Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

TSH and immunoglobulin G (IgG) preparations from patients with Graves' disease increase inositol phosphate as well as cAMP formation in Cos-7 cells transfected with rat TSH receptor (TSHR) cDNA. In a previous report, we mutated alanine 623 of the third cytoplasmic loop (residues 605-625) of the TSHR and showed it was critical for TSH and Graves' IgG initiation of phosphatidylinositol bisphosphate (PIP2) but not cAMP signaling. In this report, we substituted residues in the third loop of the TSHR with sequences from the N- and C-termini of the third loop of the alpha 1- and beta 2-adrenergic receptors (ARs), which computer analysis has identified as homologous to those in the TSHR. Alanine 623 is conserved in most ARs as well as in glycoprotein hormone receptors; there is, therefore, no change in alanine 623. After transfection of the mutant TSHR cDNAs into Cos-7 cells, we show that the mutant proteins are normally synthesized, processed, and incorporated into the membrane bilayer by Western blotting with a specific receptor antibody. We also show that the dissociation constant for TSH binding in all mutants is the same or lower than wild type TSHR. We then evaluated the ability of TSH or Graves' IgG to increase PIP2 and cAMP signals in each transfectant. Mutants A622 and B621 replace, respectively, residues 622-625 and 621-625 of the TSHR with alpha 1- and beta 2-AR residues from the C-terminus of the third cytoplasmic loop; mutants A607 and B605 replace, respectively, TSHR residues 607-609 and 605-609 with N-terminus residues from alpha 1- and beta 2-AR. All four mutants, like the alanine 623 mutant, result in transfected cells which lose TSH and Graves' IgG initiation of PIP2 but not cAMP signalling. Like the alanine 623 mutation to glutamic acid, the A607, B605, A622, and B621 mutants also result in decreased basal cAMP, but not inositol phosphate levels, relative to wild type receptor. In contrast to these results, mutants A610, B610, A617, and B617, which replace residues 610-613 or 617-620 of the TSHR with corresponding residues of the alpha 1- and beta 2-AR, retain TSH and Graves' IgG responsiveness in both inositol phosphate and cAMP assays. Mutation of residues 610-613, in fact, potentiates TSH-increased inositol phosphate production, despite having no effect on TSH-increased cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1993 Aug
PMID:Substitutions of different regions of the third cytoplasmic loop of the thyrotropin (TSH) receptor have selective effects on constitutive, TSH-, and TSH receptor autoantibody-stimulated phosphoinositide and 3',5'-cyclic adenosine monophosphate signal generation. 790 57

We have examined the role of the 2nd cytoplasmic loop of the TSH receptor (TSHR) in TSH- and TSHR autoantibody-stimulated cAMP and inositol phosphate formation using mutants created by substituting sequences from the alpha 1- or beta 2-adrenergic receptors (AR). Unlike similar substitution mutants involving the 3rd cytoplasmic loop that lose agonist-induced inositol phosphate but not cAMP increase after transfection into Cos-7 cells, mutants involving the 2nd loop showed significant change in generating both signals. Mutant B525, which substitutes residues 525-527 with a comparable beta 2-AR sequence, exhibited a complete loss in TSH- or Graves' immunoglobulin G-increased cAMP signaling and a lesser loss in phosphoinositide signaling. This is a unique mutant in which cAMP response was completely lost in all those involving the 2nd or 3rd cytoplasmic loop. On the other hand, mutant B528, in which residues 528-532 are substituted with a comparable beta 2-AR sequence, exhibited the most profound loss in phosphoinositide signaling. Mutants involving portions surrounding residues 528-532 in the 2nd cytoplasmic loop had milder losses in agonist-increased phosphoinositide signaling and much lesser losses in agonist-increased cAMP generation. The transfection efficiency of all transfectants was the same. All transfectants with mutant or wild type TSHR had a similar amount and identical profile of TSHR mRNA in Northern blots and TSHR forms on Western blots. Thus, the 2nd cytoplasmic loop is important for agonist-induced cAMP as well as for phosphoinositide signal generation, whereas the 3rd loop appears to be important only for the latter. The most important determinant for agonist-increased cAMP signal generation is in the middle of the 2nd loop, around residues 525-527. In contrast, the determinants most critical for agonist-induced phosphoinositide signaling are also located in the middle of the 2nd loop, around residues 528-532, and those with less importance are broadly distributed.
Mol Endocrinol 1994 Apr
PMID:The middle portion in the second cytoplasmic loop of the thyrotropin receptor plays a crucial role in adenylate cyclase activation. 791 49

Thyroid peroxidase (TPO) autoantibodies, a hallmark of human autoimmune thyroid disease, may have kappa or lambda light chains. Monoclonal human TPO autoantibodies with kappa light chains have previously been developed by cloning and expressing "combinatorial" libraries of immunoglobulin genes in bacteria. In the present study, an IgG1/lambda combinatorial library was generated from thyroid cDNA of a Graves' patient whose serum contained lambda TPO antibodies. Screening the bacteriophage library with 125I-TPO yielded one clone, TR1.41. The oligonucleotide sequence of TR1.41 was determined and the nature of its interaction with TPO was investigated. The affinity of TR1.41 for TPO is high (Kd approximately 10(-9) M), comparable to that of monoclonal kappa TPO autoantibodies derived from the same patient. The genes encoding the heavy and light chains of TR1.41 differ in a number of respects from the closest available germline genes. Such differences are consistent with somatic mutation in a high-affinity antibody. An important characteristic of TR1.41 is its interaction with the immunodominant domain on TPO recognized by approximately 80% of serum TPO autoantibodies. The frequency of TPO-specific F(ab) generated from the thyroid gland of patient TR was much lower for F(ab) with lambda light chains (1:150,000) than for F(ab) with kappa light chains (1:13,000). Despite this low frequency, the high affinity of TR1.41 and its recognition of the immunodominant region on TPO indicate that lambda autoantibodies of this type may represent an important constituent of the TPO autoantibody response in man. In conclusion, this is the first report on the molecular cloning and characterization of a thyroid autoantibody of lambda L chain type by the combinatorial library approach.
Mol Cell Endocrinol 1994 Jun
PMID:Isolation and characterization of a monoclonal human thyroid peroxidase autoantibody of lambda light chain type. 792 68

Graves' ophthalmopathy, a human autoimmune disease of unknown etiology, is strongly associated with autoimmune hyperthyroidism. A major controversy is whether retro-ocular muscle or orbital fat/connective tissue is the target of the immune response. Previously, we observed preferential PCR amplification of lambda (relative to kappa) light chain DNA from cDNA of Graves' orbital tissue-infiltrating B cells/plasma cells. There is little information on V lambda gene usage in man and none in diseased tissue. To characterize the orbital lambda light chains, we constructed cDNA libraries using PCR-amplified DNA from three tissues and sequenced the variable region genes from randomly selected clones. Analysis of 27 clones from orbital fat/connective tissue libraries from two patients with acute inflammatory eye disease, and 15 clones from orbital muscle of one of these patients, revealed a diverse spectrum of lambda V region genes. The nucleotide sequences of these 42 clones were most homologous to 12 different germline genes: four family I (subfamilies I-a, -b and -c), three family II, two family III and one family VII germline genes. Each orbital tissue had a distinct profile of V lambda sequences. However, all clones used J lambda 2/3 and all three orbital tissues contained clones related to family II genes. Although some clones had V region sequences in near germline conformation, the majority differed from the closest germline gene in both framework and complementarity determining regions. Whether or not these differences result from multiple germline gene usage or somatic mutation of a smaller number of germline genes cannot be determined until information on the V lambda repertoire and its polymorphisms is complete. However, the V lambda gene diversity we observed in both orbital muscle and orbital fat/connective tissue suggests a role for lambda autoantibodies in the pathogenesis of Graves' ophthalmopathy.
Mol Immunol 1994 Aug
PMID:Profile of lambda light chain variable region genes in Graves' orbital tissue. 804 71

Using mutants of the N-terminal region (residues 30-76) of the rat TSH receptor (TSHR), which substitute corresponding segments of rat gonadotropin receptors or hydrophilic (serine) and hydrophobic (alanine) amino acids as appropriate, we show that residues 30-33, 34-37, 42-45, 52-56, and 58-61, in addition to threonine-40, are determinants for the interaction of thyroid-stimulating autoantibodies (stimulating TSHRAbs) with the TSHR. The most important, residues 34-37, 42-45, and 52-56, whose mutants lose stimulating TSHRAb activity with at least 11 of 12 (> 90%) of the Graves' immunoglobulins G tested, are, like threonine-40, in regions of the TSHR that are nonhomologous with gonadotropin receptors. These data establish at least in part, therefore, the basis for the thyroid-specific effects of stimulating TSHRAbs. In no case do the same mutants lose their reactivity with TSH or blocking-type TSHR autoantibodies (blocking TSHRAbs) from hypothyroid patients with idiopathic myxedema. Since the latter have been shown to interact with high affinity TSH-binding sites on the C-terminal portion of the external domain of the TSHR, stimulating TSHRAbs and blocking TSHRAbs react with different receptor determinants, which can be presumed to have different roles in receptor function. This can explain the hyper- or hypothyroidism of different thyroid autoimmune diseases with receptor antibodies. Residues 30-33, 42-45, and threonine-40 appear to be related to the agonist action of TSH, since in each case mutation results in low affinity TSH binding, but normal TSH-increased cAMP activity, similar, for example, to a beta-adrenergic agonist. Using a receptor antibody to identify different receptor forms in the membrane, we can also identify determinants in this N-terminal region (residues 30-76) whose mutation results in a loss of all activities without apparently altering receptor synthesis, processing, or integration within the bilayer. These are residues 38 and 39, cysteine-41, residues 46-51, leucine-57, threonine-62, and, within residues 66-76, serine-69, alanine-71, phenylalanine-72, serine-74, leucine-75, and proline-76. We suggest that these residues are at the very least important in the conformational array of receptor determinants necessary for interactions with TSH and stimulating TSHRAbs.
Mol Endocrinol 1993 Jan
PMID:Identification of thyroid-stimulating antibody-specific interaction sites in the N-terminal region of the thyrotropin receptor. 809 22

We produced rabbit antibodies against mutated thyrotropin (TSH) receptor peptide TSH-R-HIS17 in comparison with non-mutated TSH receptor peptide TSH-R-ASP17. All the antibodies raised against TSH-R-HIS17 showed thyroid stimulating antibody (TSAb), thyroid stimulation blocking antibody (TSBAb) and/or TSH binding inhibiting immunoglobulin (TBII) activities and also those serum T3 levels were all in thyrotoxic levels associated with TSAb activities. However, all the antibodies raised against TSH-R-ASP17 had no activities of TSAb, TSBAb and/or TBII and those serum T3 levels were all within normal range. The data suggest that the mutation in the TSH receptor gene may have a possible relevance for the pathogenesis of Graves' disease.
Biochem Mol Biol Int 1993 Mar
PMID:Production of bioactive rabbit antibodies against the mutated receptor peptide TSH-R-HIS17 and detection of hypertriiodothyroninemia in the rabbit. 809 36

We investigated the role of the 1st cytoplasmic loop of the thyrotropin receptor (TSHR) on signal transduction using mutants as we did the 2nd and 3rd cytoplasmic loops [Kosugi S. et al. (1994) Mol. Endocrinol., in press; and 7, 1009-1020]. Five substitution mutants involving the first cytoplasmic loop showed a TSH- or Graves' IgG-stimulated cAMP response despite the low TSH binding Bmax. All the mutants completely lost or markedly decreased the TSH- or Graves' IgG-stimulated inositol phosphate increase. These findings suggest that the 1st cytoplasmic loop of the TSHR does not play a crucial role in agonist-induced adenylate cyclase activation but that it is important for phosphoinositide signaling.
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PMID:The first cytoplasmic loop of the thyrotropin receptor is important for phosphoinositide signaling but not for agonist-induced adenylate cyclase activation. 813 33

Residue 113 of the thyrotropin receptor (TSHR) is a possible asparagine-linked glycosylation site in the human TSHR, but not in rat or dog TSHR. Russo et al. (Mol Endocrinol 5:29-33) reported that mutation of this residue in the human TSHR diminished TSH binding activity after transfection. To investigate the difference in the role of residue 113 of the TSHR among species, we created a mutant at residue 113 in the rat TSHR, transfected Cos-7 cells with the mutant DNA and measured TSH binding and TSH- and Graves' IgG-stimulated cAMP and phosphoinositide signals. No difference was found in the activities of the mutant transfectant from the wild type receptor transfectant. These results might suggest a real difference in glycosylation of the TSHR among species.
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PMID:Possible difference in glycosylation of the thyrotropin receptor among species. 818 69

We have cloned a cDNA whose mRNA levels are increased in malignantly transformed rat thyroid FRTL cells (FRTL-Tc cells). We constructed a cDNA library from FRTL-Tc cells in lambda gt10 and screened the cDNAs by differential plaque filter hybridization. Twenty-five thousand clones were screened and one cDNA (C140) was selected which corresponded to a mRNA whose expression was 5.8 times higher in FRTL-Tc cells than in FRTL cells. A 0.8 kb specific C140 mRNA was detected by Northern blot analysis of FRTL-Tc and FRTL mRNAs. The C140 cDNA was sequenced and found to encode a protein of 227 amino acids. We have found that C140 mRNA is conserved in human thyroid cells, but it is encoded by a smaller 0.7 kb transcript. C140 mRNA was highly expressed in neoplastic thyroid tissues and weakly in normal thyroid tissues in the same patients. Additionally, we found that C140 mRNA was also increased in the thyroid tissue of a patient with Graves' disease. These results suggest that C140 expression might be higher in rapidly growing thyroid cells than in normal cells, and might provide a new aspect for the study of thyroid tumours.
J Mol Endocrinol 1994 Feb
PMID:Isolation of a cDNA whose expression is markedly increased in malignantly transformed FRTL cells and neoplastic human thyroid tissues. 818 17

The baculovirus expression system was used to overexpress recombinant human thyroid peroxidase. Sf-9 cells infected with the recombinant virus AcMNPV-hTPO synthesized hTPO protein (hTPO-bac) immunogenic on Western blots when probed with either rabbit anti-TPO peptide sera or pooled human anti-TPO sera (MS12/89). hTPO-bac was a major constituent of the membrane fraction from the infected cells, constituting 14.9% and 10.1% of the 1% deoxycholate-soluble and insoluble fractions, respectively, as judged by densitometry. Recombinant hTPO-bac was extracted from cellular membranes with 1% deoxycholate and partially purified by Sepharose 6B column chromatography. Specific immunoreactivity of MS12/89 to hTPO-bac on microtiter plates was seen using ELISA. Detergent extract from wild-type virus-infected Sf-9 cells was used as background control antigen; no specific reactivity to either hTPO-bac or control antigen was seen with control sera. To determine antigenic potency, MS12/89 was incubated with increasing concentrations of various preparations of hTPO antigen and with ovalbumin as control. The capacity of the partially purified hTPO-bac to immunoneutralize human anti-hTPO standard at 50% inhibition of binding was 0.01 U/microgram hTPO-bac (NIBSC Units), compared with 0.5 U/microgram and 0.06 U/microgram for natural hTPO and CHO-hTPO, respectively. When ELISA was performed using clinical samples of human sera to detect hTPO autoantibodies, results using hTPO-bac correlated well with those using hTPO from Graves' thyroid tissue (r = 0.85, p = 0.02) and those using recombinant hTPO from Chinese hamster ovary cells (hTPO-CHO) (r = 0.85, p = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1993 Jun
PMID:Expression of human thyroid peroxidase in insect cells using recombinant baculovirus. 834 29


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