Biomedical subjects

T Akamizu

Publications and source records attributed to T Akamizu.

At least 73 records · Page 4Linked to original sources

PubMed1993

Use of thyrotropin receptor (TSHR) mutants to detect stimulating TSHR antibodies in hypothyroid patients with idiopathic myxedema, who have blocking TSHR antibodies.

Deletions of residues 295-306, 299-301, and 387-395 of the TSH receptor, as well as point mutations of cysteine 301 or 390 to serine, and tyrosine 385 to phenylalanine or alanine, markedly diminish the ability of a transfected receptor to measure the activity of blocking TSH receptor autoantibodies (TSHRAbs) in patients with idiopathic myxedema and hypothyroidism, but not stimulating TSHRAbs in Graves' patients. This has allowed us to use these mutants to detect stimulating TSHRAb activity in the sera of hypothyroid patients with idiopathic myxedema who have blocking TSHRAbs. In 7 such patients, we show that 50% or more have significant stimulatory activity in cells transfected with mutant receptors, as evidenced by the ability of the immunoglobulin G to directly increase cAMP levels or to enhance the ability of TSH or a Graves' stimulating TSHRAb to increase cAMP levels. Three of the TSH receptor mutants, deletions of residues 295-306 and 387-395 and the point mutation of cysteine 301 to serine, are shown to be particularly useful in these assays and may be useful to clarify the pathogenetic role and clinical significance of stimulating TSHRAbs in patients with autoimmune thyroid disease who also have blocking TSHRAbs.

Animals↗

PubMed1993

Apparent genetic difference between hypothyroid patients with blocking-type thyrotropin receptor antibody and those without, as shown by restriction fragment length polymorphism analyses of HLA-DP loci.

HLA types in Japanese patients with primary hypothyroidism were analyzed to see whether those with blocking-type TSH receptor antibody (TSH-R BAb M) differed genetically from those with idiopathic myxedema (IM). HLA typings of -A, -B, -C, -DR, and -DQ (73 antigens) were performed serologically, and those of -D and -DP (29 antigens) were analyzed by the restriction fragment length polymorphism method. Thirty patients were studied with TSH-R BAb M, and 28 with IM. The data were analyzed and compared with our previous results from 88 Graves' patients, 46 Hashimoto patients, and 186 control subjects. Overall, 192 patients with 4 autoimmune thyroid disorders showed a decrease in -Aw19 and an increase in -DQw4 (corrected P < 0.05) and significant associations of -Aw33, -Bw46, -Cw3, -DRw8, -DR9, and -DQw3. In TSH-R BAb M patients, increases in -B35, -Bw60, and -Dw8 and decreases in -DR4 and -DPw2 were seen, whereas IM patients showed increased -DPw2, -Bw61, and -Dw23. In comparisons between TSH-R BAb M and IM, the difference in -DPw2 was highly significant. HLA-B35 differed significantly in these 2 types of hypothyroidism. In conclusion, TSH-R BAb M patients have decreased frequency of -DPw2 and are genetically similar to Graves' disease, whereas IM patients are characterized by high frequency of -DPw2 and are genetically similar to Hashimoto's thyroiditis.

Adult↗

PubMed1993

Chimeric studies of the extracellular domain of the rat thyrotropin (TSH) receptor: amino acids (268-304) in the TSH receptor are involved in ligand high affinity binding, but not in TSH receptor-specific signal transduction.

A series of chimeric TSH-LH/CG receptors were constructed by substituting homologous segments of the extracellular domain of the rat TSH receptor with corresponding segments of rat LH/CG receptor: C1 (amino acids 37-123 substituted), C2 (91-112), C3 (173-234), C4 (233-266), C5 (268-304), C6 (112-305) and C7 (36-404). After transfection in Cos-7 cell, TSH- and LH/CG-receptor activities of these chimeras were evaluated and compared with those of deletion mutants involving the same residues [Kosugi et al. Thyroid 1:321 (1991)]. Western blot analyses revealed that most of the chimeric receptor proteins were normally synthesized and integrated in the membrane of transfected Cos-7 cells: an antibody to a TSH receptor specific synthetic peptide (residues 352-366) identified 170-190kDa and 90-100kDa TSH receptor structures in the plasma membrane fractions of Cos-7 cells transfected with wild-type TSH receptor cDNA and the C1 to C6 chimeras, but not C7 or wild LH/CG receptor cDNA. Despite this, no receptor except C5 exhibited any significant TSH receptor activities either in [12I]TSH binding or in cAMP responses to TSH and thyroid-stimulating antibodies (TSAbs) from Graves' patients. The chimeric receptor C5 exhibited only low affinity TSH binding (Kd = 3.5 x 10(-8) M), as did its counterpart the M2C mutant with residues 268-304 deleted. However, unlike M2C, C5 demonstrated a significant cAMP response to TSH as well as to TSAbs. The cAMP increase in response to TSH in the wild type receptor was observed at 10(-11) M TSH. In C5 the response was first evident at 10(-10) M TSH, but the maximum cAMP stimulation by TSH and TSAbs in C5 (EC50 = 6.7 x 10(-10) M) was approximately the same as the wild type receptor (EC50 = 1.5 x 10(-10) M). Inhibition of either TSH- or TSAb- stimulated cAMP increase by thyroid-stimulating blocking antibodies (TSBAbs) was also preserved in C5. These results suggest that amino acids 268-304 do not include an important determinant required for signal transduction, since a significant cAMP response to TSH and TSAbs was observed in the C5 receptor with these residues substituted. Additionally, these residues appear to be involved in ligand high affinity binding because high affinity TSH binding was lost in the chimeric receptor C5.

Amino Acid Sequence↗

PubMed1992

Role of cysteine residues in the extracellular domain and exoplasmic loops of the transmembrane domain of the TSH receptor: effect of mutation to serine on TSH receptor activity and response to thyroid stimulating autoantibodies.

The extracellular domain of the thyrotropin (TSH) receptor is the primary site with which TSH and receptor autoantibodies interact. Cysteines 494 or 569 in the 1st and 2nd exoplasmic loops, respectively, of the transmembrane domain of the TSH receptor are important in this process or in coupling ligand binding to signal generation. Thus, when either is mutated to serine, a receptor results which has no detectable TSH binding and no cAMP response to TSH or thyroid stimulating autoantibodies after transfection, despite the fact the mutant receptor is normally synthesized, processed, and integrated in the membrane, as evidenced by Western blotting using a TSH receptor-specific antibody. Additional site directed mutagenesis studies are performed in order to identify cysteine residues in the extracellular domain of the receptor which, with cysteines 494 and 569, are important for tertiary structure and receptor bioactivity.

Animals↗

PubMed1992

Regulation of thyrotropin receptor gene expression in rat FRTL-5 thyroid cells.

TSH receptor mRNA levels in FRTL-5 thyroid cells are autoregulated at a transcriptional level by the same hormones required for the growth and function of the cells: TSH, insulin, and insulin-like growth factor-I (IGF-I). Thus, the ability of TSH, via its cAMP signal, to down-regulate steady state receptor mRNA levels is preceded by the action of TSH to decrease pre-mRNA levels in nuclear run-on assays to the same quantitative level as evident in Northern analyses. In contrast, the receptor mRNA half-life is shown not to change when down-regulation is reversed by withdrawing TSH in the presence or absence of actinomycin-D. Evidence is additionally provided that TSH receptor mRNA levels are increased by insulin, IGF-I, or calf serum in both Northern and run-on assays. This action cannot be duplicated by hydrocortisone and is evident at more than 20-fold lower concentrations of IGF-I than insulin. Moreover, insulin, IGF-I, and/or calf serum are required for the autoregulatory negative transcriptional regulation of the TSH receptor by TSH/cAMP, as is the case for thyroglobulin. This occurs despite the opposite actions of TSH/cAMP on the two genes, positive in the case of thyroglobulin and negative with TSH receptor. The positive and negative regulatory actions, respectively, of insulin/IGF-I and TSH on receptor gene expression are associated with coincident increases or decreases in cell surface receptors measured by [125I]TSH binding. The autoregulation additionally involves the interplay of a second cAMP-modulated regulatory factor, one which up-regulates TSH receptor mRNA levels rather than causing down-regulation. Thus, cycloheximide inhibits the transcriptional action of both TSH/cAMP and insulin/IGF-I/serum within 4 h, i.e. a rapidly synthesized protein is an intermediate in both cases. The presence of cycloheximide for as little as 1 h, however, uncovers the ability of TSH/cAMP to increase TSH receptor mRNA levels. This activity is the result of the action of a stable cAMP-induced activator which can be detected physiologically, i.e. in the absence of cycloheximide. For example, low levels of a cAMP analog (0.2 mM), as opposed to high levels (greater than 1 mM), can increase TSH receptor RNA levels. Low levels also accelerate the insulin/IGF-I-dependent return of receptor mRNA to normal levels after TSH withdrawal.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Opposite regulation of deoxyribonucleic acid synthesis and iodide uptake in rat thyroid cells by basic fibroblast growth factor: correlation with opposite regulation of c-fos and thyrotropin receptor gene expression.

Basic fibroblast growth factor (bFGF) increases DNA synthesis in rat FRTL-5 thyroid cells, as measured by increased incorporation of tritiated thymidine into DNA. We show that this action is associated with the ability of bFGF to increase cytosolic Ca2+ levels and transiently increase c-fos mRNA levels. Other agents that increase c-fos mRNA levels and DNA synthesis in FRTL-5 cells include TSH, insulin, insulin-like growth factor-I, phorbol esters, A23187, and alpha 1-adrenergic agents; the last two agents also act by increasing cytosolic Ca2+ levels. Despite its enhancement of DNA synthesis, however, bFGF decreases TSH-induced cAMP-mediated iodide uptake. This action appears to reflect two separate actions of bFGF. First, bFGF decreases TSH receptor mRNA levels and the ability of TSH to acutely increase cAMP levels in FRTL-5 cells. The ability of bFGF to negatively regulate TSH receptor mRNA levels is additive to and independent of the ability of TSH and its cAMP signal to negatively autoregulate TSH receptor mRNA levels. This is consistent with the effect of bFGF on cytosolic Ca2+ levels and the ability of increased cytosolic Ca2+ to decrease TSH receptor mRNA levels. Second, bFGF inhibits cAMP signal expression, as evidenced by its ability to inhibit (Bu)2cAMP-induced iodide uptake in FRTL-5 cells. Both effects are, presumably, associated with the ability of bFGF to counteract TSH/cAMP-induced increases in thyroid peroxidase mRNA levels, which we demonstrate. We suggest, therefore, that bFGF causes opposite effects on DNA synthesis and iodide uptake because of its effect on cytosolic Ca2+ levels and because increases in cytosolic Ca2+ can have opposite effects on gene transcription, particularly in the case of the TSH receptor and c-fos genes.

Animals↗

PubMed1992

Identification of separate determinants on the thyrotropin receptor reactive with Graves' thyroid-stimulating antibodies and with thyroid-stimulating blocking antibodies in idiopathic myxedema: these determinants have no homologous sequence on gonadotropin receptors.

Deletions, substitutions, or mutations of the rat TSH receptor extracellular domain between residues 20 and 107 (all residue numbers are determined by counting from the methionine start site) have been made by site-directed mutagenesis of receptor cDNA. After transfection in Cos-7 cells, constructs were evaluated for their ability to bind [125I]TSH or respond to TSH and thyroid-stimulating antibodies (TSAbs) from Graves' patients in assays measuring cAMP levels of the transfected cells. Assay results were compared to results from Cos-7 cells transfected with wild-type receptor constructs or vector alone. We identify threonine-40 as a TSAb-specific site whose mutation to asparagine, but not alanine, reduces TSAb activity 10-fold, but only minimally affects TSH-increased cAMP levels. We show that thyroid-stimulating blocking antibodies (TSBAbs), which block TSH or TSAb activity and are found in hypothyroid patients with idiopathic myxedema, continue to inhibit TSH-stimulated cAMP levels when threonine-40 is mutated to asparagine or alanine, suggesting that TSBAbs interact with different TSH receptor epitopes than the TSAb autoantibodies in Graves' patients. This is confirmed by the demonstration that these TSBAbs interact with high affinity TSH-binding sites previously identified at tyrosine-385 or at residues 295-306 of the extracellular domain of the TSH receptor. This is evidenced by a loss in the ability of TSBAbs to inhibit TSAb activity when these residues are mutated or deleted, respectively. Since the TSAb and TSBAb epitopes are in regions of the extracellular domain of the TSH receptor that have no homology in gonadotropin receptors, these data explain at least in part the organ-specific nature of TSH receptor autoantibodies in autoimmune thyroid disease. Data are additionally provided which indicate that residues 30-37 and 42-45, which flank the TSAb epitope at threonine-40, appear to be ligand interaction sites more important for high affinity TSH binding than for the ability of TSH to increase cAMP levels and that cysteine-41 is critical for TSH receptor conformation and expression on the surface of the cell. Thus, despite unchanged maximal values for TSH-increased cAMP levels, substitution of residues 42-45 or deletion of residues 30-37 results in receptors, which, by comparison to wild-type constructs, exhibit significantly worsened Kd values for TSH binding than EC50 values for TSH- or TSAb-increased cAMP activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗

PubMed1992

Characterization of the 5'-flanking region of the rat thyrotropin receptor gene.

Genomic clones containing 1.7 kilobases of the 5'-flanking region of the rat TSH receptor (TSHR) plus coding sequence from the ATG initiation codon [1 basepair (bp)] to the start of the first intron (170 bp) have been isolated and characterized. RNAase protection, primer extension, and cDNA sequences cloned by the anchored polymerase chain reaction identified multiple transcriptional start sites, the major ones clustered between -89 to -68 bp. This portion of the 5'-flanking region has neither a TATA nor a CCAAT box, is GC rich but has no GC box motif, and has features of promoters seen in "housekeeping" genes. Chimeras containing 1.7 kilobases (-1707 to -2 bp) of the 5'-flanking region, or deletions thereof, and the bacterial chloramphenicol acetyltransferase (CAT) gene expressed significant CAT activity when transfected into rat thyroid cell lines, FRTL-5 and FRT, but not BRL rat liver or HeLa cells. TSH decreased CAT activity in the FRTL-5 thyroid cells that had been stably transfected with the TSHR-CAT chimeric constructs. Negative regulation of promoter activity by TSH was duplicated by 10 microM forskolin in FRT thyroid cells, which express no TSHR mRNA. Deletion analyses indicated that a "minimal" region, exhibiting promoter activity, tissue specificity, and negative regulation by TSH, is located between -195 and -39 bp; this region is highly conserved in rat and human TSHR genes. Differential digestion of genomic DNA by MspI and HpaII revealed that the TSHR promoter is methylated in FRT, but not FRTL-5, cells; methylation of the promoter may be associated with loss of endogenous TSHR gene expression in FRT cells.

Amino Acid Sequence↗

PubMed1992

Anaplastic thyroid carcinoma with prominent cardiac metastasis, accompanied by a marked leukocytosis with a neutrophilia and high GM-CSF level in serum.

Cardiac metastasis of thyroid carcinoma is extremely rare. We treated a case of anaplastic thyroid carcinoma with prominent cardiac metastasis. The 61-year-old male was admitted because of high fever. Investigations revealed a cardiac mass and anaplastic thyroid carcinoma. Resection of the cardiac mass revealed that it was metastasis from the thyroid carcinoma. After 4 months, he died in spite of intensive therapy. Marked leukocytosis was observed during the clinical course, and a concomitant increase of granulocyte macrophage-colony stimulating factor (GM-CSF) level was demonstrated in the sera. It was suggested that the high GM-CSF level in serum contributed to leukocytosis.

Carcinoma↗

PubMed1992

Molecular basis for the autoreactivity against thyroid stimulating hormone receptor.

The present report identifies an important immunogenic region of the TSH receptor and determinants on the TSH receptor for the two types of autoantibodies seen in hyperthyroid Graves' disease and hypothyroid idiopathic myxedema, TSAbs and TSBAbs, respectively. The immunogenic domain with no important functional determinants, is contained within residues 303-382 and involves residues 352-366 in particular. There are determinants flanking the immunogenic domain on the C-terminal portion of the receptor which are the TSBAb and high affinity TSH binding sites: residues 295-306, 387-395, and tyrosine 385. Determinants on the N-terminal portion of the external domain, centered on residues 38-45, are TSAb interactions linked to low affinity TSH binding important for signal generation: threonine 40 and residues 30-33, 34-37, 42-45, 52-56, and 58-61. These determinants are conserved in human and rat receptors, are not present in gonadotropin receptors, and are each related to separate actions of TSH: binding vs. signal generation. They can, therefore, account for organ specific autoimmunity and the different disease expression effected by TSBAbs vs TSAbs, i.e. hypo- vs. hyperthyroidism, respectively. It is proposed that, in the thyroid, hormonal (TSH, insulin, hydrocortisone, IGF-I) suppression of class I genes might be one means of preserving self-tolerance in the face of the hormone action to increase the expression of tissue specific genes such as thyroglobulin and thyroid peroxidase. Inappropriately high class I expression in the thyroid, i.e. if induced by interferon, viruses, or some as yet unknown agent, would contribute to the generation of autoimmune disease. Thus, it would result in increased antigen presentation to the immune system, particularly those autoantigens increased by TSH and its cAMP signal such as thyroglobulin or thyroid peroxidase, or whose turnover is increased by TSH and its cAMP signal, such as the TSH receptor. In the case of the latter, peptide 352-366, known to be near a protease sensitive site on the receptor [41,49], would now act as a potent self-antigen and induce the formation of receptor autoantibodies. It is further proposed that methimazole and high doses of iodide are therapeutically effective agents in thyroid autoimmune disease because they, in part, decrease MHC class I gene expression. Speculation is presented which suggests that elimination of negative regulation of MHC class I and the TSH receptor is an important factor in the development of autoimmune thyroid disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Further characterization of a high affinity thyrotropin binding site on the rat thyrotropin receptor which is an epitope for blocking antibodies from idiopathic myxedema patients but not thyroid stimulating antibodies from Graves' patients.

Cysteine 390 of the rat thyrotropin (TSH) receptor, when mutated to serine, results in a receptor with a reduced ability of TSH to bind and increase cAMP levels but a preserved ability of thyroid stimulating autoantibodies (TSAbs) from hyperthyroid Graves' patients to increase cAMP levels. The ability of receptor autoantibodies from hypothyroid patients with idiopathic myxedema to inhibit the TSAb activity which is preserved is, however, like TSH binding, significantly reduced. Cysteine 390, together with tyrosine 385, thus appears to be an important determinant in a high affinity TSH binding site which is an epitope for receptor autoantibodies which block TSH or TSAb action and cause hypothyroidism rather than TSAbs which increase cAMP levels and are associated with hyperthyroidism. Threonine 388 and aspartic acid 403 may contribute to this ligand interaction site.

Amino Acid Sequence↗

PubMed1991

Site-directed mutagenesis of a portion of the extracellular domain of the rat thyrotropin receptor important in autoimmune thyroid disease and nonhomologous with gonadotropin receptors. Relationship of functional and immunogenic domains.

Residues 287 to 404 of the rat thyrotropin (TSH) receptor exhibit little homology to gonadotropin receptors. A large segment of this region, residues 303-382, has no determinants important for TSH to bind or elevate cAMP levels nor for the activity of thyroid-stimulating autoantibodies (TSAbs) from the sera of Graves' patients, i.e. deletions, substitutions, or mutations in this segment do not result in a loss of any of these activities in transfected Cos-7 cells. Critical residues for these activities do, however, flank both sides of this segment. Of particular interest, deletion or mutation of residues 299-301 and 387-395 results in a marked decrease in high affinity TSH binding but preserves the ability of a TSAb to increase cAMP levels. Tyrosine 385 is also of particular interest since its mutation to phenylalanine, alanine, threonine, or glutamine results in a receptor with a 20-fold decrease in the ability of TSH to bind or increase cAMP levels, but one whose TSAb activity is, once again, preserved. Because one activity is preserved, we can conclude that (a) the receptor must be fully integrated within the membrane of the cell without malfolding, (b) these sequences represent determinants involved in the high affinity TSH binding site, and (c) separate determinants exist for high affinity TSH binding and TSAb activity, consistent with the existence of autoantibodies in Graves' sera which inhibit TSH binding (TBIAbs) or which increase cAMP levels (TSAbs). Additionally, we show that a 16-mer peptide (residues 352-367), which reacts with the sera of greater than 80% of patients with Graves' disease, can induce the formation of antibodies to a peptide with no sequence homology, residues 377-397. This peptide flanks the region, residues 303-382, with no determinants important for TSH receptor binding or activity. As noted above, it contains residues involved in the high affinity TSH binding site but whose deletion or mutation has no effect on TSAb activity, i.e. residues which would appear to be required at an epitope important for TBIAb but not TSAb antibody activity.

Amino Acid Sequence↗

PubMed1991

Prokaryotic expression of the thyrotropin receptor and identification of an immunogenic region of the protein using synthetic peptides.

Graves' disease is characterized by hypersecretion of thyroid hormones due to binding of autoantibodies to the thyrotropin receptor (TSHR). In order to study immunological aspects of the TSHR we expressed the extracellular domain of the rat TSHR (ETSHR) as a fusion protein with beta-galactosidase in a prokaryotic system. The identity of this ETSHR-fusion protein was confirmed by Western blot, using antibodies to synthetic peptides derived from TSHR. Patients' sera reacted to a significantly greater extent with the affinity purified ETSHR relative to control sera. Similarly, sera from patients with Graves' disease displayed significant reactivity with only one of five peptides, RH2 (residues 352-366), when compared with normal sera. These data, together with the predicted hydrophilicity of the peptide RH2, suggest that amino acids 352-366 which lie within one of the unique regions of the extracellular domain of the TSHR may be important for antibody binding.

Amino Acid Sequence↗

PubMed1991

Increases in cytosolic Ca++ down regulate thyrotropin receptor gene expression by a mechanism different from the cAMP signal.

Thyrotropin (TSH) receptor mRNA levels in rat FRTL-5 thyroid cells are decreased by treatment with the calcium ionophores, A23187 or ionomycin, as well as with TSH, cholera toxin, forskolin, and 8-bromo-cAMP. Down regulation is, in each case, associated with a decrease in [125I]TSH binding and a decreased ability of TSH to increase cAMP levels. The ionophore does not alter cAMP levels and ethylene glycol-bis-(beta-aminoethyl ether) N, N'-tetraacetic acid (EGTA) in the medium prevents down regulation of TSH receptor mRNA levels by the ionophore, but not by TSH; the EGTA action is reversed by the simultaneous addition of Ca++. Whereas down regulation by TSH and its cAMP signal requires the presence of insulin and/or serum in the medium; down regulation by a calcium ionophore is still evident in their absence. Down regulation of TSH receptor mRNA levels and receptor desensitization by TSH/cAMP or an ionophore is lost in cells transfected with a full length TSH receptor cDNA devoid of regulatory elements, but able to reconstitute TSH receptor signal generation.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

The TSH receptor in autoimmune Basedow's disease.

The cloning approaches of the past two years have opened new doors to the pursuit of our understanding Basedow's disease. The cloning of the TSH receptor is the most dramatic step; nevertheless, all the proteins mentioned in the following appear to be important molecules in the bioactivity of the thyroid cell and are implicated as autoantigens.

Amino Acid Sequence↗

PubMed1991

The extracellular domain of the TSH receptor has an immunogenic epitope reactive with Graves' IgG but unrelated to receptor function as well as determinants having different roles for high affinity TSH binding and the activity of thyroid-stimulating autoantibodies.

The possibility that thyroid-stimulating antibodies (TSAbs) might interact with receptor determinants different from those important for high affinity TSH binding has been evaluated. Deletion mutants of the extracellular domain of the rat TSH receptor as well as point mutations of potential N-linked glycosylation sites were created. TSH binding and the ability of TSH or a TSAb to increase cAMP levels after transfection in Cos-7 cells were then measured. Mutation of two glycosylation sites (residues 77 and 198) was shown to significantly decrease high affinity TSH binding but not the activity of a TSAb. A third glycosylation site mutant (residue 302) was identified that enhanced TSAb activity but had no effect on high affinity TSH binding, and a deletion mutant (residues 308-410) lost TSAb activity but preserved TSH binding. The last two mutations are within a region having low homology with gonadotropin receptors. This same region has, in addition, a determinant that is not important for receptor activity, yet is reactive with Graves' IgG. Thus, a deletion of residues 339-367 has no effect on TSH binding or TSH/TSAb activity, yet contains a peptide (residues 352-367) reactive in ELISA assays with IgG from greater than 80% of Graves' patients but not with IgG from normal individuals, patients with nonautoimmune thyroid disease, or patients with autoimmune disease not related to the thyroid. We, therefore, identify different receptor determinants for TSAb and high affinity TSH binding, consistent with predictions from TSH receptor monoclonal antibody studies. In addition, we identify a receptor peptide that is reactive with TSH receptor antibodies in Graves' patients, despite its having no determinants important for TSH or autoantibody activity in functional assays.

Amino Acid Sequence↗

PubMed1990

Thyrotropin receptor gene expression in oncogene-transfected rat thyroid cells: correlation between transformation, loss of thyrotropin-dependent growth, and loss of thyrotropin receptor gene expression.

Rat FRTL-5 and PC-Cl-3 thyroid cells are continuously cultured, clonal lines which require thyrotropin to grow and function. Both can be efficiently transformed when infected with RNA or DNA viruses carrying oncogenes or when directly transfected with activated oncogenes. Transformation, assayed by the appearance of cell growth in agar and by tumorigenicity in syngeneic rats or nude mice, is associated with the loss of thyrotropin-dependent cell division and thyrotropin-regulated functions such as thyroglobulin synthesis. In 16 clones of FRTL-5 or PC-Cl-3 cells transformed with different oncogenes, we show that loss of thyrotropin-dependent growth and function correlates with the loss of thyrotropin receptor gene expression, measured with a rat thyrotropin receptor cDNA probe.

Animals↗

PubMed1990

A microsequencing approach to identify proteins which appear to interact with thyrotropin in rat FRTL-5 thyroid cells.

In order to resolve questions concerning the in situ structure of the thyrotropin (TSH) receptor, [35S]methionine-labeled thyroid cell preparations were detergent solubilized and proteins exhibiting TSH-dependent binding to TSH-Sepharose were identified. Two such proteins, 43 and 70 kd, are identified in this report as gamma-actin and a member of the heat shock 70 protein family, respectively, based on the microsequence of two peptides from each. Identification of the former was confirmed by Western blotting and immunostaining using anti-actin, the latter by its ability to bind [32P]ATP, a characteristic feature of this family of proteins. The results suggest that TSH-cross linking reports defining TSH receptor subunits should be viewed with caution in the absence of comparative sequence data; consideration must, however, be given to the existence of receptor associated proteins.

Actins↗