Fig. 2
ACVR2A forms mutual heteromeric complexes with multiple type I receptors. COS7 cells were cotransfected with pairs of expression vectors encoding myc-ACVR2A along with an HA-tagged type I receptor (ALK4, ALK2, ALK3, or ALK6). After 24 h, live cells were subjected to the IgG-mediated patching/crosslinking (CL) protocol (“Methods”), resulting in the HA-type I receptor patched and labeled by Alexa 488-GαR IgG (designated “IgG αHA”), whereas myc-ACVR2A is labeled exclusively by monovalent Fab’ (with Alexa 546-GαM Fab’ as a secondary antibody). In control experiments without HA-type I receptor crosslinking, the IgG labeling of the HA tag was replaced by exclusive Fab’ labeling. Where indicated, ligand (4 nM ActA or BMP9, or 10 nM BMP2) was added during the last fluorescent labeling step for the FRAP experiment, and maintained throughout the measurement. FRAP studies were conducted as in Fig. 1. a–c Representative FRAP curves of myc-ACVR2A coexpressed with uncrosslinked (Fab’-labeled) HA-ALK4 (a), of HA-ALK4 immobilized by IgG crosslinking (b), and of myc-ACVR2A coexpressed with IgG-crosslinked HA-ALK4 (c). d–k Average Rf (d, f, h, j) and D values (e, g, i, k) depicting the effects of coexpression with various type I receptors and their crosslinking on the lateral diffusion of myc-ACVR2A. The bars depict the average values (mean ± SEM); the number of measurements (each conducted on a different cell) is shown on each bar. Some of these numbers are lower in the D value panels, since only Rf can be extracted from FRAP curves yielding less than 20% recovery. Asterisks indicate significant differences between the Rf values of the pairs indicated by brackets (*, P < 0.05; **, P < 5 × 10−4; ***, P < 10−4; one-way ANOVA and Bonferroni post hoc test. n.s. = non-significant). A similar analysis of the D values showed no significant differences (P > 0.2)