A Novel TIP30 Protein Complex Regulates EGF Receptor Signaling and Endocytic Degradation

Abstract

Activated epidermal growth factor receptor (EGFR) continues to signal in the early endosome, but how this signaling process is regulated is less well understood. Here we describe a protein complex consisting of TIP30, endophilin B1, and acyl-CoA synthetase long chain family member 4 (ACSL4) that interacts with Rab5a and regulates EGFR endocytosis and signaling. Receptor-mediated endocytosis is a mechanism utilized by eukaryotic cells to rapidly take up specific nutrients and reduce receptor signaling at the plasma membrane. Internalized ligand-receptor complexes are enclosed in early endosomes, also called sorting endosomes, where they are either recycled or delivered to lysosomes for destruction (1, 2). Signaling receptors continue to activate certain downstream pathways from early endosomes (3-6) until ligand-receptor complexes dissociate due to lower luminal pH created by vacuolar V-ATPases, 2 the major proton pump responsible for endosomal and lysosomal acidification (2, 7-9). Inactivation of V-ATPases blocks the transition from early to late endosomes (10). Therefore, the proper endosomal targeting and activity of V-ATPases contribute to the tight regulation of both endocytic trafficking and receptor endosomal signaling.Acidic luminal pH in early endosomes is the driving force for receptors to release their ligands, such as insulin and low density lipoprotein (LDL) (1, 2). On the basis of individually tracking EGF and EGFR, it has long been considered that EGFR travels together with EGF until they reach lysosomes (11, 12). However, it is unclear whether they remain bound to each other on the way to lysosomes. Recent studies have suggested that EGFR is inactivated before being degraded and that EGF dissociates from EGFR prior to lysosomal transfer (6, 13-15).Rab5a is a small GTPase that regulates early endosome fusion in vitro (16), motility of early endosomes on microtubules (17), and the traffic between endosomes and lysosomes (18). Deletion of Rab5a in cells inhibits the transport of EGFR from early endosomes to lysosomes and consequently causes sustained EGFR signaling and delayed EGFR degradation (19). Despite its importance to endocytic transport, how Rab5a mediates downregulation of receptor signaling remains unclear.TIP30, also known as HTATIP2 or CC3 (20, 21), is a tumor suppressor that has been demonstrated to act as a transcription cofactor to repress transcription in the nucleus (22,23) and to localize at the nuclear envelope to block nuclear importing (24). However, TIP30 also localizes in the cytoplasm, where its function is not known (25-28).Here we report that a newly identified protein complex containing TIP30, ACSL4, and Endo B1 drives EGF-EGFR complex endocytic trafficking by facilitating the localization of Rab5a and V-ATPases to early endosomes. Rab5a and V-ATPase reside in vesicles devoid of the early endosomal marker EEA1 and the recycling endosomal marker transferrin receptor (TfR), suggesting that these vesicles are post-trans-Golgi network vesicles responsible for the transport of i...

Role of autophagy, SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

“…28 It is noteworthy that the rate of colocalization between SH3GLB1 and CHRN as well as between MAP1LC3A and CHRN was significantly decreased in the trim63 −/ − under control conditions. This is in agreement with earlier data that connected SH3GLB1 to endocytic processing rather than autophagy assistance, [51][52][53] and also suggests that TRIM63 controls a pool of autophagic carriers under unchallenged conditions.…”

Bif-1 suppresses breast cancer cell migration by promoting EGFR endocytic degradation

“…Further, Bif-1 has been suggested to play an important role in controlling the size of early endosomes as suppression of Bif-1 promotes the formation of enlarged early endosomes following NGF or EGF treatment. 29,30 Consistently, our data reveal that suppression of Bif-1 promoted the accumulation of enlarged Rab5-positive endosomes (Fig. 3A).…”

“…2A and D). Similarly, loss of Bif-1 decreases EGFR degradation rates in HeLa, 28 PLC-PRF-5 29 and HCT116 (data not shown) cancer cells, confirming that these findings are not cell type specific. Further, while Bif-1 suppression did not alter EGFR internalization, co-localization of EGFR with the lysosomal membrane protein LAMP-1 was dramatically reduced in Bif-1 knockdown Rab5-GTP mediated recruitment of Rab7 onto early endosomes followed by Rab7 activation and association with effector proteins such as Rab7 interacting lysosomal protein (RILP).…”

“…29 V-ATPase trafficking to early endosomes is proposed to enhance early endosome acidification, EGF-EGFR dissociation and EGFR endocytosis. 29 While our findings in MDA-MB-231 cells suggest a dispensable role of Bif-1 in mediating Rab5 recruitment to EGF-positive vesicles, we did observe an increase in intracellular pH and altered acidic vesicle localization in Bif-1 knockdown cells. In contrast, Bif-1 negatively regulates endocytic trafficking of the NGF/TrkA receptor in neuronal PC12 cells through an interaction with EEA1, suggesting an alternate function for Bif-1 in neurons.…”

Association of Endophilin B1 with Cytoplasmic Vesicles

“…Endogenous EndoB1 has been localized by immunofluorescence staining to the Golgi complex (25), mitochondria (26)(27)(28), and Rab7-positive late endosomes (29,30). Functional studies have implicated EndoB1 in vesicle budding from the trans-Golgi network and degradative endocytosis of the epidermal growth factor (EGF) receptor (31).…”