The thyrotropin (TSH) receptor (TSHR) is a member of the heterotrimeric G protein-coupled family of receptors whose main function is to regulate thyroid cell proliferation as well as thyroid hormone synthesis and release. In this study, we generated a TSHR knockout (TSHR-KO) mouse by homologous recombination for use as a model to study TSHR function. TSHR-KO mice presented with developmental and growth delays and were profoundly hypothyroid, with no detectable thyroid hormone and elevated TSH. Heterozygotes were apparently unaffected. Knockout mice died within 1 week of weaning unless fed a diet supplemented with thyroid powder. Mature mice were fertile on the thyroid-supplemented diet. Thyroid glands of TSHR-KO mice produced uniodinated thyroglobulin, but the ability to concentrate and organify iodide could be restored to TSHR-KO thyroids when cultured in the presence of the adenylate cyclase agonist forskolin. Consistent with this observation was the lack of detectable sodium-iodide symporter expression in TSHR-KO thyroid glands. Hence, by using the TSHR-KO mouse, we provided in vivo evidence, demonstrating that TSHR expression was required for expression of sodium-iodide symporter but was not required for thyroglobulin expression, suggesting that the thyroid hormone synthetic pathway of the mouse could be dissociated into TSHR-dependent and -independent steps.

Intrathyroidal T-cell accumulation in autoimmune thyroid disease is highly restricted and points to the primacy of T cells in causing thyroid disorders. These results present the possibility of using antibodies to the T-cell receptor for the specific inhibition of abnormal T-cell function in autoimmune thyroid disease.

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Human skin was labeled with purified antibodies against type I and m collagens and against their extension aminopropeptides by using the ferritin technique. Both aminopropeptides were visualized mainly along thin collagenous fibrils (diameter, 20-40 nm) and rarely in nonfibrillar regions of the skin. The labeling showed a periodicity of 60-65 nm resembling the D (67 nm) stagger of collagen molecules. Blocking of antibodies with aminopropeptides and treatment of tissues with procollagen NH2-terminal protease abolished labeling. Antibodies against type I collagen uniformly labeled -80% of the fibrils (diameter, 20-80 nm), while reaction with antibodies against type m collagen was restricted to thin fibrils. It is currently thought that the aminopropeptides of procollagen molecules are cleaved after they are released from the cell and before fibril formation. Our data indicate that aminopropeptides are removed at the fibrillar level and that fibril growth can be regulated by extracellular procollagen processing. Type I and III collagens are major components of the dermis and are organized into fibrils varying considerably in diameter. Distinct growth of fibrils is observed during development and may be disturbed in certain skin diseases (1). The mechanism of controlling this process is unknown. It has also not been established whether type I and III collagens are present in the same fibrils. Both collagens can form D-staggered fibrils in vitro showing identical cross-striations when examined in the electron microscope (2). Because specific antibodies against various collagens and procollagens are available (3, 4), it is feasible to identify distinct types of collagenous proteins at the ultrastructural level in situ, as was recently done for type III collagen (5).Interstitial collagens are synthesized in the form of procollagens that possess additional extension amino-and carboxypropeptides. Each precursor-specific peptide is removed by specific proteases (procollagen NH2-terminal and COOH-terminal proteases) presumably after release of procollagens from the cell (6). Cell and organ culture studies indicated different kinetics in the processing of type I and III procollagens (7-9). After cleavage, the aminopropeptides persist for some time in the extracellular space, as shown by immunofluorescence staining with antibodies against these peptides (3, 4).A functional role for extension aminopropeptides in the control of fibril growth was first suggested in studies of dermatosparactic animals, which, due to a defective NH2-terminal protease, accumulate an intermediate form of type I procollagen (pN-collagen) in the skin and other organs (10, 11). The collagenous fibrils appeared thin and hieroglyphic (12, 13) and could be stained with antibodies against the aminopropeptide by using a ferritin label (13). Small amounts of type I and III pN-collagens could also be extracted from the skin of growing animals (14-18). It was suggested (3, 14, 19) that these aminopropeptides are structural elements ofimmature or th...

Recently, several studies have reported oligomerization of G protein-coupled receptors, although the functional implications of this phenomenon are still unclear. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation (COIP), we previously reported that the human thyrotropin (TSH) receptor tagged with green fluorescent protein (TSHR(GFP)) and expressed in a heterologous system was present as oligomeric complexes on the cell surface. Here, we have extended this biophysical and biochemical approach to study the regulation of such oligomeric complexes. Co-expression of TSHR(GFP) and TSHR(Myc) constructs in Chinese hamster ovary cells resulted in FRET-positive cells. The specificity of the FRET signal was verified by the absence of energy transfer in individually transfected TSHR(GFP) and TSHR(Myc):Cy3 cells cultured together and also by acceptor photobleaching. Occupation of the receptor molecule by the ligand (TSH) resulted in a dose-dependent decrease in the FRET index from 20% in the absence of TSH to <1% with 10(3) microunits/ml of TSH. Such reduction in oligomeric forms was also confirmed by coimmunoprecipitation. Exposure of TSHR(GFP/Myc) cells to forskolin or cytochalasin D caused no change in the FRET index, confirming that the decrease in the oligomeric complexes was a receptor-dependent phenomenon and free of energy or microtuble requirements. The TSH-induced decrease in TSHR oligomers was found to be secondary to dissociation of the TSHR complexes as evidenced by an increase in fluorescent intensity of photobleached spots of GFP fluorescence with 10(3) microunits/ml of TSH. These data indicated that the less active conformation of the TSHR was comprised of receptor complexes and that such complexes were dissociated on the binding of ligand. Such observations support the concept of a constitutively active TSHR dimer or monomer that is naturally inhibited by the formation of higher order complexes. Inhibition of these oligomeric forms by ligand binding returns the TSHR to an activated state.

Organic anion transporters play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites from kidney, thereby preventing their potentially toxic effects within the body. The goal of this study was to extend our previous study on the functional characterization and posttranslational modification of a mouse kidney organic anion transporter (mOAT), in a mammalian cell system, COS-7 cells. The transporter-mediated p-aminohippurate (PAH) uptake was saturable, probenecid-sensitive, and inhibited by a wide range of organic anions including vitamins, antihypertensive drugs, anti-tumor drugs, and anti-inflammatory drugs. Tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited the transport activity. Immunofluorescence provided evidence that most of the protein remained in the intracellular compartment in tunicamycin-treated cells. Diethyl pyrocarbonate (DEPC), a histidine residue-specific reagent, completely blocked PAH transport. The inhibitory effect by DEPC was significantly protected (90%) by pretreating the cells with excess unlabeled PAH, suggesting that the histidine residues may be close to the PAH binding sites. Finally, in situ mRNA localization was studied in postnatal mouse kidney. The expression was observed in proximal tubules throughout development. We conclude that COS-7 cells may be useful in pharmacological and molecular biological studies of this carrier. The carbohydrate moieties are necessary for the proper trafficking of mOAT to the plasma membrane, and histidine residues appear to be important for the transport function.

We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2(b) and H-2(d)) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22-35 (A1O and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and A11 was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and A11, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.

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