Fig. 3: Receptor switching due to HER2 dimerization domain mutations (DDMs) in breast cancer cells changes downstream signaling.

A Western blot showing phospho- and panAKT, phospho- and panERK level in ZR-75-1 cells with overexpression of H2-WT and the DDMs as marked. HER2 and α-tubulin control blots were shown as well. Heatmap showing ERK activation measured using p-ERK bioluminescence resonance energy transfer (BRET) sensor (B) and AKT activation measured using p-AKT BRET sensor (C) after heregulin-1β stimulation in ZR-75-1 cells overexpressing wild-type HER2 (H2-WT) and DDMs. Each cell represents the mean of 3 replicates. D, E Representative array and heatmap showing the fold-change in phosphorylated proteins in H2-WT and its DDMs expressing cells. Respective arrays involved candidate proteins of MAPK and AKT signaling cascade. F Heatmap showing the corresponding transcript level measured for MAPK and AKT signaling molecules in H2-WT and its DDMs harboring patients with breast cancer; each column represents a patient from METABRIC dataset. G Co-immunoprecipitation result using HER2 mAb showing physical interaction specificity of HER2 DDMs with HER3 after HER3-TRIM-ing in ZR-75-1 clonal cells as marked. Loading amount was one-fifth of the amount used for immunoprecipitation; BO: bead only. Bottom immunoblot panels showing levels of phospho- and total AKT and ERK in H2-WT and its DDMs expressing cells after ligand stimulation and HER3-TRIM-ing; Control-SS: serum starved ZR-75-1 cells, EV: pcDNA-3.1 empty vector.