The ErbB family of tyrosine kinase receptors mediates the activation of a wide network of signaling pathways, which are important for cell growth, differentiation and survival. The four different ErbB proteins, EGFR, ErbB2, ErbB3 and ErbB4, are dependent on dimerization for transmitting signal into the cell. The ErbB mediated signaling is negatively regulated by endocytosis of the receptors from the plasma membrane, followed by degradation. Many types of cancers are associated with deregulation of ErbBs. ErbB3 has a weak kinase activity, however, the receptor has six docking sites for the p85 subunit of PI3K, making ErbB3 an important contributor in proliferative signaling. Recent studies suggests that ErbB3 contributes to tumor progression and drug resistance in cancers driven by EGFR and ErbB2. The degradation of ErbB3 is so far poorly understood.
In the present study, two variants of fluorescently tagged ErbB3 were generated: one with the EGFP-tag on the C-terminal (intracellular) end and one with the EGFP-tag on the N-terminal (extracellular) end of the receptor. The constructs were characterized by confocal microscopy analysis and biochemical analysis upon transient transfection of PAE cells. ErbB3 with the N-terminal (extracellular) tag was not activated upon Hrg stimulation, indicating that the tag inhibited ligand binding or sterically prevented dimerization. However, the ErbB3 with the EGFP-tag on the C-terminal (intracellular) end behaves similarly to the untagged wild-type ErbB3 with respect to ligand-induced phosphorylation, and internalization. In addition, ErbB3 with the C-terminal EGFP-tag was found to be ubiquitinated both in the presence and absence of added ligand.