Complementary DNA clones, encoding the LH-hCG (luteinizing hormone-human choriogonadotropic hormone) receptor were isolated by screening a lambda gt11 library with monoclonal antibodies. The primary structure of the protein was deduced from the DNA sequence analysis; the protein contains 696 amino acids with a putative signal peptide of 27 amino acids. Hydropathy analysis suggests the existence of seven transmembrane domains that show homology with the corresponding regions of other G protein-coupled receptors. Three other types of clones corresponding to shorter proteins were observed, in which the putative transmembrane domain was absent. These probably arose through alternative splicing. RNA blot analysis showed similar patterns in testis and ovary with a major RNA of 4700 nucleotides and several minor species. The messenger RNA was expressed in COS-7 cells, yielding a protein that bound hCG with the same affinity as the testicular receptor.

The extracellular and intracellular domains of the human thyrotropin receptor were expressed in Escherichia coli and the proteins were used to produce monoclonal anti-receptor antibodies. Immunoblot studies and immunoaffinity purification showed that the receptor is composed of two subunits linked by disulfide bridges and probably derived by proteolytic cleavage of a single 90-kDa precursor. The extracellular a subunit (hormone binding) had an apparent molecular mass of 53 kDa (35 kDa after deglycosylation with N-glycosidase F). The membrane-spanning (3 subunit seemed heterogeneous and had an apparent molecular mass of 33-42 kDa. Human thyroid membranes contained a 2.5-to 3-fold excess of (3 subunits over a subunits. Immunocytochemistry showed the presence of both subunits in all the follicular thyroid cells, and both subunits were restricted to the basolateral region of the cell membrane.The thyrotropin receptor (TSHR) has been the subject of great interest for many years due to its physiological importance and its implication in Graves disease (1, 2). However, its rarity and fragility have precluded its purification, and indirect evidence has led to conflicting reports on its molecular structure (1-13). Total molecular masses of 90-500 kDa, with subunits varying in number from one to three and in mass from 15 to 90 kDa, have been reported. TSHR cDNAs have been cloned by cross-hybridization with related G-protein-coupled receptor cDNAs or by PCR amplification with homologous primers (14-17). The primary structure of the encoded protein (molecular mass, 84.5 kDa) has been deduced. The high sequence homology with the lutropin receptor, which is composed of a single polypeptide chain (18), led most researchers to hypothesize a similar structure for the TSHR.We have used Escherichia coli to express fragments corresponding to the extracellular and intracellular domains of the TSHR. Immunization of mice led to the production of monoclonal antibodies that were used for the immunochemical characterization ofthe receptor. Here we report evidence for the heterodimeric structure of the TSHR. MATERIALS AND METHODSPreparation of Anti-TSHR Monoclonal Antibodies. cDNA fragments encoding amino acids 19-243 or 604-764 of the human TSHR were introduced into the polylinker of the vector pUR292 (19) or pNMHUB (20). Fusion proteins of TSHR with f3-galactosidase and ubiquitin, respectively, were produced in E. coli. Cell lysates were prepared in buffer A (20 mM sodium phosphate/0.3 M NaCl/10 mM MgCl2/1% Triton X-100, pH 7.4) containing lysozyme at 5 mg/ml. After two freeze-thaw cycles, the lysate was treated with DNase I (0.1 mg/ml) at 20°C for 20 min. After centrifugation at 10,000x g for 30 min, the pellets were washed twice with buffer A containing 0.5% sodium deoxycholate and twice with 2 M guanidinium chloride and solubilized in 6 M guanidinium chloride/0.5 M dithiothreitol. Samples were dialyzed for 48 hr in 10 mM sodium phosphate/150 mM NaCl/8 M urea/10 mM dithiothreitol, pH 8.0. For immunization of mice, the samples ...

The nuclear localization of the progesterone receptor is mediated by two signal sequences: one is constitutive and lies in the hinge region (between the DNA and steroid binding domains), the other is hormone dependent and is localized in the second zinc finger of the DNA binding domain. The use of various inhibitors of energy synthesis in cells expressing permanently or transiently the wild‐type receptor or a receptor mutated within the nuclear localization signals, demonstrated that the nuclear residency of the receptor reflects a dynamic situation: the receptor diffusing into the cytoplasm and being constantly and actively transported back into the nucleus. The existence of this nucleo‐cytoplasmic shuttle mechanism was confirmed by receptor transfer from one nucleus to the other in heterokaryons. Preliminary evidence was obtained, using oestrogen receptor, that this phenomenon may be of general significance for steroid receptors.

Deletion mutants of the rabbit progesterone receptor were used to identify two major mechanisms of its nuclear localization. A putative signal sequence, homologous to that of the SV40 large T antigen, was localized around amino acids 638-642 and shown to be constitutively active. When amino acids 638-642 were deleted, the receptor became cytoplasmic but could be shifted into the nucleus by the addition of hormone (or anti-hormone); it was almost fully active. The second mechanism consisted of the activation of the DNA binding domain. By deleting epitopes recognized by monoclonal antibodies, it was possible to follow different receptor mutants inside the same cells. In the absence of ligand, the receptor was transferred into the nucleus as a monomer. After administration of hormone (or anti-hormone) a "cytoplasmic" monomer was transferred into the nucleus through interaction with a "nuclear" monomer. These interactions occurred through the steroid binding domains of both monomers.

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...

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The thyrotropin (TSH) receptor belongs to a subfamily of G protein-coupled receptors, which also includes luteinizing hormone and follicle-stimulating hormone receptors. The TSH receptor (TSHR) differs from the latter by the presence of an additional specific segment in the C-terminal part of its ectodomain. We show here that this insertion is excised in the majority of receptor molecules. Preparation of specific monoclonal antibodies to this region, microsequencing, enzyme-linked immunosorbent assay, and immunoblot studies have provided insight into the mechanisms of this excision. In the human thyroid gland, N termini of the transmembrane receptor ␤ subunit were found to be phenylalanine 366 and leucines 370 and 378. In transfected L cells a variety of other more proximal N termini were found, probably corresponding to incomplete excisions. The most extreme N terminus was observed to lie at Ser-314. These observations suggest that after initial cleavage at Ser-314 the inserted fragment of TSHR is progressively clipped out by a series of cleavage reactions progressing up to amino acids 366 -378. The impossibility of recovering the excised fragment from purified receptor, cell membranes, or culture medium supports this interpretation. The cleavage enzyme has previously been shown to be inhibited by BB-2116, an inhibitor of matrix metalloproteases. However, we show here that it is unaffected by tissue inhibitors of metalloproteases. The cleavage enzyme is very similar to TACE (tumor necrosis factor ␣-converting enzyme) in both these characteristics. However, incubation of the TSH receptor with the purified recombinant catalytic domain of TACE, cotransfection of cells with TACE and TSHR expression vectors, and the use of mutated Chinese hamster ovary cells in which TACE is inactive suggested that the TSHR cleavage enzyme is different from TACE. TACE and TSHR cleavage enzyme may thus possibly be related but different members of the adamalysin family of metzincin metalloproteases. The thyrotropin receptor (TSHR)1 plays a key role in thyroid growth and function (reviewed in Refs. 1 and 2). This receptor is the target of stimulating or blocking autoantibodies produced in patients with autoimmune diseases (reviewed in Refs. 3 and 4). The TSHR was initially cloned by cross-hybridization with the luteinizing hormone receptor (5), or by polymerase chain reaction using degenerate primers (6 -8). Expression of the cloned receptor in Escherichia coli allowed its use as an immunogen to prepare monoclonal antibodies. These were used for immunoblotting and immunoprecipitation experiments which showed that the TSH receptor in human thyroid membranes underwent a post-translational cleavage event yielding two subunits: a ϳ53-kDa ␣ extracellular subunit and a ϳ38-kDa broad ␤ membrane spanning subunit. The subunits are held together by disulfide bridges (9). This maturation is unique among G protein-coupled receptors.In human thyroids, cleavage of the TSHR is almost complete. By contrast, in heterologous transfected cells monomeric ...