Fig. 4: ENG enhances complex formation between NRP1 and VEGFR2.
From: NRP1 interacts with endoglin and VEGFR2 to modulate VEGF signaling and endothelial cell sprouting
To study whether ENG can participate in complexes with both NRP1 and VEGFR2, we conducted patch/FRAP studies on COS7 cells expressing myc-NRP1 with HA-VEGFR2 (or empty vector) as described in Fig. 3. Where indicated, HA-VEGFR2 was immobilized by IgG as in Fig. 2. The lateral mobility of Fab’-labeled myc-NRP1 was measured by FRAP. FRAP studies on NRP1/VEGFR2 complex formation, depicting the average Rf (a) and D values (b) in the absence or presence of VEGF-A. c, d Studies on the modulation of NRP1/VEGFR2 interactions by ENG. The average Rf (c) and D values (d) of myc-NRP1 were measured. Where indicated, untagged ENG was coexpressed along with the tagged receptors (myc-NRP1 and HA-VEGFR2), followed by IgG crosslinking (CL) of HA-VEGFR2. Bars are mean ± SEM; the number of measurements (on different cells) appears under each bar. The reduction in Rf of myc-NRP1 was significantly reduced upon IgG CL of HA-VEGFR2, an effect enhanced by VEGF-A, either in the absence (a) or presence (c) of untagged ENG. Coexpression with ENG by itself (without IgG CL of HA-VEGFR2) already induced a mild reduction in Rf of myc-NRP1, which was significantly more pronounced upon immobilization of HA-VEGFR2 (compare a, c). These results demonstrate that the presence of ENG enhances NRP1/VEGFR2 interactions, and may serve as a bridge to form a ternary complex. Asterisks indicate significant differences between the Rf values of the pairs indicated by brackets (*p < 0.05; ***p < 10−3; ****p < 10−4; one-way ANOVA and Bonferroni post-hoc test. ns = not significant). A similar analysis of the D values showed no significant differences in all cases.
