Biomedical subjects

L D Kohn

Publications and source records attributed to L D Kohn.

At least 109 records · Page 6Linked to original sources

PubMed1994

Induction of autoimmunity by immunization of mice with human thyrotropin receptor.

The development of autoimmunity was investigated after repeated immunizations with human thyrotropin receptor (hTSH-R) of five congenic strains of female and male mice. After each immunization, free T3 levels and antibodies to hTSH-R and to six peptides of the hTSH-R were assayed. Our results showed that H-2s and H-2q female mice developed features of autoimmunity such as antibody responses to hTSH-R and to hTSH-R peptides, transient variations in the levels of free T3 thyroid hormone, and lymphocytic infiltrations in their thyroid glands. Concerning the antibody responses to hTSH-R peptides, we found that peptide P1 (352-366) contained a major B cell epitope. Furthermore, strain-specific B cell epitope was exemplified by peptide 92 (12-30) and two male- and female-specific B cell epitopes were located in peptides 91 (32-46) and 93 (316-330), respectively. These features appeared rather related to hyperthyroidism.

Amino Acid Sequence↗

PubMed1994

Methimazole prevents induction of experimental systemic lupus erythematosus in mice.

Experimental SLE can be induced in mice by immunization with a human mAb to DNA (16/6Id). Immunized mice develop Abs to the 16/6Id immunogen, DNA, and nuclear Ags. Subsequently, clinical manifestations of disease develop, including leukopenia, proteinuria, and immune complex deposits in the kidney. MHC class I Ags play a critical role in the induction of experimental SLE, as demonstrated by the finding that class I-deficient mice are resistant to disease induction. This finding suggested that agents that reduce MHC class I expression might mitigate experimental SLE in normal mice. These studies report that methimazole, which has been shown to repress class I transcription in some cell lines, reduces class I expression on PBLs in vivo and prevents the development of clinical manifestations of SLE in 16/6Id-immunized mice. These data suggest that methimazole, which has been used in the treatment of Graves' disease, may be useful in the clinical treatment of SLE and other autoimmune diseases.

Animals↗

PubMed1994

Further studies of amino acids (268-304) in thyrotropin (TSH)--lutropin/chorionic gonadotropin (LH/CG) receptor chimeras: cysteine-301 is important in TSH binding and receptor tertiary structure.

Our previous study of chimeric TSH-LH/CG receptors showed that substituting amino acid residues 268-304 of the TSH receptor with homologous residues from the LH/CG receptor markedly decreased high affinity TSH binding as evidenced by ligand displacement assays [Akamizu et al. Endocr J 40:363-372, 1993]. Despite this change in TSH binding, there was a minimal change in TSH-stimulated activity in cAMP assays. To explain this dissociation of TSH binding and function, further characterization of individual residues within the 268-304 segment was performed. Five additional chimeric TSH-LH/CG receptors within this region were constructed by substituting corresponding residues of rat LH/CG receptor. After transfection in Cos-7 cells, TSH receptor activities of these chimeras were evaluated. A single point mutation of cysteine-301 to glutamate resulted in a mutant receptor that exhibited the same receptor bioactivity as the chimeric receptor with amino acids 268-304 substituted by LH/CG receptor residues: apparent low affinity TSH binding in ligand displacement assays but significant retention of the cAMP response to TSH. The four other chimeric substitutions yielded cDNAs coding for receptors that behaved like wild-type receptors. Western blot analyses using a TSH receptor-specific antibody showed that all five of the new mutants were synthesized and integrated in the membrane. These results suggest that cysteine-301 is the critical residue whose mutation accounts for the original chimeric results and the dissociation of binding and functional activity. The possibility must be considered that cysteine-301 is involved in disulfide bond formation and is important in tertiary structure and that conformational changes in the receptor result from its mutation.

Amino Acid Sequence↗

PubMed1994

Thyrotropin stimulation of the lutropin/choriogonadotropin receptor: different sites mediate agonist activity and high affinity binding.

Surprisingly, thyrotropin (TSH) can increase cAMP and inositol phosphate (IP) levels in Cos-7 cells transfected with the lutropin (LH)/choriogonadotropin (CG) receptor (LH/CGR) as well as LH or CG, as evidenced by similar EC50 and maximal stimulation values. Additionally surprising, TSH activation is evident, despite markedly reduced levels of high affinity TSH binding by comparison to CG (Hidaka A, et al. 1993 Biochem Biophys Res Commun 196:187-195). In this report, we questioned whether the unusual TSH activity, as well as the discrepancy between TSH activity and binding, might reflect the existence of distinct agonist and binding sites on the LH/CGR extracellular domain and the ability of TSH to interact with the former despite a minimal interaction with the latter. We evaluated this possibility by using two chimeras spanning the extracellular domain of the TSHR and the LH/CGR:Mc1 + 2, where residues 8-165 of the TSHR are substituted, and Mc2 + 3 + 4, where residues 90-370 are replaced with the corresponding peptide segment from the LH/CGR. After transfection in Cos-7 cells, Mc2 + 3 + 4 exhibits higher affinity for CG than wild-type LH/CGR, but has no CG agonist response in assays measuring cAMP or inositol phosphate (IP) levels. Conversely, the Mc1 + 2 chimera exhibits significantly decreased affinity for CG, but CG agonist activity is comparable to wild-type LH/CGR in cAMP and IP assays. These data show that the extracellular domain of the LH/CGR does have distinct sites for CG binding and agonist activity: the C-terminus in Mc2 + 3 + 4 is important for high affinity CG binding, whereas the N-terminus in Mc1 + 2 is able to exhibit a CG agonist response, despite low affinity binding. When evaluated using TSH, Mc1 + 2, with the C-terminus of the TSHR present, exhibits high affinity TSH binding comparable to wild-type TSHR. Unexpectedly, Mc1 + 2, with the substitution of the N-terminus of the extracellular domain of the LH/CGR, exhibits even better TSH agonist activity than wild-type TSHR, not a loss of activity. Thus, the N-terminus of the extracellular domain of the LH/CGR can couple TSH binding to signal transduction events even better than the N-terminus of the TSHR. This may, in part, explain why TSH has an unusual agonist activity in cells transfected with LH/CGR, despite relatively low affinity binding. Although distinct agonist and binding sites exist in the linear sequence of the extracellular domain, the activity of the two sites is interdependent.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

A sporadic case of male-limited precocious puberty has the same constitutively activating point mutation in luteinizing hormone/choriogonadotropin receptor gene as familial cases.

Familial male-limited precocious puberty (FMPP) is an autosomal dominant disorder characterized by marked elevation of serum testosterone despite low levels of gonadotropin. Recently, a single point mutation in the LH/hCG receptor (LH/CGR) gene was found in FMPP families that constitutively activates the LH/CGR, causing Leydig cell activation and precocious puberty. Among the Japanese population, only four sporadic cases of male-limited precocious puberty have been reported. In the current study, we examined one of the four reported Japanese patients with sporadic male-limited precocious puberty and found the same mutation as that in the FMPP families. Genomic DNA was isolated, and the polymerase chain reaction (PCR) was performed to amplify a fragment of LH/CGR DNA encoding amino acid residues that include transmembrane helixes 5 and 6. Sequencing of the PCR products revealed a heterozygous adenosine-guanine transition at nucleotide 1733 in codon 578. The mutation encodes an aspartic acid578-glycine substitution in transmembrane helix 6. The mutant LH/CGR, created by site-directed mutagenesis in vitro, exhibited constitutively higher cAMP levels in transfected COS-7 cells than the wild-type LH/CGR, as described previously; however, basal inositol phosphate levels were not increased by transfection with complementary DNA for the mutant receptor. The concentration and affinity of [125I]hCG-binding sites were similar in cells transfected with the mutant and wild-type LH/CGR complementary DNAs, indicating that the mutant did not alter the production of receptor or its ability to bind human LH/CG. The sporadic occurrence of this case was confirmed by further studies. The mutation creates a recognition site for the restriction endonuclease MspI. Restriction digestion was positive for the mutant not digested by MspI, indicating that the patient's mutant allele was not inherited from his parents. DNA analysis of the patient and the parents, using microsatellite repeat markers, was compatible with biological paternity and maternity. We conclude that the aspartic acid578-->glycine mutation in the LH/CGR has arisen in the Japanese population and is the cause of a sporadic case of male-limited precocious puberty.

Aspartic Acid↗

PubMed1994

The middle portion in the second cytoplasmic loop of the thyrotropin receptor plays a crucial role in adenylate cyclase activation.

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.

Adenylyl Cyclases↗

PubMed1994

Thyroid-specific expression and cyclic adenosine 3',5'-monophosphate autoregulation of the thyrotropin receptor gene involves thyroid transcription factor-1.

The chimeric chloramphenicol acetyltransferase (CAT) construct, pTRCAT5'-199, containing the TSH receptor (TSHR) minimal promoter, -199 to -39 base pairs (bp), exhibits the thyroid specificity and TSH/cAMP autoregulation evident in TSHR gene expression. The present report shows that a cis-acting element between -189 and -175 bp, which binds thyroid transcription factor-1 (TTF-1), is involved in both activities. The 22 bp between -199 and -178 contains a positive element important for expression of the TSHR minimal promoter in rat FRTL-5 thyroid cells. DNAase I footprinting shows that extracts from functioning FRTL-5, but not non-functioning FRT thyroid or Buffalo rat liver (BRL) cells, protect a region between -189 and -175 bp. The protection is duplicated by TTF-1, and the protected element has only a two-base mismatch from the consensus TTF-1 element identified in the thyroglobulin (TG) and thyroid peroxidase minimal promoters. Gel mobility shift analyses reveal that FRTL-5 thyroid cell nuclear extracts form a specific protein/DNA complex with this region, which is prevented by the TTF-1 binding element from the TG promoter; FRT and BRL cell nuclear extracts do not have TTF-1 and do not form this complex. A role for the TSHR/TTF-1 binding element in thyroid-specific expression of the TSHR gene is evidenced as follows. Overexpression of TTF-1 in FRT or BRL cells, which have no TTF-1, increased the activity of pTRCAT5'-199, but not pTRCAT5'-177, which has no TTF-1 binding element. A nonsense mutation of the TTF-1 binding element eliminated TTF-1-induced activation of TSHR promoter activity in FRT or BRL cells and reduced TSHR promoter activity in FRTL-5 thyroid cells. In contrast, mutation of this element to the TTF-1 consensus sequence of the TG or thyroid peroxidase promoter had no significant influence on TSHR promoter activity. The activity of the TSHR/TTF-1 binding element requires a functioning cAMP response element (CRE). Thus, TTF-1 activity is lost when the CRE site is mutated to a nonfunctional, nonpalindromic sequence; it is, in contrast, maximized when CRE activity is maximized by its mutation to a consensus AP1 element. TTF-1 phosphorylation is important for binding and activity. Thus, binding of TTF-1 to the TSHR/TTF-1 element is phosphatase-sensitive and is increased by treating nuclear extracts with the catalytic subunit of protein kinase A. Overexpression of the catalytic subunit of PKA enhances TTF-1-increased activity of the TSHR minimal promoter.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

[Anti-thyrotropin (TSH) receptor antibody binding epitopes of TSH receptor: site-directed mutagenesis approach].

Anti-TSH receptor antibodies are thought to be involved in the expression of autoimmune thyroid diseases, especially Graves' disease and idiopathic myxedema. Cloning of TSH receptor gene allowed us to study aspects of its structure and function at the molecular level, including autoantibody-binding sites. The long extracellular domain of TSH receptor was presumed to contain antibody-binding site. Site-directed mutagenesis of this domain defined regions important for autoantibodies. The C-terminal region of the extracellular domain, residues 295-306, 387-395 and tyrosine 385 were determinants of the "blocking-type" antibody, which were present in patients with primary hypothyroidism. And the N-terminal region, residues 34-37, 40, 42-45 and 52-56 were the site of the "stimulatory-type" antibody interactions, important in patients with Graves' disease.

Amino Acid Sequence↗

PubMed1993

The cAMP response element in the rat thyrotropin receptor promoter. Regulation by each decanucleotide of a flanking tandem repeat uses different, additive, and novel mechanisms.

A decanucleotide tandem repeat (TR) sequence, between -162 and -140 base pairs (bp) of the minimal thyrotropin receptor promoter, decreases gene expression by repressing constitutive enhancer activity of its cAMP response element (CRE). Each decanucleotide acts additively. CRE-binding proteins and liver or thyroid nuclear extracts footprint a region including the CRE and the 3' decanucleotide, -148 to -124 bp; nuclear proteins interacting with the 3' decanucleotide protect a smaller region -148 to -135 bp. Separate groups of nuclear proteins interact with the CRE and the 3' decanucleotide; mutations of the CRE affect protein interactions with the 3' decanucleotide and the converse. Nuclear proteins bind to single- or double-stranded 3' decanucleotide DNA; those interacting with the CRE bind only double-stranded DNA. The repressor action of the 5' decanucleotide is associated with an interaction between the coding strand and a single-stranded binding protein in liver and thyroid nuclear extracts. The 5' decanucleotide is in a CT-rich region with S1 nuclease hypersensitivity, near perfect mirror images, and direct repeats. The data therefore indicate that each TR decanucleotide modulates CRE constitutive enhancer activity by different but additive mechanisms, competition versus interaction with a single-stranded binding protein, and each interacts with different nuclear proteins that are not thyroid-specific. The same region in the human thyrotropin receptor represses CRE constitutive enhancer activity by the same mechanisms, despite a nonidentical sequence and no overt TR.

Animals↗

PubMed1993

Thyrotropin, like luteinizing hormone (LH) and chorionic gonadotropin (CG), increases cAMP and inositol phosphate levels in cells with recombinant human LH/CG receptor.

Glycoprotein hormones and their receptors are each structurally related; thus, ligand-receptor cross reactivity may exist in pathologic situations, i.e., high human chorionic gonadotropin (hCG) levels in patients has been suggested to activate the thyrotropin receptor (TSHR). Studies with Cos-7 cells transfected with human CG and TSH receptor cDNAs suggest the converse may be more likely. Thus, in cells with TSHR, about 3 x 10(-11) and 3 x 10(-10) M TSH cause half maximal increases in cAMP and inositol phosphate (IP) levels, respectively, whereas 10(-6) M hCG has no effect on either. In cells with CGR, about 10(-11) and 10(-9) M CG or lutropin (LH) significantly increase cAMP and IP levels. Surprisingly, however, 10(-11) and 10(-9) M TSH are similarly effective in the two assays, respectively, and TSH increases cAMP and IP levels to the same extent as CG and LH. LH contamination of TSH is unlikely given similar results with highly purified TSH preparations from different sources, including recombinant TSH, and the specificity of simultaneously measured binding data. Thus, TSH binds with high affinity (Kd = 7 x 10(-11) M) to the human TSHR; hCG (up to 10(-7) M) does not displace TSH binding. Similarly, hCG binds with high affinity (Kd = 5 x 10(-10) M) to the hCGR and TSH is only a weak inhibitor (Ki = 1 x 10(-8) M). Stimulating TSHR autoantibodies, with no epitopes on the CGR, do not duplicate TSH action. The unusual agonist action of TSH with recombinant CGR is consistent with TSHR models describing separate agonist and antagonist determinants; it may be a factor in the precocious puberty of juvenile hypothyroidism with high TSH levels

Animals↗

PubMed1993

Resistance of MHC class I-deficient mice to experimental systemic lupus erythematosus.

Experimental systemic lupus erythematosus (SLE) can be induced in mice by immunization with a human monoclonal antibody to DNA that bears a common idiotype (16/6Id). These mice generate antibodies to 16/6Id, antibodies to DNA, and antibodies directed against nuclear antigens. Subsequently, manifestations of SLE develop, including leukopenia, proteinuria, and immune complex deposits in the kidney. In contrast, after immunization with 16/6Id, mice lacking major histocompatibility complex (MHC) class I molecules generated antibodies to 16/6Id but did not generate antibodies to DNA or to nuclear antigen. Furthermore, they did not develop any of the above clinical manifestations. These results reveal an unexpected function of MHC class I in the induction of autoimmune SLE.

Animals↗

PubMed1993

Effects of cycloheximide and tunicamycin on lysosomal cystine transport in rat FRTL-5 cells.

Rat thyroid FRTL-5 cells were employed to study the synthesis and degradation of a functional integral lysosomal membrane protein, the lysosomal cystine transporter. This carrier exhibited countertransport and closely resembled the cystine transport system of human leucocytes, fibroblasts, and lymphoblasts shown to be defective in the lysosomal storage disease, nephropathic cystinosis. Using cycloheximide to prevent new protein synthesis, the half-life of the FRTL-5 cell lysosomal cystine carrier was determined to approximate 21 h. Carrier function was not influenced by the N-glycosylation inhibitor tunicamycin, nor by the oligosaccharide processing inhibitors castanospermine and deoxymannojirimycin. The data suggest that the lysosomal cystine carrier is a protein without strict functional requirements for N-linked oligosaccharides, and that rat FRTL-5 cells can be employed in future investigations into the structure and function of other integral lysosomal membrane proteins as well.

Animals↗

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

Dual mechanism of perturbation of thyrotropin-mediated activation of thyroid cells by antibodies to the thyrotropin receptor (TSHR) and TSHR-derived peptides.

To further define the epitopes with which anti-TSH receptor (anti-TSHR) antibodies react and mediate their biological effects, we used antibodies against the extracellular domain of TSHR (ETSHR) protein and nine peptides derived from the ETSHR. Peptides were chosen based on their predicted immunogenicity as well as their uniqueness to the TSHR. Antipeptide antibodies showed varying degrees of reactivity against ETSHR, with antipeptide-2-(352-366) and -3A-(357-372) showing relatively stronger reactivity with the receptor. Antibodies were tested for their ability to stimulate thyroid cells and were found to be ineffective in causing both cAMP release and iodide uptake. However, anti-3A and anti-ETSHR showed blocking TSHR antibody (TSHRAb) activities of 76.9% and 79.7%, respectively, which were significantly different (P < 0.005) compared to that of preimmune serum. Anti-2 and -91 (AA 32-46) also showed blocking TSHRAb activities of 37.5% and 35.6%, respectively (P < 0.05). Antisera were also tested for their ability to block TSH binding to thyroid membranes in a RRA. Anti-ETSHR, but not any of the antipeptide antibodies, displayed TSH binding inhibitory immunoglobulin activity. These findings suggest that there might be different mechanisms that mediate blocking TSHR antibody activity. One mechanism involves the inhibition of TSH binding to the receptor, and the other probably involves a step subsequent to TSH binding.

Adenylyl Cyclases↗

PubMed1993

Receptor cross-talk can optimize assays for autoantibodies to the thyrotropin receptor: effect of phenylisopropyladenosine on adenosine 3',5'-monophosphate and inositol phosphate levels in rat FRTL-5 thyroid cells.

Immunoglobulins (IgG) from patients with Graves' disease increase inositol phosphate (IP) as well as cAMP production in rat thyroid FRTL-5 cells; IgGs from normal control subjects do not. Graves' IgG-and TSH-induced IP formation is inhibited by blocking TSH receptor (TSHR) antibodies from hypothyroid patients with primary myxedema, as is the cAMP response; this suggests that the Graves' IgG are acting through the TSHR to induce both the cAMP and phosphatidyl-inositol 4,5-biphosphate signal cascades in FRTL-5 thyroid cells as in cells with recombinant TSHR. Optimal conditions for measuring the Graves' IgG-induced IP increase include a NaCl-free Hanks' Balanced Salt Solution (HBSS) buffer system and a P1 purinergic receptor agonist; the action of each is additive. Optimization by NaCl-free HBSS is similar to that observed in cAMP assays and is specific for TSH or Graves' IgG; thus, NaCl-free HBSS did not affect ATP-induced, and actually inhibited norepinephrine-induced, IP production in FRTL-5 cells. The P1 purinergic receptor agonist acts via receptor cross-talk, which also allows further optimization of cAMP assays. Thus, adenosine deaminase improves Graves' IgG-induced cAMP production by removing adenosine from the medium. Although NaCl-free HBSS improved TSH- or Graves' IgG-induced IP and cAMP production in cells with recombinant TSHR; the modulatory action of phenylisopropyladenosine was lost.

Animals↗

PubMed1993

Identification of thyroid-stimulating antibody-specific interaction sites in the N-terminal region of the thyrotropin receptor.

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.

Amino Acid Sequence↗

PubMed1993

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.

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)

Amino Acid Sequence↗

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↗