RAB7 and TSG101 are required for the constitutive recycling of unliganded EGFRs via distinct mechanisms

Abstract

Both constitutive and ligand-mediated membrane trafficking regulate Epidermal Growth Factor Receptor (EGFR) signaling. The constitutive endocytosis and recycling of the unliganded EGFR is a critical determinant of cell surface EGFR expression and the cell’s sensitivity to ligands. We report that two proteins with established roles in trafficking the EGF:EGFR complex to the lysosome also regulate the recycling of the unliganded EGFR. Knock down of either Tumor suppressor gene 101 (TSG101) or RAB7 causes the endosomal accumulation of the inactive, unliganded receptor in morphologically and biochemically distinct organelles. Knock down of TSG101 causes the EGFR to accumulate in low density endosomes whereas RAB7 knock down results in EGFR accumulation in high density endosomes. Knock down of either protein caused the receptor to co-localize primarily with LAMP-1, but not EEA1. These two proteins regulate EGFR slow, perinuclear recycling, via distinct mechanism and are new molecular targets that regulate cell surface EGFR expression.

Rab Proteins and the Compartmentalization of the Endosomal System

“…Rab4 activity like that of Rab5 appears subject to growth factor signaling through the activation of regulatory proteins (Hellberg et al 2009;Goueli et al 2012). Rab7 is activated by virus infection and modulated by EGFR signalingalthough the mechanisms are incompletely clarified (Buranda et al 2013;Rush and Ceresa 2013). Analyses of four single-point mutants of Rab7, which are associated with a peripheral neuropathy CMT2B, revealed profound deficits in receptor trafficking in conjunction with defects in cytosolic and nuclear signaling (BasuRay et al 2010McCray et al 2010).…”

Isolation of EGFR-containing early endosomes.

“…As such, the use of a sucrose step gradient prior to Percoll gradient fractionation yields reduced plasma membrane contamination and a distribution of early endosome proteins that corresponds with proper early endosome density. These results also correlate with the distribution of EGFR in Percoll gradients within the literature [60,76].…”

“…In fact, depending on the scientific question being asked, this step is frequently omitted altogether. In many cases, the separation and enrichment of target organelles with density gradients is sufficient for further study with biochemical techniques [60,61]. However, obtaining a pure organelle sample is essential for analyzing the proteome of a compartment.…”

Targeted Noninvasive Imaging of EGFR-Expressing Orthotopic Pancreatic Cancer Using Multispectral Optoacoustic Tomography

“…Western blot was performed as previously described (24). Proteins were resuspended in Lamelli sample buffer and resolved by 7.5% SDS-Page gel prior to transfer onto nitrocellulose membrane.…”