Proteomics reveals novel protein associations with early endosomes in an epidermal growth factor–dependent manner

Abstract

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is an integral component of proliferative signaling. EGFRs on the cell surface become activated upon EGF binding and have an increased rate of endocytosis. Once in the cytoplasm, the EGF·EGFR complex is trafficked to the lysosome for degradation, and signaling is terminated. During trafficking, the EGFR kinase domain remains active, and the internalized EGFR can continue signaling to downstream effectors. Although effector activity varies based on the EGFR's endocytic location, it is not clear how this occurs. In an effort to identify proteins that uniquely associate with the internalized, liganded EGFR in the early endosome, we developed an early endosome isolation strategy to analyze their protein composition. Post-nuclear supernatant from HeLa cells stimulated with and without EGF were separated on an isotonic 17% Percoll gradient. The gradient was fractionated, and early endosomal fractions were pooled and immunoisolated with an EEA1 mAb. The isolated endosomes were validated by immunoblot using antibodies against organelle-specific marker proteins and transmission EM. These early endosomes were also subjected to LC-MS/MS for proteomic analysis. Five proteins were detected in endosomes in a ligand-dependent manner: EGFR, RUFY1, STOML2, PTPN23, and CCDC51. Knockdown of RUFY1 or PTPN23 by RNAi indicated that both proteins play a role in EGFR trafficking. These experiments indicate that endocytic trafficking of activated EGFR changes the protein composition, membrane trafficking, and signaling potential of the early endosome.

The metastasis suppressor NDRG1 down-regulates the epidermal growth factor receptor via a lysosomal mechanism by up-regulating mitogen-inducible gene 6

“…It has been established that a major process by which EGFR undergoes degradation is through trafficking to lysosomes (29,33,46,47). Considering this, it was important to investigate whether NDRG1 promoted EGFR processing through this mechanism.…”

Dissecting the role of His domain protein tyrosine phosphatase/PTPN23 and ESCRTs in sorting activated epidermal growth factor receptor to the multivesicular body

“…Furthermore, the C-terminal region of HD-PTP has an extended PRR containing multiple SH3-binding sites that bind Grb2 and potentially other SH3 domain signalling proteins, as well as an important PTPase domain [ 39 ]. It could thus engage an EGFR signalling core, consistent with the marked recruitment of HD-PTP to endosomes upon EGFR activation [ 78 ]. This may allow ESCRT assembly, and thus MVB sorting, to be coupled to the signalling status of EGFR, a prerequisite for carefully orchestrated receptor down-regulation.…”

Interactions at the cell membrane and pathways of internalization of nano-sized materials for nanomedicine

“…Other recently developed techniques that are available in cell biology to characterize pathways and that have not yet been used to study the uptake of nano-sized materials may provide novel insights into this difficult question. For instance, so-called OMICS approaches based on large-scale proteomics and full genome screenings could be of particular use [217][218][219]. While most of the "classical" methods mentioned so far require previous knowledge on the mechanisms of uptake by cells, a reverse approach could allow for discovering novel targets not yet associated with the endocytosis of nanomaterials.…”