MT1-MMP cleaves EphA2 at the Fibronectin type-III domain on the cell surface in cis. (A) Schematic representation of MT1-MMP and mutant proteins encoded by the used cDNA constructs. (B and C) MDA-MB-453 cells were cotransfected with EphA2 and MT1-MMP mutants or HA-tagged MT-MMPs followed by immunoprecipitation and immunoblotting of cell lysates and conditioned media as indicated (n = 3). Arrowheads indicate the truncated ∼60-kD C-terminal EphA2 fragments. The asterisk indicates IgG. (D) Determination of the MT1-MMP cleavage sites in EphA2. Lysates of COS-1 cells coexpressing EphA2 and MT1-MMP or MT1-E/A were subjected to immunoprecipitation, immunoblotting, and silver staining. Approximately 60-kD EphA2 fragments (1 and 2) were subjected to mass spectrometry (see Tables S1 and S2). (D, right) Schematic picture of EphA2. LBD, ligand-binding domain; CRD, cysteine-rich domain; FN, Fibronectin domain; TM, transmembrane region; JM, juxtamembrane region; KD, kinase domain; SAM, sterile α motif; PDZ, Psd-95, Dlg, and ZO1 domain. (E) Crystal structure of FN1 and amino acid sequence of the cleavage area. Red, cleavage area; blue, R³⁵⁷, R³⁸⁴, and R³⁹⁴; green, D³⁵⁹ and G³⁹¹ (green). (F) Lysates of MDA-MB-453 cells expressing EphA3 alone or in combination with the HA-tagged MT-MMPs were subjected to immunoblotting as indicated (n = 3). (G) Relative processing of EphA2 mutants was quantified and normalized with tubulin (error bars indicate mean ± SEM, n = 3). *, P < 0.05, Student’s t test. (H) Structural changes of EphA2-D/I and EphA2-G/R. Green, D³⁵⁹ or G³⁹¹; dark blue, newly generated side chains. Short green lines and disks indicate atoms in contact or slightly overlapping, and red disks indicate significant van der Waals overlap. All possible rotamers at the mutation points are shown Video 1. (I) Cell-surface EphA2, EphA2-D/I, and EphA2-G/R in control or MT1-MMP knockdown MDA-MB-231 cells were detected by cell surface biotinylation followed by immunoprecipitation. Cell lysates were subjected to immunoblotting as indicated (n = 2). Where indicated, EphA2-expressing cells were treated with 1 µM GM6001 for 16 h. The arrowhead indicates ∼60-kD EphA2 fragments. The asterisk indicates IgG. (J) Quantitative assessment of EphA2 and the mutant protein localization by immunofluorescence (error bars indicate mean ± SEM; n = 3). **, P < 0.01, Mann–Whitney U test. (K) EphA2 and MT1-MMP were expressed either by cotransfection or co-culture of single transfected MDA-MB-453 cells. Cell lysates were subjected to immunoblotting as indicated (n = 3). The arrowhead indicates ∼60-kD EphA2 fragments. The asterisk indicates IgG. Tubulin served as loading control.