| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Division of Reproductive Biology (Y.O., S.-G.L., A.J.W.H), Department of Gynecology/Obstetrics, Stanford University School of Medicine, Stanford, California 94305; Department of Pediatrics (J.S.D.), University of Texas Medical Branch, Galveston, Texas 77555; and Division of Endocrinology, Department of Medicine (C.W.), Harbor-UCLA Medical Center, Torrance California 90502
Address all correspondence and requests for reprints to: Aaron J. W. Hseuh, M.D., Stanford University School of Medicine, Department of Gynecology/Obstetrics, 300 Pasteur Drive, Stanford, California 94305-5317.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Attempts have been made to obtain soluble ectodomains of TSH receptors. However, recombinant receptor fragments produced by bacterial, insect, and mammalian cells were either inadequately folded and required refolding or were trapped intracellularly as partially glycosylated low-affinity binders (3, 4, 6). Our recent study indicated that functional ectodomains of gonadotropin and TSH receptors could be expressed on the cell surface by attaching them to a heterologous membrane anchor with a cleavable linker (7). After enzymatic treatment, soluble binding proteins specific for gonadotropins or TSH could be generated. Here, a mutant of the ectodomain of the human TSH receptor was generated with the deletion of a stretch of nine amino acids known to be important for TSH binding (2). The mutant molecule was defective in TSH binding but retained the ability to neutralize the stimulatory effects of Graves’ IgGs in in vitro assays. The present approach provides the possibility of dissociating the actions of TSH and stimulatory thyroid autoantibodies.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
), the ectodomain of human TSH receptor
(amino acids 1–390, Ref.8) was fused to the single transmembrane and
cytoplasmic region of CD8 (amino acid 162 to C terminus, Ref.9)
through a stretch of the thrombin receptor sequence using the plasmid
SK-ATE-CD8 (10) containing the thrombin cleavage site (amino acids
36–66 of thrombin receptor, Ref.11). A Flag epitope for monoclonal
antibody M1 followed by six histidine residues was also fused to the N
terminus of this construct to allow efficient purification and
immunoblotting. The resulting junctions encoded the following
sequences: between Flag epitope and TSH receptor,
—DDDDVD/HHHHHH/GMGCSSP—; between TSH receptor and thrombin receptor,
—FNPCED/ATLDP—; between thrombin receptor and CD8, — NESGL/IYIWA —.
We also constructed mTtCD8, a mutant of TtCD8 in which nine amino acids
(368–376: YTICGDSED, Ref.8) of the TSH receptor were deleted from
TtCD8 (Fig. 1A). In addition, the wild-type TSH receptor was appended
with the Flag epitope fused to the N terminus for antibody recognition.
To express wild-type and chimeric receptors in human embryonic kidney
293 cells, receptor complementary DNAs (cDNAs) were subcloned into
pcDNA3 (Invitrogen, San Diego, CA). Cell lines stably expressing TtCD8,
mTtCD8, or wild-type TSH receptors were prepared by selection with
geneticin (GIBCO, Gaithersburg, MD) and maintained in DMEM/F12
containing 10% FBS and 100 µg/ml geneticin.
|
-thrombin (Enzyme Research Laboratories, South Bend, IN). Three days
later, -thrombin (3 U/ml) was again added for 12 h before media
were collected, centrifuged, and filtered through 0.22-µm filters
(Corning, Cambridge, MA). Filtered media were incubated at 4 C with the
Sepharose Fast Flow resin covalently linked with nickel (Pharmacia,
Uppsala, Sweden) in Tris-HCl, pH 7.4, under high-salt conditions (500
mM NaCl and 20 mM imidazole). After 3 h of
slow rotation, the resin was washed with the same buffer before elution
of bound proteins with 200 mM imidazole at pH 8.0. The
eluted proteins were further immunopurified with an affinity column
containing the M1 antibody against the Flag epitope (Kodak, New Haven,
CT). After elution of bound proteins with 4 mM EDTA, the
eluant was concentrated using Centricon 30 (Amicon, Bedford, MA) before
protein determination using a protein assay kit (Bio-Rad, Hercules,
CA). Soluble proteins derived from TtCD8 and mTtCD8 were named TBP
(TSH-binding protein) and mutant (m)TBP, respectively (Fig. 1
).
Immunoblotting, enzymatic deglycosylation, and ligand cross-linking
analyses
Affinity-purified TBP and mTBP were separated on 7.5% SDS-PAGE
gels and stained with Coomassie brilliant blue G250 in 40% methanol
and 10% acetic acid. For immunoblotting, the proteins were transferred
to nitrocellulose membranes and incubated with the M1 antibody using
the enhanced chemiluminescence (ECL) Western blotting system (Amersham,
Buckinghamshire, U.K.). For deglycosylation with endoglycosidase F
(Boehringer-Mannheim, Indianapolis, IN), aliquots were diluted 10 times
in the deglycosylation buffer (50 mM sodium phosphate
buffer, pH 7.4, 1% SDS, 1% ß-mercaptoethanol, 0.5% Nonidet-P40,
and 25 mM EDTA) and incubated with 10 U/30 µl
endoglycosidase F at 37 C for 16 h. The samples were mixed with
Laemmli buffer under reducing conditions (100 mM
dithiothreitol and 5% mercaptoethanol) for immunoblotting analysis.
Lysate of 293 cells transiently transfected with Flag-tagged wild-type
TSH receptors served as a control.
For ligand cross-linking analyses, purified TBP or mTBP (100 ng) were incubated with 5,000 cpm of bovine [125I]TSH (50 µCi/µg; Kronus, San Clemente, CA) with or without 20 µg bovine TSH in 100 µl NaCl-free HBSS containing 280 mM sucrose for 3 h at 23 C. Complexes formed between [125I]TSH and TBP or mTBP were cross-linked using disuccinimidyl suberate (2 mM) for 1 h before termination of the reaction using 3.6 mM Tris-HCl, pH 7.4. After the addition of Laemmli buffer under reducing conditions, cross-linked complexes were resolved after fractionation using polyacrylamide (7.5%) gel electrophoresis and autoradiography. In competition experiments, increasing concentrations of bovine TSH were included in the reaction mixture, and data from displacement analysis based on autoradiography were used to estimate Kd (equilibrium binding constant) values.
Preparation of IgGs and immunoprecipitation
Sera were collected from patients with Graves’ disease showing
elevated thyroid-stimulating Ig levels. All patients had clinical
symptoms of hyperthyroidism with characteristic eye signs. Serum
hormone measurement of these patients showed suppressed serum TSH
levels of <0.1 µU/ml and elevated serum T4 levels of
>11 µg/dl. All serum samples showed clear stimulation (>10-fold
higher than normal serum) of cAMP production by transfected cells
expressing recombinant human TSH receptors.
To purify IgG, sera from Graves’ patients and from normal controls were incubated under constant agitation with Protein G Sepharose Fast Flow resin (Pharmacia) for 1 h at 23 C. After washing with PBS, the IgG fraction was eluted with 0.1 M glycine-HCl (pH 3.0), followed by neutralization with 0.5 M Tris-HCl, pH 8.0. The eluant was then concentrated using Centricon 50 (Amicon) and dialyzed against PBS at pH 7.4. For immunoprecipitation of TBP and mTBP by IgG, 100 µl Protein G resin were preincubated with PBS-5% BSA, followed by 100 µl affinity-purified IgG at 4 C for 2 h with intermittent agitation. After washing, the IgG-bound resin was incubated with TBP or mTBP in PBS-5% BSA at 4 C for another 2 h before extensive washing with PBS-5% BSA and PBS (x5). Proteins bound to the resin were recovered by incubation with Laemmli buffer under reducing conditions and used for immunoblotting analysis.
TSH receptor binding and cAMP assays
The ability of TBP and mTBP to interfere with TSH binding to TSH
receptor was tested using a ligand-binding assay. The 293 cells (2
x 105) stably expressing wild-type TSH receptors were
incubated with [125I]TSH (10,000 cpm/tube) with or
without TBP or mTBP in 300 µl binding buffer (NaCl-free HBSS, 280
mM sucrose, and 0.5% BSA). After incubation for 3 h
at 23 C, cells were washed with the buffer and centrifuged before
counting radioactivity in the pellet using a 
-counter. To analyze
the ability of TBP and mTBP to interfere with signal transduction
induced by TSH or Graves’ IgGs, TSH or IgGs were premixed with TBP or
mTBP. After preincubation for 2 h at 23 C, samples were added to
293 cells (2 x 104/well) expressing wild-type TSH
receptor in 96-well plates for 3 h at 37 C. Total cAMP production
was determined by RIA (12).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). The 293 cells (7.5 x
108) stably transfected with TtCD8 or mTtCD8 were treated
with -thrombin to derive 1 liter of serum-free media containing TBP
or mTBP. The mTBP represented a protein with nine amino acids deleted
in the C terminus of the extracellular region of the human TSH
receptor. A key cysteine (amino acid 371) in this region is believed to
be important for an S-S bond that is critical for TSH binding by the
holoreceptor (2, 3, 4, 5). Taking advantage of the polyhistidine tag and Flag
epitopes added to their N terminus, 
100 µg of TBP or mTBP could be
affinity purified using sequential nickel and M1 antibody columns.
After electrophoresis, Coomassie staining allowed detection of purified
proteins mostly at an apparent molecular mass of 82 kDa with small
amounts at 60 kDa (Fig. 1B, left panel). These bands
corresponded to similar ones detected by Western blotting of the Flag
epitope present in these proteins (Fig. 1B, right panel). To
determine whether these proteins are properly glycosylated,
N-glycosidase F digestion was performed (Fig. 1C). Treatment
with the enzyme decreased the sizes of TBP and mTBP to 52 kDa and 35
kDa, showing a decrease of 25–30 kDa in size. Under the same
electrophoresis conditions, the wild-type TSH receptors showed two
bands at 115 and 98 kDa, which is consistent with the presence of a
larger protein with complex carbohydrate side chains and an immature
one with mannose-rich side chains (13). Furthermore, treatment with
N-glycosidase F decreased the sizes of both forms to yield a
single band of 84 kDa, indicating a decrease of 30 kDa for the fully
glycosylated larger form. These data suggested that TBP and mTBP are
N-glycosylated and most likely have complex carbohydrate
side chains.
mTBP does not bind TSH but retains its binding to Graves’ IgG
Despite the presence of the N-terminal epitope tags, cross-linking
analysis indicated that TBP formed complexes with
[125I]TSH and showed a single band at 115 kDa (Fig. 2A). The complex formation was blocked
with excess nonlabeled TSH. In contrast, no complex formation was
detected between mTBP and [125I]TSH under the same
conditions. These findings suggested that mTBP was not capable of
binding TSH whereas only the large molecular mass form of TBP (82 kDa)
was capable of forming complexes with labeled TSH (30 kDa). To estimate
the binding affinity between TBP and TSH, competition analyses were
performed. As shown in Fig. 2B, [125I]TSH cross-linked to
purified TBP could be displaced in a dose-dependent manner by the
inclusion of increasing concentrations of nonlabeled TSH. The
Kd value for TSH binding to TBP was estimated to be 1.5
nM. Furthermore, the ability of TBP and mTBP to interact
with TSH was evaluated indirectly in a ligand-binding assay using cells
expressing wild-type TSH receptors. As shown in Fig. 2C, addition of
increasing amounts of TBP decreased the amount of
[125I]TSH available for binding to wild-type TSH
receptors in a dose-dependent manner, reaching a level similar to that
achieved by excess TSH. The ED50 for TBP displacement was
estimated to be 100 ng/ml or 1 x 10-9
M. Again, treatment with mTBP (up to 3 µg/ml) did not
interfere with TSH binding to its receptors.
|
, both TBP and mTBP could be precipitated with affinity
beads containing Graves’ IgGs obtained from three different patients.
In contrast, neither TBP nor mTBP could interact with IgGs obtained
from different normal controls.
Effect of treatment with TBP and mTBP on cAMP production induced by
TSH and Graves’ IgGs
To study the ability of TBP and mTBP to block signal transduction
induced by TSH or Graves’ IgGs, 293 cells expressing wild-type TSH
receptors were incubated with 50 ng/ml TSH or 0.5 mg/ml Graves’ IgG
with or without increasing concentrations of purified TBP or mTBP. As
shown in Fig. 3A, treatment with TBP
dose-dependently prevented cAMP production induced by TSH with an
ED50 of 150 ng/ml. At 1 µg/ml of TBP, the stimulatory
effect of TSH was completely blocked. In contrast, treatment with up to
20 µg/ml of mTBP did not interfere with TSH action.
|
, treatment with increasing doses of either TBP or mTBP inhibited, with
similar potencies, cAMP production induced by Graves’ IgG. At 1
µg/ml of either TBP or mTBP, complete suppression of IgG action was
observed. Because IgGs from Graves’ patients might show individual
variability in their interaction with mTBP, IgGs obtained from nine
additional patients were tested. As shown in Fig. 3C, treatment with
either TBP or mTBP blocked cAMP production induced by all of the
patient IgGs tested. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-fragments
derived from wild-type receptors (13, 14, 15). Treatment with
N-glycosidase F further indicated that the high molecular
mass forms of these proteins were most likely to be fully glycosylated
because the sizes of their carbohydrate side chains were comparable to
that of the wild-type TSH receptor (Fig. 1C). TBP interacted with high
affinity to TSH as well as blocked cAMP production induced by TSH or
Graves’ IgGs. In contrast, mTBP lost its ability to bind TSH but was
still capable of binding Graves’ IgGs. The mutant protein blocked
signal transduction induced by IgGs from 10 Graves’
patients. Multiple discontinuous epitopes on the TSH receptor are required for ligand binding (2, 3, 4, 5). Although different approaches have been used to generate the ectodomain of the TSH receptor, soluble proteins with ligand-binding characteristics similar to that of the intact TSH receptors have been difficult to obtain. The ectodomains derived after in vitro protein translation (16) or produced in prokaryotic cells (17) recognize TSH poorly, primarily due to the lack of correct disulfide bonds essential for protein folding and protein aggregation. Some of the refolded molecules also lacked the ability to recognize Graves’ antibodies (18, 19, 20). Although ectodomains could also be produced in insect cells, they were not properly glycosylated and showed low affinity to TSH (21). Recently, a fragment of the TSH receptor ectodomain was shown to be cleaved from thyroid cells by a metalloprotease (15) and retained TSH binding.
The present high molecular mass form of TBP generated from mammalian
cells was likely to be folded correctly because it retained high
binding affinity for TSH (Fig. 2, A and B). In a functional bioassay,
TBP competed for the action of TSH (50 ng/ml) at comparable molar
ratios with an ED50 value of 150 ng/ml (Fig. 3A). In
addition, TBP appeared to be more potent than ectodomains derived from
insect cells in the inhibition of [125I]TSH binding to
wild-type receptors (18). The observation that treatment with
endoglycosidase H was unable to cleave the high molecular mass forms of
both TBP and mTBP (data not shown) further suggested that their
carbohydrate side chains were not of the mannose-rich type. In contrast
to the high molecular mass forms, the low molecular mass forms of TBP
and mTBP that resembled the A subunit/-fragment of TSH receptors
showed negligible binding to labeled TSH in ligand cross-linking
experiments. These fragments could have been missing essential
TSH-binding motifs near the C terminus. They also showed carbohydrate
side chains of a smaller size (25 kDa) than that of the wild-type
receptors. In addition, the ratio of low and high molecular mass forms
varied in different experiments probably due to varying levels of
endogenous proteases. The lack of TSH binding by the low molecular mass
form of TBP was consistent with the finding that mTBP, with nine amino
acids deleted near the C terminus of TBP, also could not bind TSH.
Because the -form of the TSH receptor retained TSH binding (15),
future studies to compare the characteristics of this cleaved receptor
fragment and the low molecular mass form of TBP are of interest.
Consistent with previous findings using mutant TSH receptors (2, 22), this study indicated that amino acid residues 368 to 376 in TBP are important for TSH binding. Identification of ligand-binding sites in holoreceptors was complicated by the variable expression levels of mutant receptors and possible influences by their transmembrane region. Studies based on chimeric and mutant receptors suggested the involvement of the middle portion of the TSH receptor ectodomain (23) and regions near the C-terminal of ectodomain (22) for TSH binding. In contrast, a synthetic peptide approach mapped TSH-binding sites to these regions as well as other distinct domains (24). Studies using antibodies specific for synthetic peptide fragments of TSH receptors further identified additional epitopes for TSH receptor binding (4). The present soluble ectodomain approach could be useful for further elucidation of TSH-binding epitopes.
Based on interactions between patient IgGs and the ectodomain of mouse TSH receptors, specific amino acid residues of the human TSH receptor were shown to be important for antibody binding (18, 19). In addition, correctly folded ectodomains with proper glycosylation of complex carbohydrate side chains were also shown to be essential for recognition by autoimmune antibodies (6, 20). The epitopes in the TSH receptor for binding by autoimmune antibodies varied between individual patients and might not overlap with that for TSH binding. Despite the known heterogeneity of recognition sites for Graves’ IgGs from individual patients, it is interesting to note that the present mTBP, with deletion of a small stretch of nine amino acids in the C-terminal region of TBP, still recognized Graves’ antibodies from all 10 Graves’ patients tested.
In addition to its utility as a reagent to analyze interactions between Graves’ IgGs and the ectodomain of TSH receptors (18), the soluble mTBP might also be of therapeutic value because it lost TSH-binding ability but could still block signal transduction induced by Graves’ IgGs. The present treatment for Graves’ disease includes antithyroid drugs, radioactive iodine, and, to a lesser extent, thyroid surgery (25). The first two treatments usually required several weeks to induce an euthyroid state. For pregnant patients, radioactive iodine is also contraindicated (26). Because mTBP could, in theory, rapidly block the stimulatory effects of Graves’ IgGs without disturbing the normal response to TSH, it might provide rapid alleviation of Graves’ symptoms and maintain euthyroidism. After optimization of its delivery, mTBP could provide an alternative approach to surgery in pregnant patients allergic to antithyroid medications.
Because both the etiology underlying Graves’ disease and the endogenous antigens triggering the stimulatory antibodies are still unknown, it is uncertain whether mTBP might be antigenic with long-term use. Earlier studies indicated that peptides coding suspected antigenic residues of TSH receptors had only weak stimulatory effects on the proliferation of peripheral blood lymphocytes in Graves’ patients (27), whereas EBV-transformed B cell lines transfected with TSH receptors were potent in stimulating the proliferation of cloned T cells from Graves’ thyroid (28). Attempts to develop an animal model for Graves’ disease after immunization of animals with ectodomain of TSH receptor have been difficult. Recently, immunization with fibroblasts expressing both TSH receptors and class II molecules were shown to induce Graves’ symptoms in mice (29). It is possible that treatment with mTBP alone, in the absence of other coeffectors, might not exacerbate Graves’ symptoms. In addition to its potential neutralization of Graves’ IgGs, administration of mTBP could also be used for studies on oral tolerance (30). Further studies on immune responses to mTBP and TBP will be of interest.
In summary, we have generated a soluble TSH receptor ectodomain mutant capable of blocking the stimulatory effects of Graves’ IgGs on TSH receptor activation in vitro, but not interfering with the action of TSH. The present soluble ectodomain approach could allow further mapping of binding epitopes for TSH and thyroid-stimulating or -blocking antibodies found in diseased states. Mutant mTBP could be useful for studies on the pathogenesis of Graves’ disease and may eventually provide an alternative therapy for these patients.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Y.O. is on leave from the Department of Obstetrics and Gynecology,
University of Tokyo, Tokyo, Japan.
Received March 13, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  K. J. Land, P. Gudapati, M. H. Kaplan, and G. S. Seetharamaiah Differential Requirement of Signal Transducer and Activator of Transcription-4 (Stat4) and Stat6 in a Thyrotropin Receptor-289-Adenovirus-Induced Model of Graves' Hyperthyroidism Endocrinology, January 1, 2006; 147(1): 111 - 119. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Latrofa, G. D. Chazenbalk, P. Pichurin, C.-R. Chen, S. M. McLachlan, and B. Rapoport Affinity-Enrichment of Thyrotropin Receptor Autoantibodies from Graves' Patients and Normal Individuals Provides Insight into Their Properties and Possible Origin from Natural Antibodies J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4734 - 4745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J. Land, J. S. Moll, M. H. Kaplan, and G. S. Seetharamaiah Signal Transducer and Activator of Transcription (Stat)-6-Dependent, But Not Stat4-Dependent, Immunity Is Required for the Development of Autoimmunity in Graves' Hyperthyroidism Endocrinology, August 1, 2004; 145(8): 3724 - 3730. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.-N. E. Dogan, C. Vasu, M. J. Holterman, and B. S. Prabhakar Absence of IL-4, and Not Suppression of the Th2 Response, Prevents Development of Experimental Autoimmune Graves' Disease J. Immunol., February 15, 2003; 170(4): 2195 - 2204. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Smits, C. Govaerts, I. Nubourgh, L. Pardo, G. Vassart, and S. Costagliola Lysine 183 and Glutamic Acid 157 of the TSH Receptor: Two Interacting Residues with a Key Role in Determining Specificity toward TSH and Human CG Mol. Endocrinol., April 1, 2002; 16(4): 722 - 735. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Vlaeminck-Guillem, S.-C. Ho, P. Rodien, G. Vassart, and S. Costagliola Activation of the cAMP Pathway by the TSH Receptor Involves Switching of the Ectodomain from a Tethered Inverse Agonist to an Agonist Mol. Endocrinol., April 1, 2002; 16(4): 736 - 746. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. D. Chazenbalk, S. M. McLachlan, P. Pichurin, X.-M. Yan, and B. Rapoport A Prion-Like Shift between Two Conformational Forms of a Recombinant Thyrotropin Receptor A-Subunit Module: Purification and Stabilization Using Chemical Chaperones of the Form Reactive with Graves' Autoantibodies J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1287 - 1293. [Abstract] [Full Text]
|
|
|
|
|
|
S. Kaithamana, J. Fan, Y. Osuga, S.-G. Liang, and B. S. Prabhakar Induction of Experimental Autoimmune Graves' Disease in BALB/c Mice J. Immunol., November 1, 1999; 163(9): 5157 - 5164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Lee, J. Y. Park, B. Y. Cho, and C.-B. Chae Expression of the Functional Extracellular Domain of Human Thyrotropin Receptor Using a Vaccinia Virus System: Its Purification and Analysis of Autoantibody Binding J. Clin. Endocrinol. Metab., April 1, 1999; 84(4): 1391 - 1397. [Abstract] [Full Text]
|
|
|
|
|
|
M. Hayashi, E. A. McGee, G. Min, C. Klein, U. M. Rose, M. v. Duin, and A. J. W. Hsueh Recombinant Growth Differentiation Factor-9 (GDF-9) Enhances Growth and Differentiation of Cultured Early Ovarian Follicles Endocrinology, March 1, 1999; 140(3): 1236 - 1244. [Abstract] [Full Text]
|
|
|
|
|
|
G. D. Chazenbalk, Y. Wang, J. Guo, J. S. Hutchison, D. Segal, J. C. Jaume, S. M. McLachlan, and B. Rapoport A Mouse Monoclonal Antibody to a Thyrotropin Receptor Ectodomain Variant Provides Insight into the Exquisite Antigenic Conformational Requirement, Epitopes and in Vivo Concentration of Human Autoantibodies J. Clin. Endocrinol. Metab., February 1, 1999; 84(2): 702 - 710. [Abstract] [Full Text]
|
|
|
|
 |
|
 Y. Nagayama, H. Namba, N. Yokoyama, S. Yamashita, and M. Niwa Role of Asparagine-linked Oligosaccharides in Protein Folding, Membrane Targeting, and Thyrotropin and Autoantibody Binding of the Human Thyrotropin Receptor J. Biol. Chem., December 11, 1998; 273(50): 33423 - 33428. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Rapoport, G. D. Chazenbalk, J. C. Jaume, and S. M. McLachlan The Thyrotropin (TSH)-Releasing Hormone Receptor: Interaction with TSH and Autoantibodies Endocr. Rev., December 1, 1998; 19(6): 673 - 716. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
