IgFLNc21-migfilin-N interaction. A, 600-MHz two-dimensional ¹H-¹⁵N HSQC of 0.2 mm ¹⁵N-labeled IgFLNc21 in the absence (black) and presence (red) of 0.4 mm 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 mm ¹⁵N-labeled migfilin-(1-85) in the absence (black) and presence (red) of 0.4 mm IgFLNc21 at identical experimental conditions, showing that IgFLNc21 induced substantial chemical shift changes of a dozen residues as consistent with the result in A.

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.

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 μm. 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.

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.

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.

Migfilin-N dissociates IgFLN21 from integrin β CTs and promotes talin-integrin interaction. A, two-dimensional HSQC of 0.1 mm β3 CT (black) and β3 CT in the presence of 0.2 mm 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 μm), but such blocking effect was significantly reduced (blue) upon addition of migfilin-N (talin-PTB:β7-CT:IgFLNa21:migfilin, n = 50:125:250:500 μm).

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.