Thierry Planchenault

CXCR4 dimerization and beta-arrestin-mediated signaling account for the enhanced chemotaxis to CXCL12 in WHIM syndrome.

WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is an immune deficiency linked in many cases to heterozygous mutations causing truncations in the cytoplasmic tail of CXC chemokine receptor 4 (CXCR4). Leukocytes expressing truncated CXCR4 display enhanced responses to the receptor ligand CXCL12, including chemotaxis, which likely impair their trafficking and contribute to the immunohematologic clinical manifestations of the syndrome. CXCR4 desensitization and endocytosis are dependent on beta-arrestin (betaarr) recruitment to the cytoplasmic tail, so that the truncated CXCR4 are refractory to these processes and so have enhanced G protein-dependent signaling. Here, we show that the augmented responsiveness of WHIM leukocytes is also accounted for by enhanced betaarr2-dependent signaling downstream of the truncated CXCR4 receptor. Indeed, the WHIM-associated receptor CXCR4(1013) maintains association with betaarr2 and triggers augmented and prolonged betaarr2-dependent signaling, as revealed by ERK1/2 phosphorylation kinetics. Evidence is also provided that CXCR4(1013)-mediated chemotaxis critically requires betaarr2, and disrupting the SHSK motif in the third intracellular loop of CXCR4(1013) abrogates betaarr2-mediated signaling, but not coupling to G proteins, and normalizes chemotaxis. We also demonstrate that CXCR4(1013) spontaneously forms heterodimers with wild-type CXCR4. Accordingly, we propose a model where enhanced functional interactions between betaarr2 and receptor dimers account for the altered responsiveness of WHIM leukocytes to CXCL12.

HIV-1 Entry into T-cells Is Not Dependent on CD4 and CCR5 Localization to Sphingolipid-enriched, Detergent-resistant, Raft Membrane Domains

The contribution of raft domains to human immunodeficiency virus (HIV) 1 entry was assessed. In particular, we asked whether the CD4 and CCR5 HIV-1 receptors need to associate with sphingolipid-enriched, detergent-resistant membrane domains (rafts) to allow viral entry into primary and T-cell lines. Based on Triton X-100 solubilization and confocal microscopy, CD4 was shown to distribute partially to rafts. In contrast, CCR5 did not associate with rafts and localized in nonraft plasma membrane domains. HIV-1-receptor partitioning remained unchanged upon viral adsorption, suggesting that viral entry probably takes place outside rafts. To directly investigate this possibility, we targeted CD4 to nonraft domains of the membrane by preventing CD4 palmitoylation and interaction with p56 lck . Directed mutagenesis of both targeting signals significantly prevented association of CD4 with rafts, but did not suppress the HIV-1 receptor function of CD4. Collectively, these results strongly suggest that the presence of HIV-1 receptors in rafts is not required for viral infection. We show, however, that depleting plasma membrane cholesterol inhibits HIV-1 entry. We therefore propose that cholesterol modulates the HIV-1 entry process independently of its ability to promote raft formation.

CD4 and CCR5 Constitutively Interact at the Plasma Membrane of Living Cells A CONFOCAL FLUORESCENCE RESONANCE ENERGY TRANSFER-BASED APPROACH

Human immunodeficiency virus entry into target cells requires sequential interactions of the viral glycoprotein envelope gp120 with CD4 and chemokine receptors CCR5 or CXCR4. CD4 interaction with the chemokine receptor is suggested to play a critical role in this process but to what extent such a mechanism takes place at the surface of target cells remains elusive. To address this issue, we used a confocal microspectrofluorimetric approach to monitor fluorescence resonance energy transfer at the cell plasma membrane between enhanced blue and green fluorescent proteins fused to CD4 and CCR5 receptors. We developed an efficient fluorescence resonance energy transfer analysis from experiments carried out on individual cells, revealing that receptors constitutively interact at the plasma membrane. Binding of R5-tropic HIV gp120 stabilizes these associations thus highlighting that ternary complexes between CD4, gp120, and CCR5 occur before the fusion process starts. Furthermore, the ability of CD4 truncated mutants and CCR5 ligands to prevent association of CD4 with CCR5 reveals that this interaction notably engages extracellular parts of receptors. Finally, we provide evidence that this interaction takes place outside raft domains of the plasma membrane.

Changes of surface glycoproteins after retinoic acid-dibutyryl cAMP-induced differentiation of teratocarcinoma stem cells☆

Retinoic acid induces differentiation of embryonal carcinoma F9 cells into parietal endoderm. The surface proteins of F9 cells from induced and control cultures were labeled with the 125I-lactoperoxidase system and analyzed by two-dimensional gel electrophoresis. Their quantitative comparison has shown an 11-fold increase of protein p220 of apparent MW 220,000 and isoelectric point 5.6. Among other enhanced surface proteins, 3.5-fold increases of p50, p45, and p40 of MW 50,000-40,000 and isoelectric point 5.1-5.3 were observed. Simultaneously another surface protein, p70 of MW 70,000 and isoelectric point 6.1-6.3, disappeared. The quantitative changes of surface proteins produced after treatment with retinoic acid were enhanced in the presence of dibutyryl cAMP. Analysis of lectin-binding proteins demonstrated that increasing proteins p220, p50, p45, and p40 have an affinity for concanavalin A, whereas p70, which decreases, has an affinity for wheat germ agglutinin. Antibodies raised against p70 from undifferentiated cells have shown a specific immunoreaction with p220 from differentiated cells and also with the subunit B of purified laminin. The electrophoretic mobilities of p220 and of the B subunit of laminin are similar. It is suggested that p70, p220, and laminin B subunit share structural homology.

Plasmin regulating system from embryonal carcinoma F9 cells: plasminases A, B and embrinogen.

Two plasmin inactivators, plasminase A and B, and their inhibitor embrinogen were isolated from embryonal carcinoma F9 cells by preparative two-dimensional electrophoresis. Plasminases A and B have molecular weights of 160,000 and 82,000, respectively. Both are serine proteinases which digest the light chain of plasmin in a time dependent inactivation process. The heavy chain of plasmin is not affected by this action. Plasminases A and B show similar specificity towards synthetic and natural polypeptide inhibitors. The interaction of the two enzymes leads to their inhibition. Embrinogen (m.w. 84,000) inhibits both plasminases A and B as well as urokinase and plasmin. Its activation by trypsin creates embrin, a proteinase directed against plasmin heavy chain.