Sodium iodide symporter-mediated radioiodide imaging and therapy of ovarian tumor xenografts in mice.

Ovarian cancer represents the fifth leading cause of cancer death among women in the United States, with >16 000 deaths expected this year. This study was carried out to investigate the potential of sodium iodide symporter (NIS)-mediated radioiodide therapy as a novel approach for ovarian cancer treatment. Radioiodide is routinely and effectively used for the treatment of benign and malignant thyroid disease as a result of native thyroidal expression of NIS, which mediates iodide uptake. In vitro gene transfer studies in ovarian cancer cells revealed a 12- and five-fold increase in iodide uptake when transduced with Ad/CMV/NIS or Ad/MUC1/NIS, respectively. Western blot/immunohistochemistry confirmed NIS protein expression. In vivo ovarian tumor xenografts were infected with the adenoviral constructs. (123)I imaging revealed a clear image of the CMV/NIS-transduced tumor, with a less intense image apparent following infection with MUC1/NIS. Therapeutic doses of (131)I following CMV/NIS infection caused a mean 53% reduction in tumor volume (P<0.0001). MUC1/NIS-transduced tumors did not regress, although at 8 weeks following therapy, tumor volume was significantly less that of control animals (166 versus 332%, respectively, P<0.05). This study represents a promising first step investigating the potential for NIS-mediated radioiodide imaging and therapy of ovarian tumors.

Radioiodine therapy of colon cancer following tissue-specific sodium iodide symporter gene transfer.

We investigated the feasibility of using radioiodine therapy in colon carcinoma cells (HCT 116) following tumor-specific expression of the human sodium iodide symporter (hNIS) using the carcinoembryonic antigen (CEA) promoter. HCT 116 cells were stably transfected with an expression vector, in which hNIS cDNA has been coupled to a CEA promoter fragment. This promoter is responsible for tissue-specific expression of CEA in gastrointestinal tract epithelium, and has been shown to target therapeutic genes to colorectal cancer cells. Functional NIS expression was confirmed by iodide uptake assay, Western blot analysis, immunostaining and in vitro clonogenic assay. The stably transfected HCT 116 cells concentrated (125)I about 10-fold in vitro without evidence of iodide organification. In contrast, transfection of control cancer cells without CEA expression did not result in iodide accumulation. Western blot analysis using a hNIS-specific antibody revealed a band of approximately 90 kDa. In addition, immunostaining of stably transfected HCT 116 cells revealed hNIS-specific membrane-associated immunoreactivity. In an in vitro clonogenic assay approximately 95% of stably transfected HCT 116 cells were killed by exposure to (131)I, while only about 5% of NIS-negative control cells were killed. Further, using an adenovirus carrying the NIS gene linked to the CEA promoter, high levels of tumor-specific radioiodide accumulation were induced in HCT 116 cells. In conclusion, a therapeutic effect of (131)I has been demonstrated in colon carcinoma cells following induction of tumor-specific iodide uptake activity by CEA promoter-directed NIS expression in vitro. This study demonstrates the potential of NIS as a therapeutic gene allowing radioiodine therapy of colon cancer following tumor-specific NIS gene transfer.

Retinoic acid-induced stimulation of sodium iodide symporter expression and cytotoxicity of radioiodine in prostate cancer cells.

We reported recently the induction of androgen-dependent iodide uptake activity in the human prostatic adenocarcinoma cell line LNCaP using a prostate-specific antigen (PSA) promoter-directed expression of the sodium iodide symporter (NIS) gene. This offers the potential to treat prostate cancer with radioiodine. In the current study, we examined the regulation of PSA promoter-directed NIS expression and therapeutic effectiveness of (131)I in LNCaP cells by all-trans-retinoic acid (atRA). For this purpose, NIS mRNA and protein expression levels in the NIS-transfected LNCaP cell line NP-1 were examined by Northern and Western blot analysis following incubation with atRA (10 (-9) to 10(-6) M) in the presence of 10(-9) M mibolerone (mib). In addition, NIS functional activity was measured by iodide uptake assay, and in vitro cytotoxicity of (131)I was examined by in vitro clonogenic assay. Following incubation with atRA, NIS mRNA levels in NP-1 cells were stimulated 3-fold in a concentration-dependent manner, whereas NIS protein levels increased 2.3-fold and iodide accumulation was stimulated 1.45-fold. This stimulatory effect of atRA, which has been shown to be retinoic acid receptor mediated, was completely blocked by the pure androgen receptor antagonist casodex (10(-6) M), indicating that it is androgen receptor dependent. The selective killing effect of (131)I in NP-1 cells was 50% in NP-1 cells incubated with 10(-9) M mib. This was increased to 90% in NP-1 cells treated with atRA (10(-7) M) plus 10(-9) M mib. In conclusion, treatment with atRA increases NIS expression levels and selective killing effect of (131)I in prostate cancer cells stably expressing NIS under the control of the PSA promoter. Therefore atRA may be used to enhance the therapeutic response to radioiodine in prostate cancer cells following PSA promoter-directed NIS gene delivery.

In vivo sodium iodide symporter gene therapy of prostate cancer.

Radioiodine therapy, the most effective form of systemic radiotherapy available, is currently useful only for thyroid cancer because of thyroid-specific expression of the sodium iodide symporter (NIS). Here we explore the efficacy of a novel form of gene therapy using adenovirus-mediated in vivo NIS gene transfer followed by (131)I administration for treatment of prostate cancer. Prostate cancer xenografts in nude mice injected with an adenovirus carrying the NIS gene linked to the cytomegalovirus (CMV) promoter revealed highly active uptake of radioiodine. Following administration of 3 mCi of (131)I, we observed an average tumor volume reduction of 84 +/- 12%. These results show for the first time that in vivo NIS gene delivery into non-thyroidal tumors is capable of inducing accumulation of therapeutically effective radioiodine doses and might therefore represent an effective and potentially curative therapy for prostate cancer.

Immunohistochemical analysis of sodium iodide symporter expression in metastatic differentiated thyroid cancer: correlation with radioiodine uptake.

The ability of thyroid cancers to concentrate radioiodine (RAI) is dependent, in part, upon the expression and functional integrity of the sodium iodide symporter (NIS). However, some differentiated thyroid carcinomas (DTCs) and most undifferentiated thyroid carcinomas lack the ability to concentrate iodide and are thereby insensitive to 131I therapy. Variation of NIS protein expression may be an important factor in this behavior. We wished to determine whether NIS protein expression in primary DTC tumors correlated with the subsequent RAI uptake by metastatic lesions in the same patients. We obtained paraffin-embedded tissue specimens from 60 patients with metastatic thyroid cancer who had undergone total or near-total thyroidectomy at the Mayo Clinic for DTC and had known presence or absence of RAI uptake in their tumor deposits determined by total body scanning after thyroid hormone withdrawal. Tissue sections from the primary intrathyroidal tumors were subjected to immunostaining (IS) using a monoclonal antibody against human NIS. Slides were subsequently examined for specific IS by two independent reviewers. For each patient, whole body scan (WBS) uptake was recorded, and correlation between results of IS and WBS was analyzed. Of 43 patients with a positive WBS, 37 also had positive IS of their tumors. In six patients with negative IS, a positive WBS was documented, and in three of these cases TSH at the time of surgery was less than 0.3 mIU/liter. Of the 17 patients with negative WBS, 10 were also negative on IS. Positive IS accurately predicted a positive scan in our study in 84% of cases; the ability of the IS to detect all cases with a positive scan was 86%, and it increased to 90% when patients who were receiving thyroid hormone therapy at the time of surgery were excluded from the analysis. Overall, the results of our retrospective study suggest that NIS IS of the thyroidal primary tumor in patients with papillary and follicular thyroid cancers has substantial ability to predict the behavior of subsequent deposits of metastatic and recurrent cancer with respect to iodine trapping and concentration. Our findings require confirmation in prospective studies to more accurately determine the predictive ability of the test and its role in the postoperative management of patients with DTC. If confirmed, NIS IS of DTC primary lesions may prove useful in the management of patients with known or suspected metastatic thyroid cancer.

Cloning of the mouse sodium iodide symporter.

The iodide-concentrating ability of the thyroid gland is essential to the production of thyroid hormone. We report the nucleotide and amino acid sequence of the mouse sodium iodide symporter (mNIS), which mediates this activity within the thyroid gland. An open reading frame of 1,857 nucleotides codes for a protein of 618 amino acids with 95% identity to rat NIS and 84% identity to human NIS. Transient expression of the mNIS cDNA in Chinese hamster ovary (CHO) cells, a nonthyroid cell line, resulted in sodium-dependent, perchlorate-sensitive iodide uptake. Western blot analysis of membrane preparations of CHO cells transiently transfected with mNIS cDNA showed a band of 90 kd when probed with an antibody directed against rat NIS. mNIS will serve as an important reagent in determining the role of NIS in experimental thyroid diseases and for monitoring the immune response to in animal models of NIS-mediated gene therapy.

Expression of the sodium iodide symporter in human kidney.

BACKGROUND: The human sodium iodide symporter (hNIS) is a transmembrane protein that mediates the active transport of iodide in the thyroid gland. Following cloning of NIS, NIS expression has been detected in a broad range of nonthyroidal tissues, suggesting that iodide transport in these tissues is conferred by the expression of functional NIS protein. METHODS: The aim of this study was to examine functional hNIS expression in kidney by reverse transcription-polymerase chain reaction (RT-PCR), ribonuclease protection assay (RPA), immunohistochemistry, and Western blot analysis accompanied by iodide accumulation studies in kidney cells. RESULTS: Using a pair of full-length hNIS-specific oligonucleotide primers, RT-PCR followed by Southern hybridization revealed hNIS mRNA expression in normal human kidney tissue. The PCR products were subjected to automated sequencing and revealed full identity with the published human thyroid-derived NIS cDNA sequence. Furthermore, positive protected bands indicating the presence of hNIS mRNA were apparent in RPA gel lanes corresponding to human kidney cells as well as Chinese hamster ovary (CHO) cells stably transfected with hNIS cDNA and Graves' thyroid tissue. Immunohistochemical analysis of normal human kidney tissue using a mouse monoclonal hNIS-specific antibody showed marked hNIS-specific immunoreactivity confined to tubular cells, while no hNIS-specific immunoreactivity was detected in the glomeruli. NIS protein expression in human kidney cells was further confirmed by Western blot analysis. In addition, accumulation of (125)I was detected in human kidney cells in vitro and was shown to be sodium dependent and sensitive to perchlorate. CONCLUSIONS: Functional hNIS expression was demonstrated in the renal tubular system, suggesting that renal iodide transport may be, at least in part, an active process driven by NIS.

Sodium iodide symporter (NIS) gene expression in human placenta.

The placenta must allow the passage of iodide from the maternal to the fetal circulation for synthesis of thyroxine by the fetal thyroid. The thyroid sodium iodide symporter (NIS) was cloned in 1996 and, although widely distributed among epithelial tissues, early studies failed to detect it in placenta. We demonstrated NIS mRNA in human placenta and in the human choriocarcinoma cell line, JAr. NIS protein was localized to trophoblasts, with a tendency to apical distribution, in sections of human placenta immunostained with a monoclonal antibody against hNIS. We conclude that NIS is expressed in placenta and may mediate placental iodide transport.

Treatment of prostate cancer by radioiodine therapy after tissue-specific expression of the sodium iodide symporter.

Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) by targeted NIS gene transfer might offer the possibility of radioiodine therapy of prostate cancer. Therefore, we investigated radioiodine accumulation and therapeutic effectiveness of 131I in NIS-transfected prostate cancer cells in vitro and in vivo. The human prostatic adenocarcinoma cell line LNCaP was stably transfected with NIS cDNA under the control of the prostate-specific antigen promoter. The stably transfected LNCaP cell line NP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in vitro that resulted in selective killing of these cells by 131I in an in vitro clonogenic assay. Xenografts were established in athymic nude mice and imaged using a gamma camera after i.p. injection of 500 microCi of 123I. In contrast to the NIS-negative control tumors (P-1) which showed no in vivo uptake of 123I, NP-1 tumors accumulated 25-30% of the total 123I administered with a biological half-life of 45 h. In addition, NIS protein expression in LNCaP cell xenografts was confirmed by Western blot analysis and immunohistochemistry. After a single i.p. application of a therapeutic 131I dose (3 mCi), significant tumor reduction was achieved in NP-1 tumors in the therapy group compared with P-1 tumors and tumors in the control group. In conclusion, a therapeutic effect of 131I has been demonstrated in prostate cancer cells after induction of tissue-specific iodide uptake activity by prostate-specific antigen promoter-directed NIS expression in vitro and in vivo. This study demonstrates the potential of NIS as a novel therapeutic gene for nonthyroidal cancers, in particular prostate cancer.

Monoclonal antibodies against the human sodium iodide symporter: utility for immunocytochemistry of thyroid cancer.

The recent cloning of the thyroidal protein that is responsible for iodide transport, the sodium iodide symporter (hNIS), has made possible studies designed to characterize its structure, function and expression in thyroidal tissues. Using a mannose binding protein (MBP)-hNIS fusion protein as antigen, we have developed mouse monoclonal antibodies against hNIS to utilize as tools in such studies. Twenty-four clones were initially recovered which recognized the MBP-hNIS fusion protein, but only two of them were specific for hNIS while the others recognized MBP alone. Both antibodies were found to be immunoglobulin G (IgG) 1kappa (kappa). The specificity of antibodies was tested by Western blotting using membranes prepared from COS-7 cells transiently transfected with the pcDNA3 plasmid containing the full-length hNIS cDNA, or cells transfected with the pcDNA3 vector. A major band with a molecular weight (MW) of approximately 97 kDa, and several minor bands with MW of approximately 160 kDa, approximately 68 kDa, approximately 30 kDa and approximately 15 kDa, were detected specifically in the hNIS-transfected cells. After enzymatic deglycosylation, the major band was present at 68 kDa, as expected based upon the amino acid sequence of hNIS. Immunohistochemistry was performed with several different types of thyroid tissue and non-thyroidal tissues, using the monoclonal antibodies. Strong immunostaining was observed in Graves' tissue, with intermediate staining in papillary and follicular thyroid cancers and an absence of staining in Hürthle cell cancer. The staining was specific for the follicular epithelium and was concentrated in the basolateral portion of the cell membrane. These monoclonal hNIS antibodies should prove useful in the characterization of NIS expression in benign and malignant thyroid tissue and in studies characterizing its structure and function.

Germline polymorphism of codon 727 of human thyroid-stimulating hormone receptor is associated with toxic multinodular goiter.

Toxic multinodular goiter (TMNG) represents a frequent cause of endogenous hyperthyroidism, affecting 5-15% of such patients (with higher frequencies reported in iodine-deficient areas of the world). Although mutations of human TSH receptor (hTSHR) have been described in autonomously functioning thyroid nodules (AFTN), the role of such mutations in the pathogenesis of TMNG remains unclear. To search for alterations of hTSHR in AFTN and TMNG, we performed bidirectional, dye primer automated fluorescent DNA sequencing of the entire transmembrane domain and cytoplasmic tail of hTSHR (TMD+CT-hTSHR) using DNA extracted from nodular regions of 24 patients with TMNG and 7 patients with AFTN. Eight of the 24 patients (33.3%) showed heterozygote polymorphism of codon 727 on the cytoplasmic tail of hTSHR with an amino acid substitution of aspartic acid to glutamic acid. Three of 24 (12.5%) patients with TMNG were found to carry a heterozygote mutation of codon 703, resulting in substitution of alanine with glycine. One patient had multiple heterozygote mutations including I606M (Ile to Met), A703G (Ala to Gly), Q720E (Gln to Glu), and D727E (Asp to Glu). Two patients exhibited silent polymorphism of codons 460 and 618. We found no mutation of the TMD+CT-hTSHR in 7 patients with AFTN, except for a silent polymorphism of codon 460 in 1. DNA fingerprinting of codon 727 using restriction enzyme NlaIII and genomic DNA confirmed the sequencing results in all cases, indicating that the sequence alterations were not somatic in nature. This technique was also used to examine peripheral blood genomic DNA from 52 normal individuals and 49 patients with Graves' disease; 33.3% of TMNG (P = 0.019 vs. normal subjects), 16.3% of Graves' disease patients (P = 0.10 vs. normal subjects), and 9.6% of normal individuals were heterozygous for the D727E polymorphism. Expression of the D727E hTSHR variant in eukaryotic cells (COS-7) resulted in an exaggerated cAMP response to TSH stimulation compared to that of the wild-type hTSHR. These findings indicate that a germline polymorphism of codon D727E of hTSHR is associated with TMNG, suggesting that its presence is an important predisposing genetic factor in the pathogenesis of TMNG.

Development of monoclonal antibodies against the human sodium iodide symporter: immunohistochemical characterization of this protein in thyroid cells.

The thyroid sodium-iodide symporter (NIS) is responsible for iodide concentrating ability within thyroid follicular cells. We sought to develop monoclonal antibodies against human NIS (hNIS) for use as reagents in structure-function studies of the protein, as well as potential tools in the assessment of NIS expression in benign and malignant thyroid tissues. Synthetic peptides corresponding to the second ExMD and to the carboxy-terminal ExMD of hNIS were produced and utilized as antigens to develop monoclonal antibodies, which were tested by Western blotting using membranes prepared from COS-7 cells transiently transfected with a pcDNA3 plasmid containing the gene for the full-length hNIS, or a control vector. Western blotting showed a major band with molecular weight (MW) of approximately 97 kDa and several minor bands with MW of approximately 160 kDa, 68 kDa, 30 kDa, and 15 kDa, all specific for hNIS-transfected cells. Immunohistochemistry was performed in various types of thyroid tissues and nonthyroidal tissues, using the monoclonal antibodies. Strong immunostaining was observed in Graves' tissue, intermediate staining in papillary and follicular thyroid cancer, and no staining in Hürthle cell cancer or in nonthyroidal tissue. The staining was specific for the follicular epithelium in each of the tissues and was most intense in the basolateral portion of the cell membrane. Overall, our observations indicate that the monoclonal antibodies are specific for hNIS and will be invaluable reagents for investigating the role of NIS in thyroid disease.

Prostate-specific antigen (PSA) promoter-driven androgen-inducible expression of sodium iodide symporter in prostate cancer cell lines.

Currently, no curative therapy for metastatic prostate cancer exists. Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) would enable those cells to concentrate iodide from plasma and might offer the ability to treat prostate cancer with radioiodine. Therefore, the aim of our study was to achieve tissue-specific expression of full-length human NIS (hNIS) cDNA in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP and in subcell lines C4, C4-2, and C4-2b in vitro. For this purpose, an expression vector was generated in which full-length hNIS cDNA coupled to the prostate-specific antigen (PSA) promoter has been ligated into the pEGFP-1 vector (NIS/PSA-pEGFP-1). The PSA promoter is responsible for androgen-dependent expression of PSA in benign and malignant prostate cells and was therefore used to mediate androgen-dependent prostate-specific expression of NIS. In addition, two control vectors were designed, which consist of the pEGFP-1 vector containing the PSA promoter without NIS cDNA (PSA-pEGFP-1) and NIS cDNA without the PSA promoter (NIS-pEGFP-1). Prostate cancer cells were transiently transfected with each of the above-described expression vectors, incubated with or without androgen (mibolerone) for 48 h, and monitored for iodide uptake activity. In addition, stably transfected LNCaP cell lines were established for each vector. Prostate cells transfected with NIS/PSA-pEGFP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in a range comparable to that observed in control cell lines transfected with hNIS cDNA. Perchlorate-sensitive iodide uptake was not observed in cells transfected with NIS/PSA-pEGFP-1 and treated without androgen or in cells transfected with the control vectors. In addition, prostate cancer cell lines without PSA expression (PC-3 and DU-145) did not show iodide uptake activity when transfected with NIS/PSA-pEGFP-1. Western blotting of LNCaP and C4-2b cell membranes transfected with NIS/PSA-pEGFP-1 using a monoclonal antibody that recognizes the COOH-terminus of hNIS revealed a band with a molecular weight of 90,000 that was not detected in androgen-deprived cells or in cells transfected with the control vectors, as well as a minor band at Mr 150,000 in transiently transfected LNCaP cell membranes. In conclusion, tissue-specific androgen-dependent iodide uptake activity has been induced in prostate cancer cells by PSA promoter-directed NIS expression. This study represents an initial step toward therapy of prostate cancer with radioiodine.

Binding of immunoglobulin G from patients with autoimmune thyroid disease to rat sodium-iodide symporter peptides: evidence for the iodide transporter as an autoantigen.

The recent cloning of the rat sodium-iodide symporter (rNIS) from FRTL-5 cells makes possible studies of the role of this thyroid-specific protein as an antigen in autoimmune diseases of the thyroid (AITD). We generated 21 synthetic peptides replicating the entire sequence of the extramembranous domains (ExMD) of rNIS. Each was synthesized by automated chemistry, purified by high-pressure liquid chromatography (HPLC), and characterized by mass spectroscopy. Immunoglobulins were purified using protein A from serum of 27 patients with Graves' disease (GD), 27 patients with autoimmune hypothyroidism (HT), and 20 normal controls. Binding of IgG from patients and controls to each of the rNIS peptides was measured by enzyme-linked immunosorbent assay (ELISA). Binding of patient IgG significantly greater than control was observed with six peptides: peptide 262-280 (representing ExMD 8 between transmembrane [TM] domains VII and VIII), peptide 437-444 (ExMD 11), peptides 468-487, 483-602, and 498-517 from ExMD 12, and peptides 560-579 from the proximal portion of the carboxyl terminus (ExMD 13). 63% of GD patients and 26% of HT patients immunoglobulin G (IgG) bound peptide 498-517 compared to zero controls. Similarly, 59% of GD were positive against peptide 468-487 versus zero controls. Peptide 262-280 bound IgG from 44% of GD patients, 15% of HT patients, and none of the controls. The remaining peptides showed little or no binding of patient IgG. These data indicate that patients with GD and HT possess antibodies that recognize rNIS significantly greater than do normal individuals, suggesting that the iodide transporter represents an important autoantigen in AITD. They further suggest that the incidence of the antibodies is higher in GD than HT, and that the antigenic epitopes involve ExMD 8, 11, 12, and 13.

Chimeric proteins can exceed the sum of their parts: implications for evolution and protein design.

Chimeric analogs derived from pairs of homologous proteins routinely exhibit activities found in one or both parents. We describe chimeras of two glycoprotein hormones, human chorionic gonadotropin (hCG) and human follitropin (hFSH), that exhibit activity unique to a third family member, human thyrotropin (hTSH). The results show that biological activity can be separated from hormone-specific amino acid residues. This is consistent with a model for the evolution of homologous ligand-receptor pairs involving gene duplication and the creation of inhibitory determinants that restrict binding. Disruption of these determinants can unmask activities characteristic of other members of a protein family. Combining portions of two ligands to create analogs with properties of a third family member can facilitate identifying key determinants of protein-protein interaction and may be a useful strategy for creating novel therapeutics. In the case of the glycoprotein hormones, this showed that two different hormone regions (i.e., the seat-belt and the intersubunit groove) appear to limit inappropriate contacts with receptors for other members of this family. These observations also have important caveats for chimera-based protein design because an unexpected gain of function may limit the therapeutic usefulness of some chimeras.

Receptor-specific activity of heteromeric thyrotropin (TSH) analogs: development of synthetic TSH antagonists.

In an attempt to create potent and specific inhibitors of the interaction of thyrotropin (thyroid-stimulating hormone [TSH]) with its receptor, we designed a series of 18 synthetic peptides containing sequences of both alpha and beta subunits that were shown previously to interact with the TSH receptor. These "heteromeric" peptide analogs included amino acid residues from alpha 26-46, beta 31-52, beta 88-95 and beta 101-112 that were arranged variously and were separated from each other by artificial amino acid spacers. Each peptide was tested for its ability to interact with the TSH receptor in a radio-receptor assay (TSH-RRA) using porcine thyroid membranes and a bio-assay for TSH using FRTL-5 cells. Twelve of the 18 peptides showed binding activity in the TSH-RRA. None of the analogs demonstrated thyroid stimulatory activity, but five inhibited TSH bioactivity and were, thus, pure antagonists, the most potent possessing EC50 values in the 3-5 microM range. Specificity of the antagonists was tested by measuring their ability to inhibit hCG binding to ovarian membranes, hCG-stimulated progesterone production in MA-10 rat Leydig tumor cells and FSH binding to testicular membranes. Only those peptides that included the alpha-subunit sequence CFSR or CCFSR exhibited binding activity for the heterologous receptors, and that activity was 10-fold lower than in the TSH assays. None of the heteromeric peptides showed activity in the hCG bioassays, further demonstrating their specificity as TSH antagonists. These studies illustrate the utility of a synthetic peptide approach in the development of analogs of peptide hormones. Future alterations that significantly enhance the potency of these antagonists may result in substances with clinical efficacy in diseases such as Graves' disease and differentiated thyroid cancer that involve the thyrotropin receptor.

Identification of thyroid blocking antibodies and receptor epitopes in autoimmune hypothyroidism by affinity purification using synthetic TSH receptor peptides.

UNLABELLED: To examine the interaction of immunoglobulins from patients with newly diagnosed hypothyroidism with the TSH receptor (TSHr), we tested protein-A purified IgG in an ELISA assay with a series of peptides representing the entire extracellular domain (ECD) of human TSHr. Antibodies bound, on average, 4.1 peptides (range 0-16) per patient, and antibodies from 26 of 30 patients (86.6%) demonstrated binding to at least one peptide. Six of the 20-mer peptides (61, 151, 181, 301, 361, 376) were most frequently recognized. These were used to construct affinity columns and separate IgGs from 10 patients into bound and unbound fractions. All fractions were tested for their ability to stimulate and inhibit cAMP generation in FRTL-5 cells. Inhibitory IgGs were purified from 9 patients (90%), suggesting that the incidence of blocking antibodies (TBAb) in autoimmune hypothyroidism is higher than previously reported. 7 of 10 patients had antibodies that recognized peptide 361 further supporting the importance of this epitope in TBAb binding. Anti-microsomal and anti-thyroglobulin antibodies did not co-purify with inhibitory antibodies, and were always in the unbound fractions. We found no correlation between the pattern of antibody binding or bioactivity with clinical manifestations of hypothyroidism. CONCLUSIONS: (1) The majority of patients with autoimmune hypothyroidism have antibodies against the TSHr-ECD that recognized linear epitopes. Most have antibodies directed at more than one site and the pattern is quite heterogeneous. (2) Six sites (noted above) are most frequently recognized. (3) Inhibitory antibodies are distinct from anti-microsomal and anti-thyroglobulin antibodies.

Delineation of amino acid residues within hTSHr 256-275 that participate in hormone binding.

The amino acid sequence 256-275 of the human thyrotropin (TSH) receptor extracellular domain has previously been shown to participate in a high affinity TSH binding site by a synthetic peptide approach as well as by site-directed mutagenesis. To further investigate this binding site, we synthesized a series of peptides with alanine substitutions for each residue in the native sequence. Peptides were also synthesized containing truncations or deletions of the native sequence. Each peptide was tested for its ability to inhibit 125I-bTSH binding to porcine thyroid membrane preparations, and the concentration at which 50% inhibition of binding occurred was determined (EC50). Alanine substitution at residues Tyr258, Cys262, Cys263, Phe265, Lys266, Asn267, Lys269, Lys270, and Arg272 all resulted in statistically significant decreases in activity when compared to the native sequence (p < 0.05). Alanine substitution of the remaining residues did not alter their activity. Comparison of this sequence with the corresponding sequences of the remaining glycoprotein hormone receptors (human lutropin and human follitropin receptors) reveals that these residues lie within one of the most highly conserved regions of the extracellular domain. We conclude that 9 specific amino acids within the sequence 256-275 of hTSHr (-Y--CC-FKN-KK-R--) participate in the interaction of the hTSHr-extracellular domain with TSH. This may represent a site in which the nonconserved residues are involved in the binding of the beta-subunit and the conserved residues are involved in the binding of the common alpha-subunit or a region of the beta-subunit that is common to all glycoprotein hormones.

Identification of epitopes and affinity purification of thyroid stimulating auto-antibodies using synthetic human TSH receptor peptides.

We prepared a series of overlapping peptides (29 in total, 20 amino acids each) containing the sequence of the entire extracellular domain of the human TSH receptor. Three peptides (181-200, 376-394, and EC3 (629-639)) bound IgG from patients with Graves' disease in an enzyme linked immunoassay. Peptide 181-200 bound IgG from 9 of 10, EC3 from 8 of 10, and 376-394 from 6 of 10 patients respectively, compared to 0 of 9 controls. We affinity purified TSHr auto-antibodies from four Graves' patients using the three above noted peptides bound to epoxy-activated sepharose. Thyroid stimulating activity was enriched in the bound fraction from at least two of the three peptide affinity columns in each of the four patients, although the pattern of affinity enrichment differed between patients. One patient was found to possess a combination of stimulatory and inhibitory TSHr antibodies and, after affinity purification, the anti-376-394 and anti-EC3 fractions were enriched in stimulatory activity, suggesting that those regions of the receptor were epitopes for stimulatory antibodies. However, affinity purification against peptide 181-200 produced an IgG preparation that was not stimulatory, but was a potent thyroid inhibitor. Thus, we have not only partially purified TSHr auto-antibodies, but also successfully separated stimulatory and inhibitory antibodies from a single patient using combination TSHr peptide affinity.