scholarly journals Identification of Follicle-Stimulating Hormone-Selective β-Strands in the N-Terminal Hormone-Binding Exodomain of Human Gonadotropin Receptors

2006 ◽  
Vol 20 (8) ◽  
pp. 1880-1893 ◽  
Author(s):  
Henry F. Vischer ◽  
Joke C. M. Granneman ◽  
Jan Bogerd
Keyword(s):  
Hormone Receptors ◽  
The Other ◽  
Leucine Rich Repeat ◽  
Hormone Binding ◽  
Glycoprotein Hormone ◽  
Leucine Rich Repeat Domain ◽  
Glycoprotein Hormone Receptors ◽  
Binding Interface ◽  
Large N ◽  
Repeat Domain

Abstract Glycoprotein hormone receptors contain large N-terminal extracellular domains (ECDs) that distinguish these receptors from most other G protein-coupled receptors. Each glycoprotein hormone receptor ECD consists of a curved leucine-rich repeat domain flanked by N- and C-terminal cysteine-rich regions. Selectivity of the different glycoprotein hormone receptors for their cognate hormones is exclusively determined by their ECDs and, in particular, their leucine-rich repeat domain. To identify human (h)FSH-selective determinants we used a gain-of-function mutagenesis strategy in which β-strands of the hLH receptor (hLH-R) were substituted with their hFSH receptor (hFSH-R) counterparts. Introduction of hFSH-R β-strand 1 into hLH-R conferred responsiveness to hFSH, whereas hLH-R mutants harboring one of the other hFSH-R β-strands displayed none or very limited sensitivity to hFSH. However, combined substitution of hFSH-R β-strand 1 and some of the other hFSH-R β-strands further increased the sensitivity of the mutant hLH-R to hFSH. The apparent contribution of multiple hFSH-R β-strands in providing a selective hormone binding interface corresponds well with their position in relation to hFSH as recently determined in the crystal structure of hFSH in complex with part of the hFSH-R ECD.

scholarly journals Characterization of Two Fly LGR (Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor) Proteins Homologous to Vertebrate Glycoprotein Hormone Receptors: Constitutive Activation of Wild-Type Fly LGR1 But Not LGR2 in Transfected Mammalian Cells**This study was supported by NIH Grant HD-23273. The GenBank submission number for fly LGR2 is AF274591.

Endocrinology ◽  
2000 ◽  
Vol 141 (11) ◽  
pp. 4081-4090 ◽  
Author(s):  
Shinya Nishi ◽  
Sheau Yu Hsu ◽  
Karen Zell ◽  
Aaron J. W. Hsueh
Keyword(s):  
G Protein ◽  
Hormone Receptors ◽  
Coupling Mechanism ◽  
Leucine Rich Repeat ◽  
G Protein Coupled Receptor ◽  
Glycoprotein Hormone ◽  
Glycoprotein Hormone Receptors ◽  
Signaling Mechanisms ◽  
G Protein Coupled

Abstract The receptors for lutropin (LH), FSH, and TSH belong to the large G protein-coupled receptor (GPCR) superfamily and are unique in having a large N-terminal extracellular (ecto-) domain important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of a large family of the leucine-rich repeat-containing, G protein-coupled receptors (LGRs) with at least seven members in mammals. Based on the sequences of mammalian glycoprotein hormone receptors, we have identified a new LGR in Drosophila melanogaster and named it as fly LGR2 to distinguish it from the previously reported fly LH/FSH/TSH receptor (renamed as fly LGR1). Genomic analysis indicated the presence of 10 exons in fly LGR2 as compared with 16 exons in fly LGR1. The deduced fly LGR2 complementary DNA (cDNA) showed 43 and 64% similarity to the fly LGR1 in the ectodomain and transmembrane region, respectively. Comparison of 12 LGRs from diverse species indicated that these proteins can be divided into three subfamilies and fly LGR1 and LGR2 belong to different subfamilies. Potential signaling mechanisms were tested in human 293T cells overexpressing the fly receptors. Of interest, fly LGR1, but not LGR2, showed constitutive activity as reflected by elevated basal cAMP production in transfected cells. The basal activity of fly LGR1 was further augmented following point mutations of key residues in the intracellular loop 3 or transmembrane VI, similar to those found in patients with familial male precocious puberty. The present study reports the cloning of fly LGR2 and indicates that the G protein-coupling mechanism is conserved in fly LGR1 as compared with the mammalian glycoprotein hormone receptors. The characterization of fly receptors with features similar to mammalian glycoprotein hormone receptors allows a better understanding of the evolution of this unique group of GPCRs and future elucidation of their ligand signaling mechanisms.

scholarly journals The Nematode Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor (LGR) Protein Homologous to Vertebrate Gonadotropin and Thyrotropin Receptors is Constitutively Activated in Mammalian Cells

2000 ◽  
Vol 14 (2) ◽  
pp. 272-284 ◽  
Author(s):  
Masataka Kudo ◽  
Thomas Chen ◽  
Koji Nakabayashi ◽  
Sheau Yu Hsu ◽  
Aaron J. W. Hsueh
Keyword(s):  
G Protein ◽  
Hormone Receptors ◽  
Receptor Activation ◽  
Leucine Rich Repeat ◽  
Camp Production ◽  
Glycoprotein Hormone ◽  
Lh Receptor ◽  
Glycoprotein Hormone Receptors ◽  
Leucine Rich Repeats ◽  
G Protein Coupled

Abstract The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane (TM) protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of an expanding family of homologous leucine-rich repeat-containing, G protein-coupled receptors (LGRs), including the three known glycoprotein hormone receptors; mammalian LGR4 and LGR5; and LGRs in sea anemone, fly, and snail. We isolated nematode LGR cDNA and characterized its gene from the Caenorhabditis elegans genome. This receptor cDNA encodes 929 amino acids consisting of a signal peptide for membrane insertion, an ectodomain with nine leucine-rich repeats, a seven-TM region, and a long C-terminal tail. The nematode LGR has five potential N-linked glycosylation sites in its ectodomain and multiple consensus phosphorylation sites for protein kinase A and C in the cytoplasmic loop and C tail. The nematode receptor gene has 13 exons; its TM region and C tail, unlike mammalian glycoprotein hormone receptors, are encoded by multiple exons. Sequence alignments showed that the TM region of the nematode receptor has 30% identity and 50% similarity to the same region in mammalian glycoprotein hormone receptors. Although human 293T cells expressing the nematode LGR protein do not respond to human glycoprotein hormones, these cells exhibited major increases in basal cAMP production in the absence of ligand stimulation, reaching levels comparable to those in cells expressing a constitutively activated mutant human LH receptor found in patients with familial male-limited precocious puberty. Analysis of cAMP production mediated by chimeric receptors further indicated that the ectodomain and TM region of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode LGR is responsible for constitutive receptor activation. Thus, the identification and characterization of the nematode receptor provides the basis for understanding the evolutionary relationship of diverse LGRs and for future analysis of mechanisms underlying the activation of glycoprotein hormone receptors and related LGRs.

scholarly journals Lutropins appear to contact two independent sites in the extracellular domain of their receptors

1998 ◽  
Vol 335 (3) ◽  
pp. 611-617 ◽  
Author(s):  
Michael P. BERNARD ◽  
Rebecca V. MYERS ◽  
William R. MOYLE
Keyword(s):  
Hormone Receptors ◽  
Extracellular Domain ◽  
Leucine Rich Repeat ◽  
Terminal Portion ◽  
Glycoprotein Hormone ◽  
Extracellular Domains ◽  
Affinity Ligand ◽  
Glycoprotein Hormone Receptors ◽  
Contact Residues ◽  
High Affinity Ligand

Human chorionic gonadotropin (hCG) and bovine lutropin (bLH), a hormone chemically more similar to most mammalian lutropins than hCG, interact with the extracellular domains of their gonadal lutropin receptors (LHRs). These portions of the rat and human LHRs are 85% identical and both receptors bind hCG with high, albeit not identical, affinity. However, at least 1000-fold more bLH is required to inhibit binding of radiolabelled hCG to the human LHR than to the rat LHR, a phenomenon that proved useful for identifying regions of the extracellular domain that contact lutropins. Previous studies using truncated receptors and lutropin/follitropin receptor chimaeras localized most, if not all, high-affinity ligand contacts to the N-terminal three-fifths of the rat LHR extracellular domain. We report here that 10-fold more bLH was needed to inhibit binding of labelled hCG to rat/human LHR chimaeras containing the N-terminal three-fifths of the human LHR extracellular domain than to the rat LHR. Unexpectedly, 100-fold more bLH was required to inhibit binding of labelled hCG to chimaeras containing the C-terminal one-fifth of the human LHR extracellular domain than to the rat LHR. The ability of the C-terminal portion of the human LHR extracellular domain to inhibit bLH binding suggests this region of the receptor also contacts the ligand even though it is not needed for ligand binding. The extracellular domains of all the glycoprotein hormone receptors are thought to be horseshoe-shaped, a consequence of their leucine-rich repeat motifs. Portions of the ligand that become located within the cavity created by the concave surface of the horseshoe would have the opportunity to contact residues in the C-terminal portion of the extracellular domain. Changes to the ligand or receptor that influence this interaction would be expected to alter binding and confound efforts to identify residues in key ligand–receptor contacts.

scholarly journals Autologous Biological Response Modification of the Gonadotropin Receptor

2001 ◽  
Vol 276 (15) ◽  
pp. 12410-12419 ◽  
Author(s):  
Smita D. Mahale ◽  
John Cavanagh ◽  
Anja Schmidt ◽  
Robert MacColl ◽  
James A. Dias
Keyword(s):  
Secondary Structure ◽  
Hormone Receptors ◽  
Biological Response ◽  
Cd Spectroscopy ◽  
Intrinsic Activity ◽  
Activation Process ◽  
Leucine Rich Repeat ◽  
Glycoprotein Hormone ◽  
Guanine Nucleotide Binding Protein ◽  
Glycoprotein Hormone Receptors

It is generally held with respect to heterotrimeric guanine nucleotide binding protein-coupled receptors that binding of ligand stabilizes a conformation of receptor that activates adenylyl cyclase. It is not formally appreciated if, in the case of G-protein-coupled receptors with large extracellular domains (ECDs), ECDs directly participate in the activation process. The large ECD of the glycoprotein hormone receptors (GPHRs) is 350 amino acids in length, composed of seven leucine-rich repeat domains, and necessary and sufficient for high affinity binding of the glycoprotein hormones. Peptide challenge experiments to identify regions in the follicle-stimulating hormone (FSH) receptor (FSHR) ECD that could bind its cognate ligand identified only a single synthetic peptide corresponding to residues 221–252, which replicated a leucine-rich repeat domain of the FSHR ECD and which had intrinsic activity. This peptide inhibited human FSH binding to the human FSHR (hFSHR) and also inhibited human FSH-induced signal transduction in Y-1 cells expressing recombinant hFSHR. The hFSHR-(221–252) domain was not accessible to anti-peptide antibody probes, suggesting that this domain resides at an interface between the hFSHR ECD and transmembrane domains. CD spectroscopy of the peptide in dodecyl phosphocholine micelles showed an increase in the ordered structure of the peptide. CD and NMR spectroscopies of the peptide in trifluoroethanol confirmed that hFSHR-(221–252) has the propensity to form ordered secondary structure. Importantly and consistent with the foregoing results, dodecyl phosphocholine induced a significant increase in the ordered secondary structure of the purified hFSHR ECD as well. These data provide biophysical evidence of the influence of environment on GPHR ECD subdomain secondary structure and identify a specific activation domain that can autologously modify GPHR activity.

scholarly journals Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain

2019 ◽  
Vol 62 (3) ◽  
pp. 117-128 ◽  
Author(s):  
Jennifer Miller-Gallacher ◽  
Paul Sanders ◽  
Stuart Young ◽  
Andrew Sullivan ◽  
Stuart Baker ◽  
...  
Keyword(s):  
Hormone Receptor ◽  
Thyroid Stimulating Hormone ◽  
Side Chain ◽  
Leucine Rich Repeat ◽  
Glycoprotein Hormone ◽  
Leucine Rich Repeat Domain ◽  
Ligand Free ◽  
Repeat Domain ◽  
Monoclonal Autoantibody ◽  
Chain Conformations

The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22–260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22TM) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55TM, which is approximately 900 times more thermostable than wild-type TSHR260. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22TM or K1-70TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.

scholarly journals Characterization of a Region of the Lutropin Receptor Extracellular Domain Near Transmembrane Helix 1 That Is Important in Ligand-Mediated Signaling*

Endocrinology ◽  
1999 ◽  
Vol 140 (4) ◽  
pp. 1775-1782 ◽  
Author(s):  
Carlos A. Alvarez ◽  
Prema Narayan ◽  
Jianing Huang ◽  
David Puett
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Hormone Receptors ◽  
Transmembrane Helix ◽  
Extracellular Domain ◽  
The Other ◽  
Amino Acid Residues ◽  
Glycoprotein Hormone ◽  
Glycoprotein Hormone Receptors ◽  
Lutropin Receptor

Abstract The lutropin receptor (LHR), a member of the G protein-coupled receptor family, contains a relatively large N-terminal extracellular domain, accounting for about half of the receptor and responsible for high affinity ligand binding, and a standard heptahelical portion with connecting loops and a C-terminal tail. LHR and the other two glycoprotein hormone receptors, i.e. the follitropin and TSH receptors, contain an invariant 10-amino acid residue sequence, FNPCEDIMGY (residues 328–337 in rat LHR), in the extracellular domain separated by only a few amino acid residues from the beginning of transmembrane helix 1. In view of the invariant nature of this region in the three glycoprotein hormone receptors and preliminary data in the literature on the importance of Glu332 and Asp333 in signal transduction, we undertook a systematic investigation of all 10 amino acid residues because this region may function as a switch or trigger for communicating ligand binding to the extracellular domain with a conformational change of the membrane-embedded C-terminal half of the receptor to activate G proteins, particularly Gs. A total of 36 single, double, and multiple replacements, as well as two deletions, of LHR were prepared and characterized in transiently transfected COS-7 cells. Of these mutants LHRs, 26 expressed on the cell surface in sufficient numbers that quantitative assessments could be made of human choriogonadotropin binding and ligand-mediated cAMP production. Replacements of Cys331 abolished ligand binding to intact cells, although binding could be detected after solubilization of the cells. Replacements of the other nine amino acid residues that did not interfere with receptor folding or trafficking had no significant effect on ligand binding affinity; however, replacements of Pro330, Glu332, and Asp333 resulted in diminished signaling, especially for the two acidic residues. An interesting observation was made in which replacement of Tyr337 with Ala or Asp, while having no profound change on receptor function, could overcome to some extent limited expression of replacements at positions 332 and/or 333, thus permitting a more definitive analysis of signaling. Replacement of the decapeptide sequence with Gly10 prevents expression, whereas deletion of all 10 residues and deletion of Glu332-Asp333 prevents functional expression at the cell surface. Thus, this invariant sequence in the glycoprotein hormones is required for proper folding, trafficking, and ligand-mediated signaling, but not ligand binding, in LHR. Amino acid residues, Glu332, Asp333, and to a limited extent, Pro330, are important in ligand-mediated signaling but not ligand binding.

scholarly journals Specificity and promiscuity of gonadotropin receptors

Reproduction ◽  
2005 ◽  
Vol 130 (3) ◽  
pp. 275-281 ◽  
Author(s):  
Sabine Costagliola ◽  
Eneko Urizar ◽  
Fernando Mendive ◽  
Gilbert Vassart
Keyword(s):  
Hormone Receptors ◽  
Fsh Receptor ◽  
Ovarian Hyperstimulation ◽  
High Concentration ◽  
Glycoprotein Hormone ◽  
Functional Specificity ◽  
The Past ◽  
Glycoprotein Hormone Receptors ◽  
Naturally Occurring ◽  
Available Information

The dichotomy between hormone recognition by the ectodomain and activation of the G protein by the rhodopsin-like serpentine portion is a well established property of glycoprotein hormone receptors. The specificity barrier avoiding promiscuous activation of the FSH receptor by the high concentration of human chorionic gonadotropin (hCG) prevailing during human pregnancy was thus believed to lie in the ectodomain. In the past two years, mutations responsible for rare spontaneous cases of ovarian hyperstimulation syndromes have partially modified this simple view. Five naturally occurring mutations have been identified which cause an increase in the sensitivity of the FSH receptor to hCG. Surprisingly, these mutations are all located in the serpentine portion of the receptor. In addition to their effect on sensitivity to hCG, they increase sensitivity of the FSH receptor to TSH, and are responsible for activating the receptor constitutively. Together, the available information indicates that the ectodomain and the serpentine domain of the FSH receptor each contribute to the specificity barrier preventing its spurious activation by hCG. While the former is responsible for establishment of binding specificity, the latter introduces a novel notion of functional specificity.Recent data demonstrate that LH and FSH receptors can constitute functional homo- and heterodimers. This suggests the possibility that in cells co-expressing the two receptors, such as granulosa cells, the heterodimers might be endowed with functional characteristics different from those of each homodimer.

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