, 3). The roles of these domains in hormone binding and signal transduction have been intensively investigated in the past few years (4 -6). Several mechanisms of receptor activation have been proposed (2, 6). In a widely discussed model, the ECD, particularly of the thyroid-stimulating hormone receptor (TSHR), serves as an inverse agonist, stabilizing the TMD in an inactive conformation, and the hormone binding or the activating mutations disengage the inhibitory ECD-TMD interactions (7-15). This hypothesis is supported by the observation that the mutants lacking the ECD exhibite...

Section: Constructcontrasting

confidence: 44%

“…In fact, this deletion tended to reduce the LHR basal activity. Moreover, the mutationally induced constitutive receptor activation was diminished in the absence of ECD (16,17). These observations indicate specific and distinct roles for the ECDs in TSHR and LHR.…”

mentioning

confidence: 77%

Critical Involvement of the Hinge Region of the Follicle-stimulating Hormone Receptor in the Activation of the Receptor

Agrawal

Dighe

2009

Section: Glycoprotein Hormone Receptors (Gphrs)mentioning

confidence: 82%

“…We have shown that deletion of the entire ECD did not activate the LHR, which provokes an alternative hypothesis of an "intramolecular agonistic unit" where an internal agonist within the ECD is exposed upon ligand binding at the ECD (5, 8, 18). The latter hypothesis is supported by LHR studies showing that parts of the ECD are necessary to stabilize active state conformations of the 7TM (19,20).Here we show that all GPHRs are activated by an internal peptide sequence, which is located in the C-terminal part of the * The work was supported by the Deutsche Forschungsgemeinschaft (SSP ThyroidTransAct; BR 5108/1-1 AO 619225 and SFB/Transregio 166, project C1), the BMBF (IFB AdipositasDiseases Leipzig, FZ: 01EO1001), Interdisziplinäres Zentrum fü r Klinische Forschung (IZKF) Wü rzburg (B-281) and the University of Leipzig (Junior research grant by the Medical Faculty). …”

mentioning

confidence: 82%

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

Brüser¹,

Schulz²,

Rothemund

et al. 2016

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...

“…This study supports functional-structural interrelations between different regions of the TSHR. In this respect, it has been shown several times for the TSHR that the hinge region is necessary for the stabilization of a signaling-competent basal receptor conformation (17,25,(27)(28)(29)(30)(31) and that interaction with the hormone leads to receptor activation, most probably by release of an intramolecular agonist (10,32,33).…”

mentioning

confidence: 99%

Defining Structural and Functional Dimensions of the Extracellular Thyrotropin Receptor Region

Kleinau

Mueller

Jaeschke

et al. 2011

The extracellular region of the thyrotropin receptor (TSHR) can be subdivided into the leucine-rich repeat domain (LRRD) and the hinge region. Both the LRRD and the hinge region interact with thyrotropin (TSH) or autoantibodies. Structural data for the TSHR LRRD were previously determined by crystallization (amino acids Glu 30 -Thr 257 , 10 repeats), but the structure of the hinge region is still undefined. Of note, the amino acid sequence (Trp 258 -Tyr 279 ) following the crystallized LRRD comprises a pattern typical for leucine-rich repeats with conserved hydrophobic side chains stabilizing the repeat fold. Moreover, functional data for amino acids between the LRRD and the transmembrane domain were fragmentary. We therefore investigated systematically these TSHR regions by mutagenesis to reveal insights into their functional contribution and potential structural features. We found that mutations of conserved hydrophobic residues between Thr 257 and Tyr 279 cause TSHR misfold, which supports a structural fold of this peptide, probably as an additional leucine-rich repeat. Furthermore, we identified several new mutations of hydrophilic amino acids in the entire hinge region leading to partial TSHR inactivation, indicating that these positions are important for intramolecular signal transduction. In summary, we provide new information regarding the structural features and functionalities of extracellular TSHR regions. Based on these insights and in context with previous results, we suggest an extracellular activation mechanism that supports an intramolecular agonistic unit as a central switch for activating effects at the extracellular region toward the serpentine domain.