The complementary DNA for human thyroid-stimulating hormone (TSH) receptor encodes a single protein with a deduced molecular mass of 84.5 kDa. This protein is cleaved during its maturation in the human thyroid since the receptor protein has been shown to be composed of two subunits (a subunit of = 53 kDa and p subunit of = 38 kDa) held together by disulfide bridges [Loosfelt, H., Pichon, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., Vannier, B. & Milgrom, E. (1992) Proc. Nutl Acad. Sci. USA 89, 3765-37691. A similar processing occurs in an L cell line permanently expressing the human TSH receptor. The processing is however incomplete, resulting in a permanent accumulation of a 95-kDa high-mannose precursor which is present only in trace amounts in the thyroid. Pulse-chase experiments show the successive appearance in the L cells of two precursors: initially the = 95-kDa high-mannose glycoprotein followed by a = 120-kDa species containing mature oligosaccharides. This latter precursor is then processed into the a and p subunits. In primary cultures of human thyrocytes precursors of similar size are detected.Spodopteru frugiperda insect cells (Sf9 and Sf21) infected with a recombinant baculovirus encoding the human TSH receptor synthesize a monomeric protein of about 90 kDa soluble only in denaturing conditions. Comparison with the product of in vitro transcription-translation experiments (= 80 m a ) , suggests that it may be incompletely or improperly glycosylated. The TSH receptor expressed in these cells is unable to bind the hormone.Immunoelectron microscopy studies show that in human thyrocytes most of the receptor is present on the cell surface; in L cells the receptor is detected on the cell surface, as well as in the endoplasmic reticulum and in the Golgi apparatus (this intracellular pool of receptor molecules probably corresponding to the high-mannose precursor) ; in insect cells nearly all the receptor molecules are trapped in the endoplasmic reticulum. These differences in receptor distribution are concordant with the differences observed for receptor processing.The thyroid-stimulating hormone (TSH) receptor has been the subject of extensive studies (reviews in [l, 21). Interest in this receptor stems not only from its key role in the control of thyroid function and growth (review in [3]), but also from its direct implication in autoimmune diseases. Autoantibodies against the TSH receptor display either a stimulatory effect and mimic the action of the hormone, provoking Graves' disease, or a blocking effect and lead to idiopathic myxoedema (reviews in [l, 2, 4, 51). However, due to its fragility and scarcity, attempts to purify the TSH receptor have been unsuccessful. Conflicting results have been reCorrespondence to E. Milgrom, HBpital de BicCtre, 3kme niveau, F-94275 Kremlin-Bicstre, FranceAbbreviations. TSH, thyroid stimulating hormone ; TSHR, thyroid stimulating hormone receptor; Sf, Spodopteru frugiperdu insect cells ; AcMNPV, Autogrupha Culifornicu multiple nuclear polyhedrosis virus ; D...

The discontinuous immunodominant region (IDR) recognized by autoantibodies directed against the thyroperoxidase (TPO) molecule, a major autoantigen in autoimmune thyroid diseases, has not yet been completely localized. By using peptide phage-displayed technology, we identified three critical motifs, LXPEXD, QSYP, and EX(E/D)PPV, within selected mimotopes which interacted with the human recombinant anti-TPO autoantibody (aAb) T13, derived from an antibody phage-displayed library obtained from thyroid-infiltrating TPO-selected B cells of Graves' disease patients. Mimotope sequence alignment on the TPO molecule, together with the binding analysis of the T13 aAb on TPO mutants expressed by Chinese hamster ovary cells, demonstrated that regions 353-363, 377-386, and 713-720 from the myeloperoxidase-like domain and region 766 -775 from the complement control protein-like domain are a part of the IDR recognized by the recombinant aAb T13. Furthermore, we demonstrated that these regions were involved in the binding to TPO of sera containing TPO-specific autoantibodies from patients suffering from Hashimoto's and Graves' autoimmune diseases. Identification of the IDR could lead to improved diagnosis of thyroid autoimmune diseases by engineering "mini-TPO" as a target autoantigen or designing therapeutic peptides able to block undesired autoimmune responses.Human thyroid peroxidase (TPO), 1 described previously as the "thyroid microsomal antigen" (1), is a membrane-bound enzyme expressed at the apical pole of thyrocytes (2). TPO generates the functional form of thyroglobulin by iodination and coupling of tyrosine residues (3). During autoimmune thyroid diseases (AITD), TPO represents a major target for the immune system (4, 5), leading to high titer TPO-specific autoantibodies (aAbs) in the sera of patients suffering from Hashimoto's thyroiditis and Graves' disease. Besides their role as efficient and early diagnostic markers of AITD, TPO-specific aAbs also act as effector molecules either through modulating antigen presentation to T cells or by mediating thyroid destruction after complement activation or antibody-dependent cell cytotoxicity (6 -12). Alignment studies and structural homologies have shown that TPO is formed by three distinct domains: a myeloperoxidase (MPO)-like, a complement control protein (CCP)-like, and an EGF-like domain, from the N-to the Cterminal extremities (13). Although the structure of each domain has been elucidated in part by three-dimensional modeling (13-16), the full three-dimensional structure of TPO remains unknown, even though low resolution crystals have been obtained (17,18). The flexibility observed for the hinge regions probably make difficult the exact positioning of each domain in relation to the others (15).These observations denote a highly complex structure of TPO, thus explaining the reason why TPO aAbs from patients' sera preferentially recognize discontinuous epitopes on . Different approaches have been used to determine the epitopic regions recognized by anti-TPO aAbs from ...

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Two plant (Arabidopsis thaliana) K+ transport systems, KAT1 and AKT1, have been expressed in insect cells (Sf9 cell line) using recombinant baculoviruses. Microscopic observation after immunogold staining revealed that the expressed AKT1 and KAT1 polypeptides were mainly associated with internal membranes, but that a minute fraction was targeted to the cell membrane. KAT1 was known, from earlier electrophysiological characterization in Xenopus oocytes, to be an inwardly rectifying voltage-gated channel highly selective for K+, while similar experiments had failed to characterize AKT1. Insect cells expressing KAT1 displayed an exogenous inwardly rectifying K+ conductance reminiscent of that described previously in Xenopus oocytes expressing KAT1. Under similar conditions, cells expressing AKT1 showed a disturbed cell membrane electrical stability that precluded electrophysiological analysis. Use of a baculovirus transfer vector designed so as to decrease the expression level allowed the first electrophysiological characterization of AKT1. The baculovirus system can thus be used as an alternative method when expression in Xenopus oocytes is unsuccessful for electrophysiological characterization of the ion channel of interest. The plant AKT1 protein has been shown in this way to be an inwardly rectifying voltage-gated channel highly selective for K+ ions and sensitive to cGMP.