Outside-in signal transmission by conformational changes in integrin Mac-1 - PubMed (original) (raw)

Outside-in signal transmission by conformational changes in integrin Mac-1

Craig T Lefort et al. J Immunol. 2009.

Abstract

Intracellular signals associated with or triggered by integrin ligation can control cell survival, differentiation, proliferation, and migration. Despite accumulating evidence that conformational changes regulate integrin affinity to its ligands, how integrin structure regulates signal transmission from the outside to the inside of the cell remains elusive. Using fluorescence resonance energy transfer, we addressed whether conformational changes in integrin Mac-1 are sufficient to transmit outside-in signals in human neutrophils. Mac-1 conformational activation induced by ligand occupancy or activating Ab binding, but not integrin clustering, triggered similar patterns of intracellular protein tyrosine phosphorylation, including Akt phosphorylation, and inhibited spontaneous neutrophil apoptosis, indicating that global conformational changes are critical for Mac-1-dependent outside-in signal transduction. In neutrophils and myeloid K562 cells, ligand ICAM-1 or activating Ab binding promoted switchblade-like extension of the Mac-1 extracellular domain and separation of the alpha(M) and beta(2) subunit cytoplasmic tails, two structural hallmarks of integrin activation. These data suggest the primacy of global conformational changes in the generation of Mac-1 outside-in signals.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1

FIGURE 1

A and C, PMN were treated at 37°C for 15 min with 10 µg/ml CBR LFA-1/2 or ICAM-1, both in the presence of 1 mM MnCl2 or with anti-αM Fab (Fab44) and secondary F(ab′)2 to induce Mac-1 clustering. For cells treated with CBR LFA-1/2, anti-αM Fab fragments at a concentration of 50 µg/ml were used to block potential ligand binding. In A, immunoblots of whole-cell lysates were probed for the presence of tyrosine-phosphorylated proteins and for β-actin as a protein loading control. In C, immunoblots were probed for phosphorylated (phospho-p38; pThr180/pTyr182) and total p38, or for phosphorylated (phospho-Akt; pThr308) and total Akt. Western blot results from two experiments were quantified using ImageJ software and were normalized according to the control treatment. B, Unstimulated PMNs were incubated at 37°C for 15 min with FITC-conjugated anti-αM Fab44 (5 µg/ml) in the absence (left) or presence (right) of secondary F(ab′)2 (2.5 µg/ml) to induce Mac-1 clustering. D and E, PMN were treated with 1 mM MnCl2, 10 µg/ml CBR LFA-1/2 Fab and 10 µg/ml anti-αM Fab (Fab44), 1 mM MnCl2 and 200 µg/ml ICAM-1, or 5 µg/ml anti-αM Fab (Fab44) and 2.5 µg/ml secondary F(ab′)2 to induce Mac-1 clustering. Cells were pretreated for 15 min with or without 40 µM LY294002 or 2 µM SB239063. After incubation at 37°C for 16 h, cells were labeled with FITC-conjugated annexin V to detect apoptotic PMNs. Data are means ± SEM from three (D) or five (E) experiments performed in duplicate, and are expressed as percent of annexin V-positive cells. Significantly different from control (**, p < 0.01; or *, p < 0.05).

FIGURE 2

FIGURE 2

A, Schematic depicting the loss of FRET between FITC-conjugated mAbs and ORB membrane dye during extracellular domain extension. The Abs used were: 1, FITC-ICRF44; 2, FITC-CBRM1/5; and 3, CBR LFA-1/2. B, PMNs labeled with FITC-ICRF44 and ORB (blue trace) or IgG1 isotype control (red trace) were analyzed by flow cytometry. C, PMN labeled with FITC-ICRF44 and then incubated with or without 400 nM ORB were imaged by immunofluorescence microscopy. Far right, intensity of the FITC signal converted to a rainbow scale. Bar, 10 µm.

FIGURE 3

FIGURE 3

PMN were stimulated with or without 10 nM fMLP (A and B) or with 1 mM MnCl2 and 10 µg/ml CBR LFA-1/2 mAb (C–E) for 15 min. Unstimulated cells were labeled with FITC-ICRF44, and stimulated cells were labeled with FITC-ICRF44 (A and C), FITC-CBRM1/5 (B and D), or FITC-ICAM-1 (E). Cells were then incubated with 0, 75, 200, or 400 nM ORB and then analyzed by flow cytometry. Each plot consists of measurements from a single blood donor, with each condition being performed in duplicate (two curves per condition). Data are plotted as the fraction of the donor mean fluorescence intensity in the absence of acceptor fluorophores to that in the presence of the measured fluorescence acceptor (Equation 1).

FIGURE 4

FIGURE 4

Untreated PMN or PMN treated with 1 or 10 nM IL-8 or 100 ng/ml PMA were labeled with FITC-ICRF44 mAb. Cells were incubated with ORB as described in Fig. 3 and then analyzed by flow cytometry. Data are plotted as described in Fig. 3.

FIGURE 5

FIGURE 5

Untreated PMN or PMN treated with 10 nM fMLP or 1 mM MnCl2 and 10 µg/ml CBR LFA-1/2 mAb and then labeled with FITC-VIM12 mAb. Cells were incubated with ORB as described in the legend to Fig. 3 and then analyzed by flow cytometry. Data are plotted as described in Fig. 3.

FIGURE 6

FIGURE 6

A, K562 cells expressing αM-mCFP and β2-mYFP were labeled with anti-αM ICRF44 mAb, anti-β2 TS1/18 mAb, or IgG1 isotype mAb (red trace), followed by PE-conjugated secondary Abs. Cells were analyzed by flow cytometry. B, K562 cells expressing αM-mCFP and β2-mYFP were visualized under phase contrast and immunofluorescence microscopy. C, Whole-cell lysates from K562 cells expressing αM-mCFP and β2-mYFP were analyzed by immunoblotting using an anti-GFP polyclonal Ab. D, Mac-1 was immunoprecipitated from K562 cells, K562 cells expressing wild-type αM and β2, and K562 cells expressing αM-mCFP and β2-mYFP using the anti-β2 CBR LFA-1/2 mAb. Proteins were separated on a 7.5% SDS-PAGE gel and then analyzed by silver staining. E, K562 cells expressing αM-mCFP and β2-mYFP were allowed to adhere to tissue culture plastic coated with ICAM-1 or PVP, as a control, in the presence or absence of 1 mM MnCl2, 10 µg/ml CBR LFA-1/2 mAb, and anti-αM clone 44 mAb. After a 30-min incubation, nonadherent cells were washed away, and the number of adherent cells per field was counted. Data are presented as mean ± SEM from one of three independent experiments. *, Significantly different from control (p < 0.01).

FIGURE 7

FIGURE 7

A, Schematic depicting the loss of FRET between αM-mCFP and β2-mYFP during Mac-1 cytoplasmic tail separation. B, K562 cells expressing αM-mCFP and β2-mYFP were treated with 1 mM MnCl2 in the absence or presence of 10 µg/ml CBR LFA-1/2 Fab or 200 µg/ml ICAM-1, or with 100 nM PMA. CFP and YFP images were captured of individual cells before and after photobleaching of the YFP signal. FRET efficiency was calculated as described in Materials and Methods. *, Significantly different from control (p < 0.05).

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