Migfilin, a molecular switch in regulation of integrin activation - PubMed (original) (raw)
Migfilin, a molecular switch in regulation of integrin activation
Sujay Subbayya Ithychanda et al. J Biol Chem. 2009.
Abstract
The linkage of heterodimeric (alpha/beta) integrin receptors with their extracellular matrix ligands and intracellular actin cytoskeleton is a fundamental step for controlling cell adhesion and migration. Binding of the actin-linking protein, talin, to integrin beta cytoplasmic tails (CTs) induces high affinity ligand binding (integrin activation), whereas binding of another actin-linking protein, filamin, to the integrin beta CTs negatively regulates this process by blocking the talin-integrin interaction. Here we show structurally that migfilin, a novel cytoskeletal adaptor highly enriched in the integrin adhesion sites, strongly interacts with the same region in filamin where integrin beta CTs bind. We further demonstrate that the migfilin interaction dissociates filamin from integrin and promotes the talin/integrin binding and integrin activation. Migfilin thus acts as a molecular switch to disconnect filamin from integrin for regulating integrin activation and dynamics of extracellular matrix-actin linkage.
Figures
FIGURE 1.
IgFLNc21-migfilin-N interaction. A, 600-MHz two-dimensional1H-15N HSQC of 0.2 m
m
15N-labeled IgFLNc21 in the absence (black) and presence (red) of 0.4 m
m
unlabeled migfilin-N at pH 6.5, 29 °C. Insets I-III are the zoomed region of IgFLNc21 in the presence of the different migfilin fragments. Inset I, 1-85; II, 1-36; and III, 1-24. The spectral patterns in the insets are identical indicating that migfilin-(1-24) fragment (migfilin-N) is sufficient to bind to IgFLN21.B, HSQC of 0.2 m
m
15N-labeled migfilin-(1-85) in the absence (black) and presence (red) of 0.4 m
m
IgFLNc21 at identical experimental conditions, showing that IgFLNc21 induced substantial chemical shift changes of a dozen residues as consistent with the result in A.
FIGURE 2.
A, isothermal calorimetry data demonstrating similar migfilin binding of IgFLNa21; B, IgFLNc21. C, β7 binds IgFLNa21 at an order of magnitude lower affinity than migfilin.
FIGURE 3.
Features of migfilin-N structure and its binding to IgFLN21. A, sequence comparison of integrin β CTs and migfilin-N.B, backbone superposition of the 20 lowest energy NMR structures; and_C,_ their corresponding ribbon representation (right). The bound migfilin-N is colored in pink, forming β-sheet with IgFLN21 strand C. D, surface depictions of IgFLNa21 bound to integrin β7 CT (PDB code 2brq). E, IgFLNc21 bound to migfilin-N. Comparison of D and E shows that β7 CT and migfilin-N bind to IgFLN21 in the same manner. F, detailed interface between IgFLNa21 and β7 CT (PDB code 2brq). G, detailed interface between IgFLNc21 and migfilin-N.
FIGURE 4.
Migfilin-N dissociates IgFLN21 from integrin β CTs and promotes talin-integrin interaction. A, two-dimensional HSQC of 0.1 m
m
β3 CT (black) and β3 CT in the presence of 0.2 m
m
IgFLNc21 (red) and IgFLNc21/migfilin-N (1:2) (green), pH 5.6, 29 °C. The spectra in red and_green_ are identical showing that migfilin-N-IgFLNc21 interaction prevented the IgFLN21 binding to β3 CT. B, HSQC spectra of a selected region of 15N-labeled talin-PTB in the absence (black) and presence of unlabeled β7 CT (red) showing that talin-PTB interacts with β7 CT. The representative Lys357, which is shifted upon addition of β7 CT, is known to be involved in interacting with integrin β CTs (14). Lys357 shifts back to the position of the free form talin-PTB (green) upon addition of IgFLNa21 demonstrating that the IgFLNa21 blocks the β7 CT binding to talin-PTB (talin-PTB:β7-CT:IgFLNa21 = 50:125:250 μ
m
), but such blocking effect was significantly reduced (blue) upon addition of migfilin-N (talin-PTB:β7-CT:IgFLNa21:migfilin, n = 50:125:250:500 μ
m
).
FIGURE 5.
Migfilin promotes integrin activation. A, overexpression of migfilin promotes fibronectin and HUTS-4 (a mAb specific for the active conformation of the β1 integrin) binding of A7 melanoma cells. The cells were transfected with a vector encoding for GFP and full-length migfilin, and FACS were performed as described under “Experimental Procedures.” The data shown are means ± S.E. for duplicate determinations, and the results are representative of 3-4 similar experiments. B, cell-deliverable migfilin-N (WT-Migfilin-CCR7) enhances αIIbβ3 activation, as monitored with PAC-1, a mAb specific for the active conformation of this integrin. The cells used were CHO cells stably expressing αIIbβ3 (A5 cells). The mutant (MT-MigfilinCCR7) is significantly (p value <0.05) less effective in activating the integrin. The cell-permeable peptides, as well as the tag (CR7) and the untagged migfilin peptide, were added to the cells at 10 μ
m
. After 5 min, the A5 cells were washed, and PAC-1 and an Alexa 633-conjugated secondary antibody were added. The cells were analyzed by FACS after an additional 30 min.C, cell-deliverable migfilin-N (WT-MigfilinCCR7) induces significant PAC1 binding to αIIbβ3 in platelets. In contrast, the filamin-binding defective migfilin-N mutant (MT-MigfilinCCR7) peptide had a significantly (p < 0.05) lower effect.
FIGURE 6.
Migfilin-N promotes platelet aggregation. A, addition of WT-migfilin-CCR7 to a washed platelet suspension induced significant aggregation of the cells. This effect was minimal with the MT-migfilin-CCR7 at the same concentration and was not observed at all with the tag alone or the untagged WT-migfilin peptide. Data are representative of more than 10 experiments from at least five different donors. B, co-precipitation experiment showing that migfilin-N leads to dissociation of the filamin-integrin complex in platelet lysates. Data are representative of four independent experiments. IP, immunoprecipitation.
FIGURE 7.
A model for the role of the migfilin-filamin interaction in regulating integrin activation and cytoskeleton reassembly. Migfilin is recruited by kindlin-2 (Mig-2) to integrin adhesion sites (25). The recruited migfilin promotes dissociation of filamin from integrins, which is bound to the resting integrin and inhibits the talin-integrin interaction (16) as well as the anchoring of kindlins to integrin β CTs. When filamin is displaced, talin and kindlins become more effective in mediating integrin activation. Because filamin is a major actin cross-linking protein critical for maintaining the actin network, the migfilin-induced disconnection between integrin and filamin may also cause temporal rearrangement of the integrin-actin linkage to facilitate the cytoskeleton remodeling.
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