Endocytosis of receptor tyrosine kinases

Abstract

Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work.

Figure 1.

Pathways of RTK endocytosis. RTKs are endocytosed by clathrin-mediated and clathrin-independent mechanisms. Typical rate constants (K_e) for RTK internalization through both pathways are shown. Clathrin-coated vesicles are uncoated shortly after fission from the plasma membrane and fuse with early endosomes (EEs). In the most well-studied EGFR system, EGFR-ligand complexes are detected in these highly dynamic, morphologically heterologous compartments within 2–5 min after EGF stimulation (Haigler et al. 1979; Beguinot et al. 1984; Miller et al. 1986; Hopkins et al. 1990). Ligand-RTK complexes remain intact but certain ligands dissociate from the receptor in the acidic environment of the endosomal lumen. Released ligands remain in the vesicular parts of endosomes (most of the endosomal volume), whereas unoccupied receptors are found mainly in tubular extensions (most of the membrane area). Ligand-occupied and unoccupied RTKs can rapidly recycle from EEs through the process of back fusion of peripheral EEs with the plasma membrane or via tubular carriers derived from these endosomes (retroendocytosis). EEs mature into sorting endosomes (SE) or multivesicular bodies (MVBs) in which RTKs are incorporated into intraluminal vesicles (ILVs) by inward membrane invagination. RTKs can also be delivered to the pericentriolar Rab11-containing recycling compartment. Recycling of unoccupied and ligand-occupied RTKs is slower from the SE/MVBs and recycling compartment. SE/MVBs gradually lose early endosome components, such as Rab5 and EEA.1, and recycling cargo (such as transferrin receptors) while become enriched in resident late endosomal proteins (such as Rab7), thus maturing into late endosomes. Fusion of late endosomes with primary lysosomes carrying proteolytic enzymes results in degradation of receptors and growth factors. A typical time scale of RTK endocytosis, their accumulation in EEs, and SE/MVBs is shown in minutes.

Figure 2.

Sequence motifs and posttranslational modifications of RTKs involved in endocytosis. Examples of RTKs with a single kinase domain (EGFR, Met, IR/IGF-1R, Trk subtypes) and an insert-divided kinase domain (PDGFR, FGFR, kit, CSFR, VEGFR) are shown. Sequence motifs containing phosphorylated tyrosine residues (P-Y and Y-P) serve as binding sites for SH2 domains of Grb2, Grb10, and the tyrosine kinase binding (TKB) domain of c-Cbl, Cbl-b, and Cbl-3. Proline-rich motifs in Cbl-b and c-Cbl bind to SH3 domains in Grb2. WW domains of NEDD family E3 ligases bind to PPxY motifs in RTKs, and in case of FGFR, to an unconventional motif in the juxtamembrane domain of this receptor. C2 domain of NEDD4-1 binds to the Grb10 SH2 domain that also binds to phosphotyrosine-containing motifs. Lysine residues are conjugated to ubiquitin in the kinase domains by Cbl, NEDD4, and other E3 ligases. Acetylation of distal lysines in the carboxyl terminus of EGFR is shown. Internalization motifs NPxY, YxxΘ (Θ, bulky hydrophobic residue), and LL are located in juxtamembrane domains, kinase insert, and carboxy-terminal tails of receptors.

Figure 3.

Hypothetic model of clathrin-mediated internalization of ligand-activated RTK. On ligand binding, RTKs are ubiquitinated by E3 ligases. Proteins, such as epsin, Eps15, and Eps15R, contain ubiquitin-interacting motifs (UIMs, a type of ubiquitin-binding domain [UBD]), and bind to ubiquitinated RTKs. UIM proteins bind to the appendage domains (mainly α) of AP-2, and epsin can also directly bind to the terminal domain of clathrin heavy chain (main components of clathrin triskelions). Interaction of RTKs with UIMs can occur before the recruitment of receptors into CCP and/or with UIMs present in assembled CCPs. The heterotetrameric AP-2 complex consists of four subunits: α, β2, μ2, and σ2. The trunk/core domain of AP-2 is formed by μ2, σ2, and the core domains of α and β2. YxxΘ and LL motifs directly interact with the μ2 and σ2/β2 subunits of AP-2, respectively. NPxY motifs interact with phosphotyrosine-binding domain (PTB) of adaptor proteins, such as Disabled-2, ARH, and NUMB, which themselves bind to the clathrin terminal domain and AP-2 appendage domains. The hinge region of β2 (between trunk and appendage) binds to clathrin heavy chain.

Figure 4.

Hypothetic mechanism of RTK sorting into intraluminal vesicles of MVBs. Ubiquitinated RTK is recognized by UIMs of Hrs and STAM1/2 (ESCRT-0). Hrs is anchored to endosomal membrane through the interaction of its FYVE domain with phosphatidylinositol 3-phosphate (PI3P). Hrs also contains a coiled-coil domain that interacts with a similar domain of STAM, VHS domain, and a clathrin-binding motif. Binding of clathrin triskelions to Hrs nucleates an assembly of a flat clathrin lattice that further recruits additional Hrs molecules, leading to trapping of ubiquitinated cargo in the “Hrs microdomain.” STAM contains the SH3 domain known to interact with AMSH. Accumulation of Hrs onto the endosomal membrane facilitates translocation of TSG101 and other components of ESCRT-I, and sequentially, components of ESCRT-II from cytosol to the MVB membrane. TSG101 and the ESCRT-II component EAP45/Vps36 have UBDs that may interact with ubiquitinated RTK. Ubiquitinated receptors appear to be transferred from ESCRT-0 to ESCRT-I and -II owing to increasing local concentrations of the latter complexes. ESCRT-III does not have ubiquitin-binding domains, and presumably traps receptors into forming ILVs by assembling into concentric hetero-oligomeric filaments and restricting diffusion of receptors. After formation of an ILV, ESCRT-III is disassembled by the Vps4 complex. The ESCRT model is reviewed by Teis et al. (2009). Before RTK entering the ILV, DUBs are proposed to remove ubiquitin from the receptor. Furthermore, receptor-associated proteins, such as SH2 adaptors and ubiquitin ligases, must also be removed before sequestration of receptors into ILV, possibly by means of receptor dephosphorylation by phosphotyrosine phosphatases like PTP1B (Eden et al. 2010). In addition, RTKs may facilitate ILV formation by phosphorylating annexin 1 (not shown) (White et al. 2006).