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. 2012 Nov 9;287(46):39041-9.
doi: 10.1074/jbc.M112.383851. Epub 2012 Sep 27.

Vitronectin induces phosphorylation of ezrin/radixin/moesin actin-binding proteins through binding to its novel neuronal receptor telencephalin

Yutaka Furutani  1 , Miwa Kawasaki, Hitomi Matsuno, Sachiko Mitsui, Kensaku Mori, Yoshihiro Yoshihara
Affiliations

Vitronectin induces phosphorylation of ezrin/radixin/moesin actin-binding proteins through binding to its novel neuronal receptor telencephalin

Yutaka Furutani et al. J Biol Chem. .

Abstract

Vitronectin (VN) is an extracellular matrix protein abundantly present in blood and a wide variety of tissues and plays important roles in a number of biological phenomena mainly through its binding to αV integrins. However, its definite function in the brain remains largely unknown. Here we report the identification of telencephalin (TLCN/ICAM-5) as a novel VN receptor on neuronal dendrites. VN strongly binds to TLCN, a unique neuronal member of the ICAM family, which is specifically expressed on dendrites of spiny neurons in the mammalian telencephalon. VN-coated microbeads induce the formation of phagocytic cup-like plasma membrane protrusions on dendrites of cultured hippocampal neurons and trigger the activation of TLCN-dependent intracellular signaling cascade including the phosphorylation of ezrin/radixin/moesin actin-binding proteins and recruitment of F-actin and phosphatidylinositol 4,5-bisphosphate for morphological transformation of the dendritic protrusions. These results suggest that the extracellular matrix molecule VN and its neuronal receptor TLCN play a pivotal role in the phosphorylation of ezrin/radixin/moesin proteins and the formation of phagocytic cup-like structures on neuronal dendrites.

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Figures

FIGURE 1.
FIGURE 1.
Firm adhesion between VN and TLCN. A, Western blot analysis of TLCN expression induced by IPTG in TLCN-N2a cells. The cell lysates were blotted with anti-TLCN and anti-actin antibodies. B, the band intensities of TLCN and actin were quantified. Error bars indicate S.E. (n = 3). C and D, triple immunofluorescence labeling of gapVenus-expressing N2a cells in the absence or presence of IPTG with anti-GFP (C1 and D1; blue in C4 and D4), anti-TLCN (C2 and D2; green in C4 and D4), and anti-phospho-ERM (C3 and D3; red in C4 and D4) antibodies. Scale bar, 20 μm. E, trypsin/EDTA-resistant adhesion of TLCN-N2a cells on culture substrate. Control N2a (black circles) and TLCN-N2a (green) cells were treated with trypsin/EDTA for 0, 1, 5, and 15 min. The cells remaining attached were quantified by measuring activity of mitochondrial hydrogenases. Error bars indicate S.E. (n = 3). F, TLCN-dependent adhesion of N2a cells onto VN. Control N2a (gray bars) and TLCN-N2a (green bars) cells were plated onto ECM-coated wells. Adherent cells were stained with crystal violet and quantified. Error bars indicate S.E. (n = 3).
FIGURE 2.
FIGURE 2.
Direct and specific binding between VN hemopexin domains and TLCN. A–D, interaction between VN and TCLN was assessed by surface plasmon resonance analysis. Vitronectin (A), fibronectin (B), fibrinogen (C), and laminin (D) were immobilized on sensor chips and assayed for binding to recombinant TLCN(1–9)/Fc protein at different concentrations (16.9, 33.8, 67.5, 135, and 270 nm). RU, response units. E–I, specific binding between TLCN and VN hemopexin domains. A schematic diagram of VN structure and its recombinant protein fragments (E) is shown. VN consists of the somatomedin B domain (SMB; open box), connecting region (CR; gray box) with integrin binding RGD motif, hemopexin domains (Hx1–4; green boxes), and the Lys/Arg-rich region (Lys/Arg-rich; orange box). GST-fused VN fragments (F, Fr-1; G, Fr-2; H, Fr-3; I, Fr-4) were immobilized on sensor chips and assayed for binding to TLCN/Fc protein at different concentrations (12.5, 25, 50, 100, and 200 nm).
FIGURE 3.
FIGURE 3.
Requirement of the second Ig-like domain of TLCN for VN binding. Surface plasmon resonance analysis of protein-protein interaction between recombinant ICAM family molecules and VN. A, a schematic diagram shows TLCN, TLCN(1–9)/Fc, TLCN-truncated mutants TLCN(1–3)/Fc, TLCN(1–2)/Fc, and TLCN(1)/Fc, TLCN 2nd Ig-like domain deletion and substitution mutants TLCN(1,3–9)/Fc and TLCN(1,3–9)ICAM-1(2)/Fc, and two other ICAMs, ICAM-1/Fc and ICAM-2/Fc proteins. Green, red, and yellow ovals show TLCN, ICAM-1, and ICAM-2 Ig-like domains, respectively. B–I, VN was immobilized on sensor chip and assayed for binding to TLCN(1–9)/Fc (B), TLCN(1–3)/Fc (C), TLCN(1–2)/Fc (D), TLCN(1)/Fc (E), TLCN(1,3–9)/Fc (F), TLCN(1,3–9)ICAM-1(2)/Fc (G), ICAM-1/Fc (H), and ICAM-2/Fc (I) proteins at different concentrations (16.9, 33.8, 67.5, 135, and 270 nm). RU, response units.
FIGURE 4.
FIGURE 4.
Formation of phagocytic cup-like membrane protrusions on dendrites through VN-TLCN interaction. A, accumulation of TLCN, PI(4,5)P2, and phospho-ERM to phagocytic cup-like structure was induced by VN-coated microbeads. Hippocampal neurons (15 days in vitro) expressing PI(4,5)P2-specific fluorescent probe PLCδ1-PH-GFP (A1; red in A5) were incubated with VN-coated non-fluorescent microbeads and immunostained with anti-TLCN (A2; green in A5) and anti-phospho-ERM (A3; blue in A5) antibodies. VN-coated beads were visualized by phase contrast imaging (A4). Magnified views of a boxed area in A5 in individual channels are shown (A6). Scale bars, 20 μm in A5 and 5 μm in A6. B–E, VN (B1)-, collagen I (COLI; C1)-, fibronectin (FN; D1)-, and laminin (LN; E1)-coated fluorescent microbeads were added to cultured hippocampal neurons. TLCN (B2–E2; green in B4-E4) accumulated to VN-coated beads (B) but not to collagen I-, fibronectin-, and laminin-coated beads (C, D, and E). Scale bar, 5 μm. F, numbers of VN (light green)-, collagen I (purple)-, fibronectin (brown)-, and laminin (gray)-coated beads attached onto hippocampal neurons. Error bars indicate S.E. (n = 10). G, percentages of ECM-coated beads on neurons with TLCN accumulation. Error bars indicate S.E. (n = 10). H–K, TLCN-dependent formation of phagocytic cup-like structure. Triple fluorescence images of wild-type (WT; H and J) and TLCN-deficient (KO; I and K) neurons added with VN-coated fluorescent beads (H1-K1; red in H4-K4) and labeled with anti-TLCN antibody (H2–K2; green in H4–K4), and anti-phospho-ERM antibody (H3 and I3; blue in H4 and I4) or Alexa488-phalloidin (J3 and K3; blue in J4 and K4). Scale bars, 5 μm. L, time-lapse imaging of phagocytic cup-like structure formation. A filopodia-bearing dendritic segment of TLCN/YFP-expressing hippocampal neurons was imaged every 20 min. Upper panels (L1) are merged images of a VN-coated fluorescent bead (red) and TLCN/YFP (green) showing the attachment of the bead on a filopodia and its subsequent movement toward a dendritic shaft. Lower panels (L2) show the rapid accumulation of TLCN/YFP to the bead attachment site. Scale bar, 1 μm. M, a vertical section of phagocytic cup-like structure reconstructed from confocal laser scanning microscopic data. TLCN (M1; green in M3) is enriched in the dendritic membrane protrusion that surrounds the VN-coated fluorescent bead (M2; red in M3). Scale bar, 1 μm.
FIGURE 5.
FIGURE 5.
Enlargement of spine heads in VN-deficient mice. A and B, representative images of apical dendrites of DiI-labeled CA1 pyramidal neurons in adult wild-type (A) and VN-deficient (B) mice. Scale bar, 4 μm. C, a cumulative frequency plot for spine width of CA1 pyramidal neurons. The spine width in VN-deficient mice (red; n = 556 spines) was significantly larger than that in wild-type mice (black; n = 723 spines) (analysis of variance; p < 0.01). D, box plots for spine width. Means, blue circles; medians, middle lines; 75th and 25th quartiles, top and bottom lines, respectively; whiskers show range. **, p < 0.01 (two-tailed t test). E, box plots for spine density on apical dendrites of CA1 pyramidal neurons. Spine density in VN-deficient mice (red; n = 15 neurons) was significantly lower than that in wild-type mice (black; n = 15 neurons). **, p < 0.01 (two-tailed t test).
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