Endocytic downregulation of ErbB receptors: mechanisms and relevance in cancer

Abstract

ErbB receptors (EGFR (ErbB1), ErbB2, ErbB3, and ErbB4) are important regulators of normal growth and differentiation, and they are involved in the pathogenesis of cancer. Following ligand binding and receptor activation, EGFR is endocytosed and transported to lysosomes where the receptor is degraded. This downregulation of EGFR is a complex and tightly regulated process. The functions of ErbB2, ErbB3, and ErbB4 are also regulated by endocytosis to some extent, although the current knowledge of these processes is sparse. Impaired endocytic downregulation of signaling receptors is frequently associated with cancer, since it can lead to increased and uncontrolled receptor signaling. In this review we describe the current knowledge of ErbB receptor endocytic downregulation. In addition, we outline how ErbB receptors can escape endocytic downregulation in cancer, and we discuss how targeted anti-cancer therapy may induce endocytic downregulation of ErbB receptors.

Fig. 1

Structure of EGFR. In the extracellular part of the receptor, EGFR harbours two domains (L1 and L2) that upon folding form the ligand-binding pocket. Between L1 and L2 is another domain (S1) that includes the dimerization arm. Intracellularly, EGFR has a kinase domain and a C-terminal tail with several amino acid residues that can be phosphorylated. The tyrosine residues (Y) that are involved in Cbl binding are shown. The parts of wild-type EGFR that are deleted in EGFRvIII, EGFRvIV, and EGFRvV are indicated

Fig. 2

Internalization and endosomal sorting of EGFR. Main figure: upon activation, EGFR (green) is translocated to clathrin coated pits (CCP) on the plasma membrane and internalized (grey arrows). After transport to early endosomes (EE), EGFR is either recycled back to the plasma membrane (white arrows) or taken up into intraluminal vesicles (ILVs). EE will mature to late endosomes (LE), and EGFR in ILVs will eventually be degraded in lysosomes (Lys) (black arrows). Upper insert at the plasma membrane, EGF-activated EGFR dimerize and the kinase activity of the receptors phosphorylates tyrosine residues (P) in EGFR. This creates docking sites for intracellular proteins such as Grb2. Grb2 mediates binding of the ubiquitin ligase Cbl that adds mono- or polyubiquitins (Ub) to EGFR. Activated EGFR is transported to clathrin coated pits that in addition to clathrin also consists of Eps15 and other proteins. Lower insert At the EE vacuolar membrane, EGFR destined for degradation still binds Cbl and is continuously phosphorylated and ubiquitinated. The EGFR ubiquitins are bound by Hrs that resides in at Hrs/STAM/clathrin coat, and this is followed by binding of ESCRT complexes to the ubiquitinated EGFR leading to uptake into ILVs

Text box 1

Ubiquitination

Fig. 3

EGF induces membrane ruffling and macropinocytosis. HEp2 cells expressing GPI-GFP as a marker of the plasma membrane were followed by 3D confocal microscopy over time. The upper panel shows two cells that were imaged before and 5 min after stimulation with 100 ng/ml EGF. Arrows indicate membrane ruffles formed after EGF stimulation. The lower panel shows that ruffling leads to formation of macropinosomes (arrows). The large image to the left shows a cell 7 min after stimulation with 100 ng/ml EGF, and the small images show a time sequence during which macropinosomes are formed. Bars, 20 μm

Fig. 4

Crosslinking of ErbB2 induces receptor internalization. a Fixed SK-BR-3 breast cancer cells have been incubated with a mouse monoclonal antibody against the extracellular N-terminal part of ErbB2, and subsequently with a gold-conjugated anti-mouse antibody. Thereafter the cells were embedded in Epon, sectioned and examined in the electron microscope. It is seen that ErbB2 is mainly associated with membrane protrusions. An empty clathrin-coated pit (CCP) is also seen. b, c SK-BR-3 cells have been incubated for 1 h at 37°C with the anti-ErbB2 antibody followed by the gold-conjugated antibody before fixation and embedding. Note how the antibody crosslinking drives ErbB2 down from the protrusions to the bulk membrane, where it is seen in clathrin-coated pits (CCP). Labeling of a multivesicular body (MVB) is also seen in c, showing that crosslinked ErbB2 is internalized. For details see (Hommelgaard et al. 2004). Bar 0.5 μm

Fig. 5

HSP90 inhibition stimulates ErbB2 cleavage and internalization. a ErbB2 is internalized after 2 h of Geldanamycin-stimulation (3 μM) of SK-BR-3 breast cancer cells. Sequential immunocytochemistry was used to distinguish between internalized and surface associated ErbB2. Fixed cells were stained with antibodies against ErbB2 before permeabilization (green). After permeabilization, cells were once again stained with antibodies against ErbB2 (red). Only the latter antibody staining (red) gain access to intracellular ErbB2, which therefore appears red whereas surface ErbB2 is stained both red and green and therefore appears yellow. For details see (Lerdrup et al. 2006). b YFP-ErbB2-CFP is cleaved after 2 h of Geldanamycin-stimulation of SK-BR-3 cells. A construct with YFP fused to the extracellular N-terminus and CFP fused to the intracellular C-terminus of ErbB2 was expressed in SK-BR-3 cells for 48 h and followed by incubation with 0.3 μM Geldanamycin for 2 h as indicated. YFP-ErbB2-CFP where the C-terminal tail has been cleaved off appears green. Note the increased amount of such cleavage in vesicles compared to the plasma membrane after Geldanamycin stimulation. For details see (Lerdrup et al. 2007). Bars 20 μm