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.
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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