A YPX3L motif of PAR1 mediates binding to ALIX. (A) Alignment of PAR1 and PAR2 ICL2 sequences from various species. The conserved residues of the YPX3L motif are shaded in gray. (B) HeLa cells coexpressing HA-ALIX WT and either FLAG-PAR1 WT or Y206A mutant were stimulated with 100 µM SFLLRN at 37°C, as indicated. Cell lysates were immunoprecipitated and examined as described in Fig. 4 B. IB, immunoblotting. The data (mean ± SD) represent the amount of immunoprecipitated ALIX normalized to the amount of immunoprecipitated PAR1 and were significant, as determined by Student’s t test (**, P < 0.01; n = 3). A.U., arbitrary unit. (C) HeLa cells transfected with FLAG-PAR1 and either HA-ALIX WT or F676D mutant were stimulated with agonist and processed as previously described. Cell lysates were immunoblotted with anti-HA and -actin antibodies. The data (mean ± SD) were calculated, as previously described, and significant, as determined by Student’s t test (**, P < 0.01; n = 3). (D) Biotinylated PAR1 ICL2 peptide containing the YPX3L motif was immobilized on streptavidin beads and incubated with GST, GST–ALIX V domain, GST–ALIX V F676D, or the GST–ALIX Bro1 domain. Pulldowns (PD) were analyzed for the presence of bound protein by immunoblotting. Input was analyzed by Coomassie staining.
