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

Abstract

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...

The Antibodies against the Computationally Designed Mimic of the Glycoprotein Hormone Receptor Transmembrane Domain Provide Insights into Receptor Activation and Suppress the Constitutively Activated Receptor Mutants

“…7B, the WT basal activities of GpHRs in terms of hormone-independent cAMP production vary widely, with TSHR being the "noisiest" receptor followed by FSHR and LHR. Moreover, as reported previously, removal of ECD further increased cAMP production for both TSHR and FSHR (7,35). This high basal cAMP level in TSHR/FSHR TMD mutants decreased by the introduction of HinR in the HinTMD mutants, suggesting that HinRs act as a tethered inverse agonist to the TMD, direct evidence for their roles in maintaining the basal as well as hormone-independent receptor activation (7, 24).…”

“…TSHR/LHR/FSHR Wild Type and Mutant Constructs-Stable cell lines expressing WT TSHR, LHR, and FSHR were created in HEK293 background and characterized as described previously (7,24,25).…”

“…LRR-and ECD-deleted mutants and the activating mutations of TSHR and LHR used in the this study were created as described earlier for FSHR (7). Briefly, modified pcDNA3.1-MycHis vectors (Invitrogen) were created containing the signal peptides of TSHR/LHR/FSHR (pCDNA3.1-SP) and the DNA fragments encoding the receptors without their LRRs (TSHR HinTMD, aa 261-764; LHR HinTMD, aa 265-699; FSHR HinTMD, aa 260 -695) and without their entire ECDs (TSHR TMD, aa 414 -764; LHR TMD, aa 355-699; FSHR TMD, aa 367-695) of GpHRs were cloned downstream of the respective cognate signal peptides to ensure proper translocation to the cell surface.…”

A Monoclonal Antibody against Human Notch1 Ligand–Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem–like Cells

“…Antibodies in general have proved to be excellent tools to map the ligand-receptor contact points and mechanism of receptor activation (38,45). Recently, it was shown that antibodies against Notch receptor EGF repeats inhibit ligand-receptor interactions and signal transduction (32,33).…”

Functional Differences of Invariant and Highly Conserved Residues in the Extracellular Domain of the Glycoprotein Hormone Receptors

“…FSHR/S226A did not express at sufficient levels to permit evaluation. It has been reported that the hinge region of FSHR is important in receptor activation (60,61). Although structural information is lacking beyond residue Glu 259 in FSHR (30), it appears that the last two/three strands are much shorter with fewer hydrogen bonds stabilizing the parallel strands.…”