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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 level of synthesis and extracellular release of human immunodeficiency virus type 1 Gag by insect cells was analyzed, using eight different recombinants of Autographa californica nuclear polyhedrosis virus harboring various constructs of the gag gene, cloned under the polyhedrin promoter. The results obtained suggested that gag expression was mainly regulated at the transcriptional level and was not significantly influenced by posttranslational events, e.g., Gag self-assembly, nuclear transport, or extracellular release. Two different forms of Gag were found in the culture medium of recombinant-infected cells. One form consisted of membrane-enveloped, corelike particles released by budding at the plasma membrane; the other of nonparticulate, soluble Gag polyprotein molecules. Both forms coexisted in recombinants expressing Gag with an intact N-terminal myristylation signal, whereas recombinants expressing nonmyristylated Gag released solely the soluble form. This suggested that myristylation of the N terminus was not a prerequisite for efficient extracellular release of Gag by insect cells, which could proceed via two independent but simultaneous mechanisms.

The open reading frame (ORF) III product (PIII) of cauliflower mosaic virus is necessary for the infection cycle but its role is poorly understood. We have used in vitro protein binding ('far Western') assays to demonstrate that PIII interacts with the cauliflower mosaic virus (CaMV) ORF II product (PII), a known aphid transmission factor. Aphid transmission of purified virions of the PII-defective strain CM4-184 was dependent upon added PII, but complementation was efficient only in the presence of PIII, demonstrating the requirement of PIII for transmission. Deletion mutagenesis mapped the interaction domains of PIII and PII to the 30 N-terminal and 61 C-terminal residues of PIII and PII, respectively. A model for interaction between PIII and PII is proposed on the basis of secondary structure predictions. Finally, a direct correlation between the ability of PIII and PII to interact and aphid transmissibility of the virus was demonstrated by using mutagenized PIII proteins. Taken together, these data argue strongly that PIII is a second 'helper' factor required for CaMV transmission by aphids.

It has been previously shown that the HIV-1 envelope glycoprotein 120 (gp120) activates cell signaling by CXCR4, independently of CD4. The present study examines the involvement of different intracellular signaling pathways and their physiopathologic consequences following the CD4-independent interaction between CXCR4 or CCR5 and gp120 in different cell types: primary T cells, CD4 ؊ / CXCR4 ؉ /CCR5 ؉ T cells, or glioma cells. These interactions were compared with those obtained with natural ligands, stromal cell-derived factor 1 ␣ (SDF-1␣) (CXCL12) and macrophage inflammatory IntroductionAIDS results from different actions induced by HIV on its wide variety of target cells. The CD4 molecule is the major receptor of HIV-1. 1 The high-affinity binding of CD4 to glycoprotein 120 (gp120) 2 induces conformational changes in gp120 to allow its further binding to secondary receptors, 3-5 which could result in a trimolecular complex, CD4-gp120-coreceptor. 6,7 HIV coreceptors belong to the chemokine receptor family (see Baggiolini et al 8 for review), among which CXCR4 and CCR5 are the major attachment coreceptors for T-tropic and M-tropic isolates of HIV-1, respectively. 9,10 The HIV envelope binding to target cells induces activation through receptors, 11,12 eliciting functional responses that are important in AIDS pathogenesis. Although the cell activation is induced by a CD4-transconformed gp120 through the coreceptor, 13-15 a CD4-independent activation occurs, 16,17 particularly in T cells and cells from the central nervous system (CNS). 18,19 Indeed, it has been reported that cell signaling is activated by either the X4-tropic or R5-tropic HIV-1 envelopes, leading to activation of such functional responses as proliferation, differentiation, chemotaxis, 13,16 proinflammatory cytokines secretion, or apoptosis. 20,21 Apoptosis or programmed cell death is one of the major physiopathologic mechanisms of AIDS. Cells continuously stimulated become sensitive to apoptosis. 22 Interactions between 23 or between tumor necrosis factor ␣ (TNF␣) and its receptor (TNFr), activation of caspases, and down-regulation of the proto-oncogene Bcl2 protein and interleukin-2 (IL2) are considered the major apoptotic events during HIV infection leading to destruction of T cells, 24 macrophages, dendritic cells, and neurons.In the CNS, another important factor contributing to the neurodegenerative process is the presence of extracellular proteases from the matrix metalloproteinase (MMP) family. MMPs have been shown to degrade constituents of the extracellular matrix such as collagens, 25 favoring (1) The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 1734. For personal use only. on April 4, 2019. by guest www.bloodjournal.org From destruction of the extracellular matrix in vivo or in vitro. 27 The presence of MMP-9 was reported in cerebrospinal fluid of HIVinfected patients. 26,2...