Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor

The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of ϳ330 amino acids, the N-terminal extracellular exodomain and membraneassociated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/ exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of 580 KVPLITVSKAK 590 was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH ␣ but not FSH ␤. This is in contrast to photoaffinity labeling of FSH ␤ by the peptide mimic of the N-terminal region of the receptor. The FSH1 receptor (FSHR) 1 and other glycoprotein hormone (luteinizing hormone/chorionic gonadotropin and thyroid-stimulating hormone) receptors belong to a structurally unique subfamily of G protein-coupled receptors. Unlike other receptor subfamilies, they comprise two equal halves, an N-terminal extracellular half (exodomain) and a C-terminal membraneassociated half (endodomain) (1-4). The exodomain is ϳ350 amino acids long and alone is capable of high affinity hormone binding (5-8) with hormone selectivity (9 -11) but without hormone action (7,12). Receptor activation occurs in the endodomain (13), which is structurally equivalent to the entire molecule of many other G protein-coupled receptors (14). Glycoprotein hormones initially bind to the exodomain, and then the resulting hormone/exodomain complex modulates the endodomain (13), which activates adenylyl cyclase (AC) to generate cAMP and phospholipase C␤ (PLC␤) to produce inositol phosphate and diacylglycerol. Therefore, the ternary interactions among the hormone, exodomain, and endodomain are crucial for successful signal generation. However, there is little information on the subject, particularly concerning the FSH receptor. Since the exodomain lacking the endodomain is capable of high affinity hormone binding (5-8, 15), the high affinity hormone binding appears to be independent of the endodomain. Contrary to this view, it has been reported that FSH and human chorionic gonadotropin binding to their cognate receptors is regulated by certain residues of exoloops 2 and 3 of the endodomain (15, 16). Furthermore, the hinge region of the exodomain interacts with exoloop 2 and modulates cAMP induction (17-19). These results suggest that the exodomain interacts with the exoloops and modulates them for signal generation. Yet it is unclear whether the hormone complexed with the exodomain also contacts the exoloops.In this study, we set out to investigate whether exoloops interact with the hormone at all. In a step toward this goal, we examined exoloop 3 of FSHR for its involvement in the activation of the two eff...

Section: Mutagenesis and Functional Expression Of Human Fsh Receptor-mentioning

confidence: 90%

“…Unlike other receptor subfamilies, they comprise two equal halves, an N-terminal extracellular half (exodomain) and a C-terminal membraneassociated half (endodomain) (1)(2)(3)(4). The exodomain is ϳ350 amino acids long and alone is capable of high affinity hormone binding (5)(6)(7)(8) with hormone selectivity (9 -11) but without hormone action (7,12).…”

mentioning

confidence: 99%

Follicle-stimulating Hormone Interacts with Exoloop 3 of the Receptor

Sohn

Ryu

Sievert

et al. 2002

“…Unlike other receptor subfamilies, they comprise two equal halves, an extracellular N-terminal half (exodomain) and a membrane-associated C-terminal half (endodomain) (7)(8)(9)(10)(11). The exodomain is ϳ350 amino acids long, and it alone is capable of high affinity hormone binding (12)(13)(14)(15) with hormone selectivity (16 -18) but without hormone action (14,19,20).…”

mentioning

confidence: 99%

Two Defective Heterozygous Luteinizing Hormone Receptors Can Rescue Hormone Action

Lee

Ryu

et al. 2002

Luteinizing hormone receptor is a G protein-coupled receptor and consists of two halves: the N-terminal extracellular half (exodomain) and C-terminal membraneassociated half (endodomain). Hormone binds to the exodomain, and the resulting hormone-exodomain complex modulates the endodomain to generate signals. There are mutations that impair either hormone binding or signal generation. We report that the coexpression of a binding defective mutant and a signal-defective mutant rescues signal generation to produce cAMP. This rescue requires both types of mutant receptors and is dependent on the human chorionic gonadotropin dose, the surface concentration of mutant receptors, and the amino acid position of mutations. Furthermore, random collisions among mutant receptors are not involved in the rescue. Our observations provide new insights into the mechanisms of the functional and structural relationship of the exo-and endodomain, signal transduction, and receptor genetics, in particular for defective heterozygotes.

“…Because of the importance of the TSHR as an autoantigen, a large effort has been made in the 7 years since the molecular cloning of its cDNA (3)(4)(5) to generate this protein in various expression systems, including bacteria (6 -11), insect cells (12)(13)(14)(15)(16), stably transfected mammalian cells (3,(17)(18)(19)(20)(21), and cell-free translation (22), as well as by peptide synthesis (23,24). However, the generation of effective TSHR antigen has been extraordinarily difficult.…”

mentioning

confidence: 99%

Engineering the Human Thyrotropin Receptor Ectodomain from a Non-secreted Form to a Secreted, Highly Immunoreactive Glycoprotein That Neutralizes Autoantibodies in Graves' Patients' Sera

Chazenbalk

Jaume

McLachlan

et al. 1997

Self Cite

110

Previous attempts to generate autoantibody-reactive, secreted thyrotropin receptor (TSHR) ectodomain in mammalian cells have failed because of retention within the cell of material with immature carbohydrate. We have overcome this difficulty by performing progressive carboxyl-terminal truncations of the human TSHR ectodomain (418 amino acid residues including signal peptide). Three ectodomain variants (TSHR-261, TSHR-289, and TSHR-309) were truncated at residues 261, 289, and 309, respectively. Unlike the full ectodomain, ectodomain variants were secreted with an efficiency inversely proportional to their size. Secreted ectodomain variants contained ϳ20 kDa of complex carbohydrate. TSHR-261 was chosen for further study because it was secreted very efficiently and neutralized autoantibodies in Graves' patients' sera. This ectodomain variant was partially purified using sequential lectin and nickel-chelate chromatography, permitting the first direct visualization and quantitation of the mammalian TSHR. Most important, very small (nanogram) quantities of this material neutralized 70 -100% of TSHR autoantibody activity in all 18 Graves' sera studied.In summary, carboxyl-terminal truncation of the human