Figure 5.
ErbB3 scaffolds assembly of the Arf6–GGA3–Rabaptin5 endosomal sorting complex. (A) Immunoblotting of ErbB3 immunoprecipitates or input cell lysates, after 30-min treatment with PQ, showing endogenous binding of ErbB3 with GGA3 and Rabaptin5 that increases upon PQ treatment, and the presumed accumulation of recycling endosomes (representative of three independent experiments). IgG was used as a negative control for immunoprecipitations. (B) Immunoblotting of Arf6 immunoprecipitates or input cell lysates, transfected with the indicated siRNAs, following PQ or vehicle treatment for 10 min. (C and D) Quantification of GGA3 and Rabaptin5 protein levels (C: immunoblotting) and mRNA levels (D: RT-qPCR) in ErbB3 siRNA-transfected MCF10A cells relative to control cells (n = 4 experiments for protein and n = 3 for mRNA). Data are presented as mean values ± SEM. P values were determined by unpaired Student’s t test. (E) Structural model highlighting the putative GGA3-binding motif 864-DxxLL-867 in the ErbB3 kinase domain. (F) Immunoblotting of ErbB3 immunoprecipitates or input cell lysates, after ectopic expression of ErbB3 or the ErbB3 LL866/867AA mutant with GGA3 in HEK293T cells. (G) LL866/867AA mutation compromises the ability of ErbB3 to promote assembly of the Arf6–GGA3–Rabaptin5 sorting complex: immunoblotting of Arf6 immunoprecipitates or input cell lysates, following ectopic expression of Arf6, GGA3, and Rabaptin5, with or without ErbB3 or ErbB3-LL866/867AA. Average densitometric values of at least two independent biological replicates were normalized to the respective controls and to the loading control protein when appropriate. (H) Confocal imaging of WT or LL866/7AA mutant ErbB3-mCherry and Alexa 594–conjugated Integrin β1 in MCF10A cells. The analysis of ErbB3-mCherry colocalization with Integrin β1 shows the relative ErbB3 enrichment at the Integrin β1–positive structures, as determined by the formula (a–b)/b, where a is the ErbB3-mCherry intensity of the center in Integrin β1 structures, and b is the adjacent volume (background) for each structure. Each data point represents the minimum average of 20 structures in one cell. P values were determined using unpaired two-tailed Student’s t test. Scale bars, 15 µm (H).

ErbB3 scaffolds assembly of the Arf6–GGA3–Rabaptin5 endosomal sorting complex. (A) Immunoblotting of ErbB3 immunoprecipitates or input cell lysates, after 30-min treatment with PQ, showing endogenous binding of ErbB3 with GGA3 and Rabaptin5 that increases upon PQ treatment, and the presumed accumulation of recycling endosomes (representative of three independent experiments). IgG was used as a negative control for immunoprecipitations. (B) Immunoblotting of Arf6 immunoprecipitates or input cell lysates, transfected with the indicated siRNAs, following PQ or vehicle treatment for 10 min. (C and D) Quantification of GGA3 and Rabaptin5 protein levels (C: immunoblotting) and mRNA levels (D: RT-qPCR) in ErbB3 siRNA-transfected MCF10A cells relative to control cells (n = 4 experiments for protein and n = 3 for mRNA). Data are presented as mean values ± SEM. P values were determined by unpaired Student’s t test. (E) Structural model highlighting the putative GGA3-binding motif 864-DxxLL-867 in the ErbB3 kinase domain. (F) Immunoblotting of ErbB3 immunoprecipitates or input cell lysates, after ectopic expression of ErbB3 or the ErbB3 LL866/867AA mutant with GGA3 in HEK293T cells. (G) LL866/867AA mutation compromises the ability of ErbB3 to promote assembly of the Arf6–GGA3–Rabaptin5 sorting complex: immunoblotting of Arf6 immunoprecipitates or input cell lysates, following ectopic expression of Arf6, GGA3, and Rabaptin5, with or without ErbB3 or ErbB3-LL866/867AA. Average densitometric values of at least two independent biological replicates were normalized to the respective controls and to the loading control protein when appropriate. (H) Confocal imaging of WT or LL866/7AA mutant ErbB3-mCherry and Alexa 594–conjugated Integrin β1 in MCF10A cells. The analysis of ErbB3-mCherry colocalization with Integrin β1 shows the relative ErbB3 enrichment at the Integrin β1–positive structures, as determined by the formula (a–b)/b, where a is the ErbB3-mCherry intensity of the center in Integrin β1 structures, and b is the adjacent volume (background) for each structure. Each data point represents the minimum average of 20 structures in one cell. P values were determined using unpaired two-tailed Student’s t test. Scale bars, 15 µm (H).

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