UT activation mediating GABA_AR internalization.

<p>(Aa–Ad) CHO-UT transiently transfected with cDNA encoding α₂β₃^HAγ₂ GABA_AR subunits. Internalization was controlled through translocation of β₃^HA subunit (red) in control (Aa) or after 60 min of <i>h</i>UII (10⁻⁸ M, Ab), Iso (10⁻⁴ M, Ac) or <i>h</i>UII+Iso (Ad) incubation. Fluorescence intensity plots of green and red fluorescences corresponding to the localization of GABA_AR (β₃^HA) at the plasma membrane and in the cytosol, respectively, across the regions delimited by the white line scans. A.u., arbitrary unit; scale bars, 25 µm. (B) Bar graphs of the fraction of fluorescence at the plasma membrane on CHOT-UT-GABA_AR or CHO-GABA_AR in the different conditions. Each bar corresponds to mean ± SEM percent obtained from 3 to 18 cells. ns, non significant; ***, <i>P</i><0.001 <i>versus</i> control in CHO-UT-GABA_AR; ###, <i>P</i><0.001 <i>versus</i> control in CHO-GABA_AR.</p

Co-localization of UT with γ subunits in neuron and glial components in rat cerebellum.

<p>(A, A′) Double-fluorescence staining for UT (green) and NeuN (red) showing the presence of UT in both mature (arrowhead, merge, A′) and unidentified cells (arrows, merge, A′) in the IGL. (B) Co-staining of UT and the marker of Purkinje cells, calbindin (red), in Purkinje cell soma and dendrites (arrowhead, B′). (C) Staining for UT and the marker of migrating neuroblasts doublecortin DCX (red) depicting a diffuse labeling in the ML. (C′) UT immunopositive fibers contiguous to DCX-expressing migrating granule cells (merge, yellow, arrowhead). (D, D′) Staining for UT and GFAP (red) in glial fibers (merge, yellow, arrowhead) of the ML. (E, F) Distribution of UT and the γ₁ (E) and γ₂ (F) GABA_AR subunits (red), in Purkinje cells (merge, arrowhead) and few extents of glia (merge, arrow) in the ML and IGL. Nuclei (blue) were counterstained with DAPI. Scale bars, 50 µm (A–F); 20 µm (A′–F′). EGL, external granule cell layer; IGL, internal granule cell layer; ML, molecular layer; PCL, Purkinje cell layer. (A′–F′) images of digitally zoomed regions corresponding to the white boxes in A–F.</p

Schematic model depicting the mechanism of UT-mediated GABA_AR down-regulation.

<p>UII efficiently activates the G protein-coupled receptor UT, leading to a fast short-term decrease of the chloride current not sustained by G proteins, calcium, phosphorylation and endocytosis processes. This rapid effect involves the distal 19 C-terminal amino acids of UT and the presence of γ subunits within of the GABA_AR complex (1). During the washout period, a long-term inhibition develops <i>via</i> a dynamin-, calcium- and phosphorylation-dependent endocytic mechanisms, requiring at least in part the 351–370 sequence of UT and GABA_AR γ subunits (2). It is hypothesized that the directional cross-talk between UT and GABA_AR, and the extinction of the latter at the plasma membrane, may relay transition from quiescent to proliferant astrocytes.</p

UII-induced fast current inhibition and GABA_AR desensitization.

<p>(A, B) Examples of currents recorded from CHO-UT-GABA_AR during a long desensitizing pulse (25 s) of Iso (10⁻⁴ M), in the absence (black line) or presence (green line) of <i>h</i>UII (10⁻⁸ M, 1 min). (A) Exponential fit to the desensitizing current phases were shown overlaid on the currents. Bar graphs corresponding to the average desensitization constant parameter τ in the absence (τCtrl) or presence (τ<i>h</i>UII) of <i>h</i>UII (n = 5). (B) Prolonged Iso (30 s) application eliciting current desenzitization followed by a time course of the recovery from desensitization, in the absence (control) or presence of <i>h</i>UII. Graph represents the Iso-evoked current expressed as a fraction of the peak control current induced by the long Iso application to the current amplitude elicited by each short pulse, and plotted against interpulse intervals. Data are mean ± SEM from 3 to 8 cells. *, <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001 compared with the corresponding control Iso-evoked current.</p

Receptor sequences involved in UT regulation of the GABA_AR activity.

<p>(A) Schematic diagrams mixed with sequence alignments of the HA epitope-tagged human UT, C-terminus truncated UT^HA₃₇₀, UT^HA₃₅₁, UT^HA₃₃₂, UT^HA₃₁₉ mutants, and peptidomimetics corresponding to the entire C-terminus cytosolic fragment of UT (UT^c-myc_319–389). (B and C) Traces of Iso (10^–4 M, 2 s)-evoked current before (1), during (2) a 1-min <i>h</i>UII (10⁻⁸ M) application and after 22-min washout (3). (B) Currents recorded from CHO coexpressing GABA_AR and UT^HA (Control), UT^HA₃₇₀, UT^HA₃₅₁, UT^HA₃₃₂ or UT^HA₃₁₉. Corresponding average time course of the current, in the absence or presence of UT truncated mutants. (C) Current traces recorded from CHO-UT-GABA_AR, in the absence or presence of UT^c-myc_319–389. Corresponding average time course of the Iso-evoked current, in the absence or presence of UT^c-myc_319–389. In B, significance was only annotated above the time course graph during <i>h</i>UII perfusion and after 18-min washout, for clarity. Data are mean ± SEM from 3 to 13 cells. ns, non significant; *, <i>P</i><0.05; ** <i>P</i><0.01 compared with the corresponding control Iso-evoked current.</p

UII-induced GABA_AR loss from the plasma membrane through the C-terminus fragment of UT in CHO.

<p>The effect of <i>h</i>UII on the proportion of GABA_AR and UT at the cell surface of CHO was assessed by ELISA. (A) CHO transiently transfected with cDNA encoding UT^c-myc and α₂β₃, or α₂β₃γ₂^HA GABA_AR subunits (left), or UT^c-myc, and α₂β₃γ₂^HA GABA_AR subunits cotransfected with the cDNA encoding UT_319–389YFP (right). Background bioluminescence (left) and fluorescence (right) were measured after anti-HA antibody and colorimetric alkaline phophatase substrate incubation, in the absence or presence of 30 min of <i>h</i>UII (10⁻⁸ M, left), or directly on a fluorescent plate reader (right). (B) CHO transiently transfected with cDNA encoding UT^c-myc and α₂β₃γ₂^HA GABA_AR subunits (left), or cotransfected with the cDNA encoding UT_319–389YFP, and immunodetected with anti-HA (left) or anti-c-myc (right) antibodies. Percentage of cell surface γ₂^HA GABA_AR subunit (left) or UT^c-myc (right) are represented as the proportion of receptor at the plasma membrane (non permeabilized cells) to the total expressed receptor (permeabilized cells). One hundred percent correspond to values in the absence of 30 min treatment with <i>h</i>UII (10⁻⁸ M, 37°C). Each bar corresponds to mean ± SEM percent obtained from 5 to 7 independent experiments, in triplicates. ns, non significant; *, <i>P</i><0.05; ***, <i>P</i><0.001.</p

UII-induced depression of GABA_AR in UT-expressing cerebellar astrocytes.

<p>(Aa, Ab) Double immunofluorescence labeling of UT (green) and the specific astrocyte marker GFAP (red, Aa), or the mature neuron marker NeuN (red, Ab) in astrocyte-neuron co-culture from P7 rat cerebellum. Astrocytes, recognized by strong GFAP staining show UT immunoreactivity (arrows), whereas few weaker UT-stained cells express NeuN (arrowheads), and were likely attributed to mature granule cells (arrowheads, Ab). Nuclei (blue) were counterstained with DAPI. Scale bars, 50 µm. (B) Phase contrast photomicrograph of astrocytes in mono-culture, or astrocytes and neurons in co-culture at 3 days <i>in vitro</i>. (C) Membrane depolarizations and currents evoked by the GABA_AR agonist isoguvacine (Iso, 10⁻⁴ M, 2 s for membrane potential and 5 s for chloride current) in astrocytes and cerebellar granule neurons before, during <i>r</i>UII (10⁻⁷ M, 40 s) application and after 2-min washout. Right, normalized amplitudes deduced by the mean Iso-evoked depolarization or current obtained before <i>r</i>UII application. (D) Concentration-response relationship of Iso-evoked currents from astrocytes yielding an EC₅₀ value of 43.6±23.7 10⁻¹² M. Data are mean ± SEM of 4 to 6 cells. *, <i>P</i><0.05; ** <i>P</i><0.01 compared with the corresponding control Iso-evoked current.</p

UII-evoked GABA_AR internalization in native human astrocytes and glioma.

<p>(A, B) FIow cytometric analysis of the β₃ GABA_AR subunit and UT expression in native human astrocytes (A) and human U87 glioma cell line (B). Cells were stained with the anti-human β₃ subunit or anti-human UT in permeabilized or non permeabilized conditions (membrane receptor). The black lines depict results from control staining with only secondary antibodies. The β₃ GABA_AR subunit or UT cell surface expression was evaluated in the absence or presence of <i>h</i>UII (10⁻⁸ M, 30 min) by flow cytometry. Data obtained in A and B illustrate two representative experiments showing β₃ (magenta line) and UT (yellow line) mean fluorescence in the cytosol and at the plasma membrane of a minority of non permeabilized human astrocytes (A) or U87 (B) in culture. The exposure to <i>h</i>UII induced internalization of β₃ in both cell types and of UT only in U87 glioma. (C) U87 glioma cell line expressing UT and GABA_AR composed of β₃ subunit, and transfected with the cDNA encoding UT_319–389YFP, and immunodetected with anti-β₃ (left) or anti-UT (right) antibodies. Percentage of cell surface β₃ subunit (left) or UT (right) are represented as the proportion of receptor at the plasma membrane (non permeabilized cells) to the total expressed receptor (permeabilized cells). One hundred percent correspond to values in the absence of 30 min treatment with <i>h</i>UII (10⁻⁸ M, 37°C). Each bar corresponds to mean ± SEM percent obtained from at least 3 independent experiments, in triplicates. ns, non significant; *, <i>P</i><0.05; ***, <i>P</i><0.001.</p

Role of specific UT ligands on cytosolic calcium in CHO-UT.

<p>(A, B) <i>h</i>UII (A) or URP (B) (10⁻⁸ M, each) provoked a robust increase of [Ca²⁺]_c, which remained stable (A) or recovered to the basal line level (B) during washout. (C, D) Effect of the UT antagonists [Orn⁵]-URP (10⁻⁶ M, C) or palosuran (10⁻⁶ M, D), before and during <i>h</i>UII application. Right, bar graphs represent the percent increase of the [Ca²⁺]_c during drug perfusion or during the washout period. Percent values were obtained by normalizing signals evoked during and after treatments to the value measured before ligand application. Data are mean ± SEM from 9 to 25 cells. ns, non significant; ** <i>P</i><0.01; *** <i>P</i><0.001 compared with the corresponding control Iso-evoked current. In each type of experiment, three different cells have been selected as representative exemples.</p

Chemotactic G protein-coupled receptors control cell migration by repressing autophagosome biogenesis

<p>Chemotactic migration is a fundamental behavior of cells and its regulation is particularly relevant in physiological processes such as organogenesis and angiogenesis, as well as in pathological processes such as tumor metastasis. The majority of chemotactic stimuli activate cell surface receptors that belong to the G protein-coupled receptor (GPCR) superfamily. Although the autophagy machinery has been shown to play a role in cell migration, its mode of regulation by chemotactic GPCRs remains largely unexplored. We found that ligand-induced activation of 2 chemotactic GPCRs, the chemokine receptor CXCR4 and the urotensin 2 receptor UTS2R, triggers a marked reduction in the biogenesis of autophagosomes, in both HEK-293 and U87 glioblastoma cells. Chemotactic GPCRs exert their anti-autophagic effects through the activation of CAPNs, which prevent the formation of pre-autophagosomal vesicles from the plasma membrane. We further demonstrated that CXCR4- or UTS2R-induced inhibition of autophagy favors the formation of adhesion complexes to the extracellular matrix and is required for chemotactic migration. Altogether, our data reveal a new link between GPCR signaling and the autophagy machinery, and may help to envisage therapeutic strategies in pathological processes such as cancer cell invasion.</p