Targeting of β-Arrestin2 to the Centrosome and Primary Cilium: Role in Cell Proliferation Control

International audienceBackground: The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs). Most GPCRs are regulated by b-arrestins, barr1 and barr2, which control both their signalling and endocytosis, suggesting that barrs may also function at primary cilium.Methodology/Principal Findings: In cycling cells, βarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, βarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, βarr2 was found at the basal body and axoneme of primary cilia. Interestingly, βarr2 was found to interact and colocalize with 14-3-3 proteins and Kif3A, two proteins known to be involved in ciliogenesis and intraciliary transport. In addition, as suggested for other centrosome or cilia-associated proteins, βarrs appear to control cell cycle progression. Indeed, cells lacking βarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions.Conclusions/Significance: Our results show that βarr2 is localized to the centrosome in cycling cells and to the primary cilium in quiescent cells, a feature shared with other proteins known to be involved in ciliogenesis or primary cilium function. Within cilia, βarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell “antenna”

The Phospholipid Scramblases 1 and 4 Are Cellular Receptors for the Secretory Leukocyte Protease Inhibitor and Interact with CD4 at the Plasma Membrane

Secretory leukocyte protease inhibitor (SLPI) is secreted by epithelial cells in all the mucosal fluids such as saliva, cervical mucus, as well in the seminal liquid. At the physiological concentrations found in saliva, SLPI has a specific antiviral activity against HIV-1 that is related to the perturbation of the virus entry process at a stage posterior to the interaction of the viral surface glycoprotein with the CD4 receptor. Here, we confirm that recombinant SLPI is able to inhibit HIV-1 infection of primary T lymphocytes, and show that SLPI can also inhibit the transfer of HIV-1 virions from primary monocyte-derived dendritic cells to autologous T lymphocytes. At the molecular level, we show that SLPI is a ligand for the phospholipid scramblase 1 (PLSCR1) and PLSCR4, membrane proteins that are involved in the regulation of the movements of phospholipids between the inner and outer leaflets of the plasma membrane. Interestingly, we reveal that PLSCR1 and PLSCR4 also interact directly with the CD4 receptor at the cell surface of T lymphocytes. We find that the same region of the cytoplasmic domain of PLSCR1 is involved in the binding to CD4 and SLPI. Since SLPI was able to disrupt the association between PLSCR1 and CD4, our data suggest that SLPI inhibits HIV-1 infection by modulating the interaction of the CD4 receptor with PLSCRs. These interactions may constitute new targets for antiviral intervention

Disruption of the CD4/PLSCR1 interaction by SLPI.

<p>A) In vitro inhibition of the CD4/PLSCR1 interaction by SLPI. GST or GST-PLSCR1 (left panel, coomassie blue) was incubated with equal amounts of lysates from Jurkat CD4-positive T cells in the presence of the indicated concentrations of either GST-SLPI or GST-ARF1 (left panel) used as a control. Bound proteins were analyzed by Western blot with anti-CD4 (right panel). B) Mapping of the PLSCR1 determinants required for binding to CD4 and SLPI. L40 yeast strain expressing either the cytoplasmic domain of CD4 (CD4c) or SLPI fused to LexA in combination with each of the deleted forms of the Gal4AD-PLSCR1 hybrids indicated on the left was analyzed for histidine auxotrophy and beta-gal activity. The interactions between hybrid proteins were scored as follows: (+), cell growth on medium without histidine and development of a β-gal activity; (−), no growth on medium without histidine and no β-gal activity.</p

Anti-HIV-1 activity of recombinant SLPI.

<p>A) Inhibition of virus replication. Primary T lymphocytes were isolated from peripheral blood mononuclear cells and used for infection with the HIV-1JR-CSF isolate in the presence of increasing concentrations (0–50 µg/ml) of purified GST-SLPI (see inset, coomassie blue) or GST (50 µg/ml). After washing, infected cells were cultured for 6 days and the viral production was quantified by measuring the p24 production in the cell-culture medium. B) Inhibition of virus transfer from dendritic cells to T lymphocytes. Dentritic cells were derived from purified primary monocytes, and incubated with either HIV-1Bal (R5 strain, upper panel) or HIV-1NDK (×4 strain, lower panel) for 1 h at 37°C in the presence of 50 µg/ml of purified GST-SLPI or GST. After washing, IL-2 activated-lymphocytes were added in a 1/5 ratio and maintained in co-culture for 72 h. Viral production was quantified by measuring the p24 production in the cell culture medium.</p

Mapping of the CD4 determinants required for PLSCR1 binding by co-immunoprecipitation assay.

<p>A) Schematic representation of the human CD4 mutants. The extracellular and transmembrane (TM) domains of CD4 are represented in grey and hatched, respectively. The a.a. sequences of the cytoplasmic tail of the CD4 mutants are aligned with that of the wild-type CD4 (CD4 WT). Dashes (−) indicate a.a. identities with the wild type protein and a.a. substitutions are identified. B) Co-immunoprecipitation assay. Protein extracts were prepared 48 h post transfection from 293T cells co-expressing HA-PLSCR1 together with wild type or mutated CD4 as indicated at the top. Crude extracts were then subjected to CD4 immunoprecipitation followed by Western blot analysis with anti-CD4 (upper panels) and anti-HA (lower panels).</p

Mapping of the CD4 determinants required for PLSCR1 binding by ELISA.

<p>A) Primary a.a. sequences of the CD4 long (405–433) and short (405–426) peptides used in the ELISA test. The primary sequence of the entire cytoplasmic domain of CD4 is shown at the top; the 2 Cys residues required for p56Lck binding and mutated in Ala in both peptides are indicated in red. B) ELISA interaction test. 96-well plates were coated with the CD4c long (left panel) or short (right panel) peptides at a final concentration of 0.5 µM. After washings and blocking of non-specific binding sites, GST or GST-PLSCR1 was incubated at concentrations ranging from 1 nM to 300 nM. Binding was then revealed with an anti-GST antibody and a secondary peroxidase-conjugated anti-mouse IgG. The peroxidase substrate solution was incubated for 10 min and the optical density was measured at 450 nm.</p

Interaction of PLSCR1 and PLSCR4 with the CD4 receptor.

<p>A) In vitro interaction. Lysates from Jurkat CD4-positive T cells were incubated with equal amounts of GST or GST-PLSCR1 (upper panel, coomassie blue) immobilized on GSH-sepharose beads. Bound proteins were then analyzed by immunoblotting with anti-CD4 (lower panel). B) Co-immunoprecipitation of endogenous proteins from T lymphocytes. Jurkat T cells were lyzed and CD4 was precipitated with either anti-CD4 (OKT4) or a control isotypic antibody. Precipitates were analyzed by Western blot with anti-CD4 (upper panel) or anti-PLSCR1 (lower panel). C) Co-precipitation of overexpressed CD4 and PLSCR1 proteins. 293T cells expressing HA-tagged PLSCR1 (lower panel, Cell lysate) in combination with wild-type CD4 (WT) or a mutant of CD4 deleted of its cytoplasmic domain (ΔCT) were lyzed and the CD4 forms were precipitated with anti-CD4. Precipitates were then analyzed by Western blot with anti-CD4 (upper panel) or anti-HA (middle panel). D) Co-precipitation of overexpressed CD4 and PLSCR4 proteins. 293T cells expressing GFP- PLSCR1, GFP-PLSCR3 or GFP-PLSCR4 (lower panels, Cell lysate) in combination with CD4 WT (right panels) or CD4 ΔCT (left panels) were lyzed and the CD4 forms were precipitated with anti-CD4. Precipitates were then analyzed as in (C). Of note, the differences in migration observed between PLSCR1 (318 a.a.), PLSCR3 (295 a.a.) and PLSCR4 (329 a.a.) is likely related to the respective amino acid lengths of the GFP fusion proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005006#pone.0005006-Zhou1" target="_blank">[23]</a>. E) Interactions in the two-hybrid system. L40 yeast strain expressing the LexA-CD4c hybrid in combination with the indicated Gal4AD hybrids was analyzed for histidine auxotrophy and β-gal activity. Transformants were patched on medium with histidine (upper panels) and then replica-plated on medium without histidine (middle panels) and on Whatman filter for β-gal assay (lower panels).</p

Co-distribution of scramblases and CD4 at the plasma membrane.

<p>A) Schematic representation of PLSCR1. PLSCR1 is a type-II transmembrane protein with a long N-terminal cytoplasmic domain (a.a. 1–290, grey box) containing 3 Cys-rich motifs (red boxes), and a transmembrane domain (a.a. 291–310, black box). Amino acids are numbered according to Zhou et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005006#pone.0005006-Zhou1" target="_blank">[23]</a>. B) Localization of wild type and deleted GFP-PLSCR1 forms. Jurkat CD4-positive T cells expressing the full length (left panel) or deleted forms (1–310 and 1–290, central and right panels, respectively) of GFP-PLSCR1 were fixed and directly examined. Cells were analyzed by epifluorescence microscopy, and images were acquired using a CCD camera. C) Localization of GFP-PLSCR3 and GFP-PLSCR4. Jurkat cells expressing GFP-PLSCR1 (left panel), GFP-PLSCR3 (central panel) or GFP-PLSCR4 (right panel) were fixed and directly examined as in (B). D) Subcellular distribution of CD4 and PLSCR1. Jurkat cells expressing wild type GFP-PLSCR1 (middle panel) were fixed, permeabilized and subsequently stained with an anti-CD4 (left panel). Scale bars, 10 µm.</p