Biomedical subjects

G D Chazenbalk

Publications and source records attributed to G D Chazenbalk.

At least 19 recordsLinked to original sources

PubMed1996

Is receptor cleavage into two subunits necessary for thyrotropin action?

Unlike the wild-type thyrotropin (TSH) receptor, the chimeric TSH-LH/CG receptor TSH-LHR-14 does not cleave into two subunits when cross-linked to [125I]TSH on the surface of intact cells. Immunoblotting of TSH-LHR-14 in whole cell homogenates demonstrated that only a single chain receptor was detected under reducing conditions. TSH-LHR-14, like the A subunit of another chimeric receptor (TSH-LHR-10) that does cleave into two subunits, was almost entirely resistant to endoglycosidase H, indicating that it contains predominantly complex carbohydrate. The fact that TSH-LHR-14 reaches the cell surface where it binds TSH with high affinity and transduces a signal indicates that receptor cleavage into two subunits is not a prerequisite for TSH action.

Animals↗

PubMed1996

Critical relationship between autoantibody recognition and thyrotropin receptor maturation as reflected in the acquisition of complex carbohydrate.

Generation of large amounts of recombinant TSH receptor (TSHR) protein capable of recognition by TSHR autoantibodies is a goal of clinical importance. We expressed in Chinese hamster ovary cells the human TSHR ectodomain (ECD) with a carboxyl-terminus six-histidine tag. After transgene amplification, expressing clones were selected by nickel chelate chromatography in combination with [35S] methionine precursor labeling. An approximately 74-kDa protein was detected in the culture medium, and larger quantities of an approximately 68-kDa protein were found in the cell soluble fraction. Immunoblot analysis with a rabbit antiserum revealed that most of the TSHR-ECD was not secreted, but was retained within the soluble fraction of the cell. Nickel chelate chromatography was not effective in purifying significant quantities of this material. In contrast, with Concanavalin A, but not with wheat germ agglutinin, an approximately 50-fold purification of TSHR-ECD was achieved from the cell soluble fraction. Surprisingly, this affinity-enriched TSHR, containing high mannose carbohydrate, was not recognized by human TSHR autoantibodies in sera from six individuals. By ion exchange chromatography, the autoantibody-neutralizing TSHR in the cell supernatant fraction was found to be nonidentical with TSHR-ECD protein recognized by antisera from immunized animals. The present data indicate the critical relationship between autoantibody recognition and TSHR maturation as reflected in the acquisition of complex carbohydrate. Nonsecretion of the TSHR-ECD appears to be related to the specific protein rather than to the glycosylation apparatus of the host cell. Antibodies from immunized animals may be ineffective in monitoring purification of autoantigen-competent TSHR. Finally, the data explain why soluble recombinant TSHR generated in many expression systems is not recognized satisfactorily by human autoantibodies.

Animals↗

PubMed1995

Expression of the extracellular domain of the thyrotropin receptor in the baculovirus system using a promoter active earlier than the polyhedrin promoter. Implications for the expression of functional highly glycosylated proteins.

Conventional baculovirus vectors that utilize the very late polyhedrin promoter have not proved successful for expressing a thyrotropin (TSH) receptor capable of ligand and Graves' disease autoantibody binding comparable to the receptor produced in mammalian cells. Because of the clinical importance of high level expression of this protein, we reassessed the baculovirus system using a new transfer vector (pAcMP3) containing the late basic protein promoter, which functions earlier than the classical polyhedrin promoter. Maximal synthesis of the [35S]methionine-labeled TSH receptor extracellular domain, affinity-purified using a 6-histidine tag, occurred earlier (1 day after insect cell infection) than with a vector (pVL1393) containing the polyhedrin promoter. The pAcMP3-derived TSH receptor extracellular domain was larger (approximately 68 kDa) than the pVL1393-derived protein (approximately 63 kDa). Only the 68-kDa product was secreted, albeit in trace amounts detectable only by precursor labeling. Enzymatic deglycosylation reduced both 68- and 63-kDa cellular proteins to approximately 54 kDa, indicating that the pAcMP3 vector generated a protein with greater carbohydrate content. However, despite its greater degree of glycosylation, most of the 68-kDa protein remained within the cell, almost entirely in the particulate fraction. Remarkably, the trace amounts of 68-kDa receptor protein affinity-purified from the soluble cytosolic fraction of infected insect cells completely neutralized TSH receptor autoantibodies in patients' sera and partly inhibited TSH binding. In conclusion, a baculovirus vector with a promoter active earlier than the conventional polyhedrin promoter generates a more glycosylated and functional TSH receptor extracellular domain protein, albeit at low levels. These data carry important implications for the expression by baculovirus vectors of functional, highly glycosylated proteins.

Animals↗

PubMed1995

Genetic alterations in thyroid hyperfunctioning adenomas.

Thirty-seven thyroid autonomously hyperfunctioning adenomas were screened for mutations in the TSH receptor (TSHR), G alpha s (gsp), and ras genes. Polymerase chain reaction-amplified fragments of the TSHR C-terminal part (exon 10), the G alpha s (exons 8 and 9), and the three ras genes were obtained from the genomic DNA extracted from 37 tumors and their adjacent normal tissues and were studied by direct nucleotide sequencing and hybridization with synthetic probes. A point mutation in the third intracellular loop (codon 623) of the TSHR was found in 3 of 37 adenomas studied. This mutation codes for a change (Ala to Ser) in the TSHR structure and is somatic and heterozygotic. Constitutive activation of the TSHR was demonstrated by an increase in basal cAMP levels after transfection of Chinese hamster ovary cells with a mutated Ser623-TSHR complementary DNA. Nine gsp[00ae]MDRV[00af]- and one ras-activating mutations were also detected. No simultaneous alteration of the studied genes was present. Thus, in hyperfunctioning thyroid adenomas, our data suggest that a mutational activation of the TSHR and gsp genes may play a tumorigenic role through constitutive activation of the cAMP pathway.

Adenoma↗

PubMed1994

Cleavage of the thyrotropin receptor does not occur at a classical subtilisin-related proprotein convertase endoproteolytic site.

The human thyrotropin receptor (TSHR) undergoes proteolytic cleavage closely upstream to amino acid 317. Between residues 261 and 313 are three clusters of positively charged amino acids, arginines (Arg) and lysines (Lys), which are potential subtilisin-related proprotein convertase sites. We used oligonucleotide-directed mutagenesis to perform conservative amino acid substitutions within these regions (Arg or Lys to glutamine, Gln). Chinese hamster ovary cells stably transfected with mutant receptor cDNA TSHR-CS1 (Gln261) and TSHR-CS3 (Gln312, Gln313) bound radiolabeled TSH with an affinity similar to the wild-type TSHR. Mutant cDNA TSHR-CS2 (Gln290, Gln291) and TSHR-CS4 (Gln261, Gln290, Gln291, Gln312, Gln313) did not express a protein on the cell surface capable of specific TSH binding. After covalent cross-linkage of radiolabeled TSH to TSHR-CS1 and TSHR-CS3, the mutant receptors dissociated into two subunits under reducing conditions. The most prominent cluster of basic amino acids in the TSHR extracellular region (residues 287-293) was studied in a second series of mutations designed to eliminate the classical proprotein convertase sites in this region and yet be compatible with TSHR function. All three mutant receptors, TSHR-CS5 (Gln290), TSHR-CS6 (Gln291), and TSHR-CS7 (Gln291, Gln293) bound TSH with an affinity similar to that of wild type, and none of these amino acid substitutions prevented proteolytic cleavage of the extracellular domains of the TSHR. Thus, cleavage of the TSHR extracellular domain does not involve a classical subtilisin-related proprotein convertase cleavage site, raising the possibility that TSHR cleavage occurs after processing and trafficking of the protein to the plasma membrane.

Amino Acid Sequence↗

PubMed1994

Recombinant thyroid peroxidase-specific autoantibodies. II. Role of individual heavy and light chains in determining epitope recognition.

Most thyroid peroxidase (TPO) autoantibodies in man recognize closely associated epitopes in two domains (A and B) on TPO. These epitopes were defined by recombinant monoclonal human autoantibodies expressed as antigen-binding fragments [F(ab)]. Only five heavy (H) and light (L) chain gene combinations encoded 34 F(ab), all of which have high affinity (Kd, approximately 10(-10) M) for TPO. We, therefore, investigated the roles of H and L chain genes in TPO domain recognition in two ways. First, we created hybrid F(ab) by forced recombination of H and L chain genes from 4 F(ab) recognizing the A or B domains. These hybrid F(ab) proteins, expressed in bacteria, bound extremely poorly (or not at all) to TPO, even at concentrations more than 100-fold higher than those required for detection of TPO binding by the original F(ab). Nucleotide sequencing of the cDNA as well as gel electrophoresis of the expressed proteins confirmed that poor hybrid F(ab) binding to TPO was not the result of cloning artifacts. Therefore, contrary to prevailing views on combinatorial libraries, we found no tolerance for H and L chain cross-combinations in high affinity TPO binding. These observations strengthen the likelihood that the H and L chain combinations from combinatorial libraries reflect those of TPO autoantibodies in vivo. In a second approach to examine the roles of H and L chains in TPO binding, we focused on three original F(ab) with similar L chains (encoded by KL012-like germline genes) and similar H chains (encoded by V1-3B-like germline genes), but different diversity (D) regions. All F(ab) bound predominantly to TPO domain A, as observed previously for a F(ab) with a KL012 L chain and a different H chain. Conversely, a F(ab) with a V1-3B-like H chain but a different L chain (A') bound to TPO domain B. These data indicate that the L chain plays a major role in defining TPO epitope recognition.

Amino Acid Sequence↗

PubMed1994

Studies on homologous desensitization of the thyrotropin receptor in 293 human embryonal kidney cells.

It is well known that the TSH receptor (TSHR) undergoes homologous desensitization. That is, prolonged stimulation of thyroid cells with TSH attenuates the cAMP response to subsequent TSH stimulation. However, the existence of homologous desensitization of the recombinant TSHR expressed in nonthyroidal eukaryotic cells is controversial. In the present studies, therefore, we first investigated whether or not the TSHR was desensitized by TSH in 293 human embryonal kidney cells, a cell line in which the LH/CG receptor (LH/CGR) is reported to undergo homologous desensitization. The wild type (wt) TSHR and the wt-LH/CGR stably expressed in 293 cells bound to their respective hormones with high affinity and produced a dose-dependent intracellular cAMP response to hormone stimulation. Pretreatment of cells expressing the TSHR or the LH/CGR with their respective hormones attenuated the cAMP response to subsequent hormone stimulation without down-regulation of the receptors, demonstrating that the TSHR, as well as the LH/CGR, undergoes homologous desensitization in 293 cells. With this cell type expressing mutant TSHRs, we then studied some aspects of the molecular mechanism of TSHR desensitization and compared our data to those obtained with the beta-adrenergic receptor (beta-AR), which is widely regarded as the prototype for receptor desensitization. We cotransfected the wt-TSHR and a chimeric receptor consisting of the LH/CGR extracellular ligand binding domain with the TSHR transmembrane/cytoplasmic signal transducing region. These two receptors have distinct hormone specificities but share common signal regulatory mechanisms. We observed that, like the beta-AR, only hormone-occupied receptor is likely to be involved in homologous desensitization. On the other hand, studies with a truncated TSHR indicated that, in contrast to the beta-AR, the serine/threonine-rich region in the carboxyl two thirds of the cytoplasmic tail of the TSHR is not involved in homologous desensitization.

Animals↗

PubMed1993

Lack of promiscuity in autoantigen-specific H and L chain combinations as revealed by human H and L chain "roulette".

Individual H or L chains from a human autoantibody were used to search for other L or H chains that could form antigen-binding fragments, Fab, with the same specificity. The parent Fab (SP1.2) exhibits high affinity binding for thyroid peroxidase (TPO), a 107-kDa protein that is the major autoantigen in human autoimmune thyroiditis. This autoantibody "roulette," performed by using Ig H and L chain gene libraries expressed in bacteria, increased the frequency of TPO-binding clones in the new libraries. However, the frequency was still much lower than would be the case if promiscuous combinations with a variety of H or L chains were compatible with specific Ag binding. Nucleotide sequence analysis of the H and L chains of the new TPO-binding clones revealed even more restriction. Thus, with the SP1.2 H chain, all 11 new Fab utilized L chains from the same V kappa 1 family germline gene as SP1.2 itself. Similarly, five of six H chains "captured" by the SP1.2 L chain were very closely related to the SP1.2 H chain. However, one totally different H chain was isolated: SP4.6 has a VH region that differs substantially from that of SP1.2. SP4.6 also has a distinct D region, uses a different JH, and, unlike SP1.2, which is an IgG1, belongs to subclass IgG4. The affinities for TPO of SP4.6 (with the different H chain) and SP1.20 (which had the least mutated L chain germline gene) were similar to that of SP1.2 (approximately 10(-10) M). As expected, the SP1.2 and SP1.20 Fab, which have the same H chain and closely related L chains, bound to the same domain on TPO. However, a similar domain on TPO was recognized by both SP4.6 and SP1.2, despite the fact that their V, D, and J regions are quite different. This observation raises the possibility that the L chain is critical in defining epitope specificity, even in the presence of completely different D regions and nonidentical VH regions.

Amino Acid Sequence↗

PubMed1993

Human organ-specific autoimmune disease. Molecular cloning and expression of an autoantibody gene repertoire for a major autoantigen reveals an antigenic immunodominant region and restricted immunoglobulin gene usage in the target organ.

The most common organ-specific autoimmune disease in humans involves the thyroid. Autoantibodies against thyroid peroxidase (TPO) are present in the sera of virtually all patients with active disease. We report the molecular cloning of the genes for 30 high-affinity, IgG-class human autoantibodies to TPO from thyroid-infiltrating B cells. Analysis of the putative germline genes used for the TPO human autoantibodies suggests the use of only five different H and L chain combinations involving four H chains and three L chains. In addition, the same combination of H and L chains was found in multiple patients. The F(ab) proteins expressed by these genes define two major, closely associated domains (A and B) in an immunodominant region on TPO. These A and B domains contain the binding sites of approximately 80% of IgG-class TPO autoantibodies in the sera of patients with autoimmune thyroid disease. The present information permits analysis, not previously possible, of the relationship between autoantibody H and L chain genes and the antigenic domains on an autoantigen. Our data, obtained using target organ-derived autoantibodies, indicate that there is restriction in H and L chain usage in relation to the interaction with specific antigenic domains in human, organ-specific autoimmune disease.

Antibody Affinity↗

PubMed1993

The immunodominant region on human thyroid peroxidase recognized by autoantibodies does not contain the monoclonal antibody 47/c21 linear epitope.

We performed studies to determine whether the binding sites on thyroid peroxidase (TPO) of immunoglobulin antigen binding fragments (Fabs) representing more than 80% of the human autoantibody repertoire overlap with the binding site of monoclonal antibody (Mab) 47, the only Mab whose partial epitope has been defined at the amino acid level (residues 713-721). We also investigated whether these Fabs preferentially recognize native or denatured TPO. None of the Fabs, when bound to radiolabeled TPO, interfered with the ability of Mab 47 to bind to this material. In enzyme-linked immunosorbent assay experiments, the binding of TPO autoantibody Fabs SP1.5, WR1.7, TR1.8, and TR1.9 was greatly diminished by denaturation of TPO. In contrast, binding of Mab 47 was higher to denatured TPO than to intact TPO. Our studies indicate that the Mab 47/C21 epitope lies outside the immunodominant region on TPO. Further, the data confirm that the majority of epitopes for TPO autoantibodies are highly conformational (dependent on the three-dimensional structure of the native protein). Native TPO will be needed to complete the mapping of the epitopes for TPO autoantibodies as well as to determine the amino acids at the autoantibody-antigen-binding sites.

Animals↗

PubMed1992

Cloning of a member of the arrestin family from a human thyroid cDNA library.

We used the cDNA of human retinal arrestin as a probe to screen a human thyroid cDNA library. We isolated and plaque-purified one clone (hTHY-ARRX). The nucleotide sequence of the 1.8 kb cDNA insert had an open reading frame of 1227 bp coding for a protein of 409 amino acids. Northern blot analysis revealed a single transcript of 1.7 kb in human thyroid cells. There is significant homology between amino acid sequences of human thyroid arrestin and human retinal arrestin (63%) and bovine beta-arrestin (74%), respectively. The hTHY-ARRX cDNA was stably transfected into Chinese hamster ovary cells already expressing a functional human thyrotropin (TSH) receptor. The cAMP response to TSH stimulation was unaltered in these cells, and homologous desensitization to TSH stimulation was not restored. It is not presently known whether hTHY-ARRX is human beta-adrenergic arrestin or a new member of the arrestin family.

Amino Acid Sequence↗

PubMed1992

Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase.

To characterize the nature of thyroid peroxidase (TPO) autoantibodies present in the sera of patients with autoimmune thyroid disease, we cloned three IgG1/kappa Fab fragments which bind 125I-TPO. This was accomplished by the molecular cloning and expression in bacteria of IgG gene fragments from B cells infiltrating the thyroid of a patient with Graves' disease. The three Fab fragments (SP2, SP4, and SP5) are coded for by a common heavy chain (VH1, D, JH3) and three related, but different, light chains (VK1, JK2). The SP Fab fragments bind specifically to TPO with high affinities (6 x 10(-11)-2 x 10(-10) M) comparable to those of serum TPO autoantibodies. TPO autoantibodies represented by the SP Fab fragments are present in all 11 patients studied, constitute a high proportion (36-72%) of serum TPO autoantibodies in individual patients and interact with a conformational epitope on TPO.

Amino Acid Sequence↗

PubMed1992

Role of amino acids 261-418 in proteolytic cleavage of the extracellular region of the human thyrotropin receptor.

Previous in vivo cross-linking studies of TSH to the recombinant TSH receptor revealed that the receptor exists at least in part as a single chain glycoprotein of approximately about 100 kilodaltons (kDa), with intramolecular disulfide bonds. TSH also binds to a 54-kDa amino-terminal fragment of the TSH receptor (cleaved up-stream of amino acid residue 317). In the present study in order to better understand the structure of the TSH receptor, we covalently cross-linked radiolabeled TSH to six TSH-LH receptor extracellular region chimeras and the wild-type TSH receptor expressed on Chinese hamster ovary cells in vivo. In these chimeras, different regions of the TSH receptor were substituted with the homologous regions of the LH receptor. When analyzed under nonreducing conditions by polyacrylamide gel electrophoresis, the TSH-cross-linked products were similar to all TSH-LH receptor chimeras and the wild-type receptor. In contrast, differences among the receptors were noted when the TSH-cross-linked products were examined under reducing conditions. With the exception of two chimeras, as noted previously with the wild-type receptor, two TSH-cross-linked products were observed, representing TSH cross-linked to a holoreceptor of about approximately 100 kDa and a fragment of the receptor of about approximately 54 kDa. However, in the two chimeras in which both domains D and E (amino acids 261-418) of the TSH receptor were substituted, only the holoreceptor and not the smaller fragment was detected. Substitution of domains ABC (amino acids 1-260) did not prevent proteolytic cleavage of the TSH receptor. In conclusion, amino acids 261-418 are necessary for proteolytic cleavage of the extracellular region of the human TSH receptor.

Endopeptidases↗

PubMed1992

Studies on the role of amino acids 38-45 in the expression of a functional thyrotropin receptor.

We previously reported that deletion or substitution of a unique eight-amino acid tract (residues 38-45) in the extracellular domain of the human TSH receptor led to the loss of specific ligand binding to the surface of transfected cells. In the present study we analyzed this region in more detail. Using site-directed mutagenesis of the TSH receptor cDNA, we substituted amino acid residues 38-45, either in three overlapping groups of four amino acids each or individually. The resultant TSH receptor mutant cDNAs were stably transfected into Chinese hamster ovary cells, and the cells were tested for their TSH-binding ability. Our data demonstrate that amino acid residues 38-40 and 42-45 in this region of the human TSH receptor can be substituted without alteration in receptor function and are, therefore, not critical in forming or maintaining the TSH-binding site. However, substitution of Cys41, either alone or together with adjacent amino acids, leads to the loss of TSH binding to its receptor. These data suggest a central role for the amino acid in position 41 in preserving the biological function of the TSH receptor.

Amino Acid Sequence↗

PubMed1991

A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocyte-derived immunoglobulin genes in a bacteriophage lambda library.

A human Fab fragment (SP2) which binds specifically to human thyroid peroxidase has been generated by expressing random combinations of heavy and light chain immunoglobulin genes (derived from Graves' thyroid cDNA) in a bacteriophage lambda library. In common with many serum TPO autoantibodies, the cloned Fab fragment is IgG1 kappa and has a high affinity for TPO (approximately 10(-9) M). On the basis of their nucleotide sequences, the heavy and light chain genes coding for SP2 belong to families VHI, (D), JH3 and VKI, JK2, respectively. These data provide the first characterization at a molecular level of a human thyroid peroxidase antibody associated with autoimmune thyroid disease.

Amino Acid Sequence↗

PubMed1991

Eleven amino acids (Lys-201 to Lys-211) and 9 amino acids (Gly-222 to Leu-230) in the human thyrotropin receptor are involved in ligand binding.

Our previous studies involving chimeric thyrotropin-lutropin/choriogonadotropin (TSH-LH/CG) receptors suggest that multiple segments spanning the entire extracellular domain of the human TSH receptor contribute to the TSH binding site. Nevertheless, the mid-region (segment C, amino acid residues 171-260) of the receptor extracellular domain is particularly important in TSH binding. In the present studies, we constructed seven new chimeric receptors in order to analyze segment C in further detail. Seven small segments spanning segment C of the TSH receptor were replaced with the counterpart of the rat LH/CG receptor. These mutant receptors were stably introduced into Chinese hamster ovary cells and were tested for hormone binding and cAMP responsiveness to hormone stimulation. The results indicate that 11 amino acids of the TSH receptor (Lys-201 to Lys-211) and the corresponding region of the LH/CG receptor (Thr-202 to Ile-212) are important for specific TSH and human CG binding, respectively. In addition, nine amino acids of the TSH receptor (Gly-222 to Leu-230) are also involved in TSH binding. A further conclusion from these data is that TSH and human CG bind to partially overlapping sites on their respective receptor molecules.

Amino Acid Sequence↗

PubMed1991

Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function.

To define the sites in the extracellular domain of the human thyrotropin (TSH) receptor that are involved in TSH binding and signal transduction we constructed chimeric thyrotropin-luteinizing hormone/chorionic gonadotropin (TSH-LH/CG) receptors. The extracellular domain of the human TSH receptor was divided into five regions that were replaced, either singly or in various combinations, with homologous regions of the rat LH/CG receptor. The chimeric receptors were stably expressed in Chinese hamster ovary cells. The data obtained suggest that the carboxyl region of the extracellular domain (amino acid residues 261-418) and particularly the middle region (residues 171-260) play a role in signal transduction. The possibility is also raised of an interaction between the amino and carboxyl regions of the extracellular domain in the process of signal transduction. With respect to hormone binding, substitution of the entire extracellular domain of the LH/CG receptor for the corresponding region of the TSH receptor resulted in high-affinity human CG binding with complete loss of TSH binding. Surprisingly, however, there was at least one chimera with a substitution at each of the five domains that still retained high-affinity TSH binding. Substitution of residues 1-170 of the TSH receptor with the corresponding region of the LH/CG receptor was associated with the retention of high-affinity TSH binding but ligand specificity was lost in that TSH and human CG could interact functionally with the receptor. In summary, these studies suggest that the middle region and carboxyl half of the extracellular domain of the TSH receptor are involved in signal transduction and that the TSH-binding region is likely to span the entire extracellular domain, with multiple discontinuous contact sites.

Animals↗

PubMed1991

Binding domains of stimulatory and inhibitory thyrotropin (TSH) receptor autoantibodies determined with chimeric TSH-lutropin/chorionic gonadotropin receptors.

We examined the relative effects of thyrotropin (TSH) and TSH receptor autoantibodies in the sera of patients with autoimmune thyroid disease on three TSH-lutropin/chorionic gonadotropin (LH/CG) receptor extracellular domain chimeras. Each chimera binds TSH with high affinity. Only the chimera with TSH receptor extracellular domains ABC (amino acids 1-260) had a functional (cAMP) response to thyroid stimulatory IgG. The chimeras with TSH receptor domains CD (amino acids 171-360) and DE (amino acids 261-418) were unresponsive. The lack of response of the chimera with TSH receptor domains DE was anticipated because it fails to transduce a signal with TSH stimulation, unlike the other two chimeras. A different spectrum of responses occurred when the TSH-LH/CG chimeras were examined in terms of autoantibody competition for TSH binding. IgG with TSH binding-inhibitory activity when tested with the wild-type TSH receptor also inhibited TSH binding to the chimera with TSH receptor domains DE. Dramatically, however, these IgG did not inhibit TSH binding to the chimera with TSH receptor domains CD, and had weak or absent activity with the chimera with TSH receptor domains ABC. Chimeras with TSH receptor domains ABC and DE were equally effective in affinity-purifying IgG with thyroid-stimulatory and TSH binding-inhibitory activities. Nonstimulatory IgG with TSH binding-inhibitory activity inhibited the action of stimulatory IgG on the wild-type TSH receptor, but not with the chimera containing TSH receptor domains ABC. In summary, TSH receptor autoantibodies and TSH bind to regions in both domains ABC and DE of the TSH receptor extracellular region. Stimulatory and inhibitory TSH receptor autoantibodies, as well as TSH, appear to bind to different sites in domains ABC, but similar sites in domains DE, of the receptor. Alternatively, TSH and the different TSH receptor antibodies bind with differing affinities to the same site in the ABC region.

Autoantibodies↗