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. 2007 Dec 21;364(3):540-8.
doi: 10.1016/j.bbrc.2007.10.049. Epub 2007 Oct 17.

A NPxY-independent beta5 Integrin Activation Signal Regulates Phagocytosis of Apoptotic Cells

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Free PMC article

A NPxY-independent beta5 Integrin Activation Signal Regulates Phagocytosis of Apoptotic Cells

Sukhwinder Singh et al. Biochem Biophys Res Commun. .
Free PMC article

Abstract

Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the beta chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 (beta5) integrin cDNA was expressed in alphav positive, beta5 and beta3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, beta5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing beta5 mutant (Y750A) abrogated adhesion and beta5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of beta5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a beta5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10 microm diameter microspheres developed as apoptotic cell mimetics. The critical sequences in beta5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of beta5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the beta5 cytoplasmic tail for adhesion and phagocytosis.

Figures

Figure 1
Figure 1. The proximal β5 NPxY motif regulates β5 mediated adhesion to VTN
(A). Alignment of NPxY and NxxY motifs in beta integrin cytoplasmic tails. The localization of the conserved motif is underlined. (A) Schematic of β5 intracellular domain mutants utilized in this study. Integrin surface expression was analyzed to determine if mutations alter surface expression or heterodimer formation. The β5 integrin (β5) was detected with anti-β5 clone IVF2 and the active configuration (αvβ5) was detected with anti-PIF6 mAb which detects a shared epitope. Dashed line indicates separate FACS data sets. In C, transient transfected CS-1 cells were plated on VTN (20 μg/μL) coated 96-well dishes for 24 hrs. In lanes 2-4, FAK DNA was transfected, whereas lane 1 is a control. Adhesion was determined by crystal violet staining. Adhesion was further verified by Western blotting for FAKpY397 using β5 integrin point (C) mutations (lower panel). For the pFAK/FAK ratio's, the blots were scanned by laser densitometry, and after normalization of the β5 signal to 1.0, the mutants are expressed as pFAK/FAK to yield relative specific activity.
Figure 2
Figure 2. Co-localization of β5-EGFP fusion proteins with components of the focal adhesion machinery
pCDNA-β5-EGFP or pCDNA-β5 Y750A/Y770A double tyrosine mutant (β5(AA)-EGFP) were transiently transfected into HeLa cells. After plating on VTN (20 μg/mL) coated coverslips for 4 h, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were then co-stained with Rhodamine-Phalloidin (panel A), or with a monoclonal antibody to paxillin (panel B), or with a monoclonal antibody to talin (panel C), followed by anti-Cy3 secondary antibody (see methods). In each figure, the area enclosed in the box is shown under 100X magnification, to indicate the localization of the β5 integrin with components of the focal adhesion machinery. Immunofluorescence was collected with SPOT RT Color CCD digital camera via an inverted Nikon TE300 fluorescence microscope.
Figure 3
Figure 3. Effects of NPLY mutation on β5-mediated phagocytosis of apoptotic cells
(A,B). CS-1 cells were transfected with wildtype β5 or β5 mutants. 48 h post-transfection, β5-expressing CS-1 cells were co-cultured with apoptotic CEM cells for 2 h. EGFP expressing (integrin expressing) CS-1 cells were analyzed for apoptotic cell uptake, red dye. Graphs indicate fold differences of the geometric means of each sample. Panels are represented as follows (A) Deletion mutants. (B) Point mutants. (C). Surface expression of β5 Δ741 and β5 Y742A mutants was analyzed with anti-β5 mAb and and anti-αvβ5 mAb as described in Fig. 1. (D). Mapping of the YEMAS region to determine the minimal functional unit that regulates phagocytosis.
Figure 4
Figure 4. Molecular modeling of MFG-E8 EGF-like domain I, and generation of 10 μm MFG-E8-coated microspheres as apoptotic mimetics
(A) Homology modeling was performed to predict the minimal peptide length required to produce a functional EGF-like domain I. The integrin binding motif, RGD, is highlighted in black and the side chains are shown. The interrupted region in Activated Protein C is aa 71, which was disordered. Recombinant MFG-E8 domain I, but not the RGE mutant, transmits signals through αvβ5 integrin. (B). Functionality of the recombinant MFG-E8 proteins were assessed by plating HeLa cells on recombinant protein glass cover slips coated with recombinant protein (50μg/μl) for 60 min, followed by fixation and staining for actin and nucleus (DAPI). (C) β5-mediated adhesion to recombinant MFG-E8 was blocked with an anti-αvβ5 (PIF6) function-blocking antibody. (D) β5-EGFP or β5 Y750A/Y770A (AA) EGFP double tyrosine mutant were transiently transfected into HeLa cells. Transfected cells were then co-cultured with 10 μm polystyrene beads coated with GST-fusion of recombinant MFG-E8 domain I for 60 min. Arrows indicate integrin clustering around the MFG-E8 beads. Inserts represent 100X magnifications of the same images shown in the left panels. Asterisks indicate corresponding arrows and dashed circles represent beads.

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