Fig. 3
BMP type I receptors compete with ALK4 for binding to ACVR2A. Patch/FRAP studies were carried out on COS7 cells expressing myc-ACVR2A with HA-ALK4 (or empty vector). Where indicated, untagged ALK2, ALK3, or ALK6 was coexpressed with myc-ACVR2A and HA-ALK4. Where shown, HA-ALK4 was immobilized by IgG crosslinking as in Fig. 2. The lateral mobility of Fab’-labeled myc-ACVR2A was measured by FRAP. a Average Rf values; b Average D values. Bars are mean ± SEM; the number of measurements (on different cells) appears on each bar. The full and dashed lines depict the Rf values of myc-ACVR2A coexpressed with HA-ALK4 without (full line; taken from Fig. 2 d, second bar from the left, as indicated to the right of the panel) or with (dashed line; Fig. 2 d, third bar) IgG αHA crosslinking. No significant differences were found between D values of myc-ACVR2A upon coexpression with HA-ALK4 ±IgG crosslinking, or with or without coexpression with additional type I receptors (b) (one-way ANOVA with Bonferroni post hoc test; P > 0.7). The reduction in Rf of myc-ACVR2A upon immobilization of HA-ALK4 (Fig. 2 d) disappeared when untagged ALK2, ALK3, or ALK6 were coexpressed with myc-ACVR2A and HA-ALK4. Thus, the Rf values of myc-ACVR2A became similar to that of singly expressed myc-ACVR2A (a, leftmost bar; P > 0.05, one-way ANOVA and Bonferroni post hoc test), indicating that they compete with ALK4 for binding ACVR2A. c, d Point-confocal measurements of the expression levels of coexpressed myc-ACVR2A (c) and HA-ALK4 (d) with or without untagged ALK2, ALK3, or ALK6. The measurements were as described under “Methods.” Each bar represents mean ± SEM of 30 independent measurements. No significant differences were observed between the levels of either myc-ACVR2A or HA-ALK4 upon coexpression with one of the untagged type I receptors (one-way ANOVA and Bonferroni post hoc test; P > 0.99)