Endocytic Trafficking of EGFR
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Endocytic Trafficking of EGFR
All cells need to interpret their environment. Efficient processing of signals from the extracellular milieu is achieved through dynamic signal-transduction systems. The components of this system that come into contact first with external signals are cell-surface receptors. RTKs (Receptor Tyrosine Kinases) comprise one large group of receptors that respond to polypeptide growth factors and have intrinsic tyrosine kinase activity. On ligand binding, receptor signaling is activated. The activation of RTKs initiates signal transduction and receptor endocytosis. Most activated RTKs are efficiently cleared from the cell surface by endocytosis and sorted to Lysosomes for degradation. In contrast, inactive RTKs are constitutively internalized and recycled back to the cell surface, an essential mechanism for the restoration of unused receptors. Simultaneous signal transduction and the endosomal transport of activated receptors represent a mechanism of bidirectional interplay that requires precise regulation. EGFRs (Epidermal Growth Factor Receptors) are important members of the RTK family of receptors, whose endocytosis forms an important part of their signaling outcomes (Ref.1 & 2).

The inactive EGFR localizes in steady-state equilibrium to the plasma membrane. It is internalized at low rates and efficiently recycled back via EEs (Early Endosomes) to the plasma membrane. In contrast, ligand (EGF (Epidermal Growth Factor))-induced activation of the EGFR results in its rapid and efficient internalization via CCPs (Clathrin-Coated Pits). Clathrins are the main structural coat proteins which play a key role in the intracellular transport between membranous organelles. Each molecule of Clathrin consists of three CLCs (Clathrin Light Chains) and three CHCs (Clathrin Heavy Chains) that form a structure called a triskelion. To bring EGFR from the outside of the cell inside, Clathrin molecules assemble into a dynamic two-dimensional lattice on the plasma membrane to form a CCP on the inside surface of the outer cell membrane. The pit then rounds and pinches off, trapping a section of membrane in a CCV (Clathrin-coated vesicle). The membrane section, and the associated EGFR, are then carried into the cell for processing, disassembly, or recycling (Ref.1 & 3).

Upon ligand binding and activation of EGFR, upto several 100-1000 CCVs or more per minute are generated for rapid internalization. AP2 (Adaptor Protein-2 Complex), a multi-subunit adaptor protein that binds efficiently to the activated EGFR is a central player in this regulatory step of Endocytosis. It recruits a network of essential endocytic proteins from the cytoplasm to the activated receptor. The Alpha subunit of AP2 recruits EPS15 (Epidermal Growth Factor Receptor Pathway Substrate-15) and Dynamin, whereas Alpha and Beta2 subunits, together, link the activated receptor to the nascent Clathrin coat. Activation of the EGFR also results in rapid tyrosine phosphorylation of the CHC and in recruitment of Clathrin to the plasma membrane. Receptor-induced activation of the tyrosine kinase Src (v-Src Avian Sacroma (Schmidt-Ruppin A-2) Viral Oncogene), along with the tyrosine kinase activity of the receptor is required to phosphorylate a critical tyrosine residue on the CHC. This CHC phosphorylation promotes rapid Clathrin recruitment and assembly at the plasma membrane. The nucleation of Clathrins causes self-assembly into a lattice-like network, thereby generating force on the plasma membrane to bud into a curvature, the CCP. The GTPase Dynamin is required to promote vesicle fission from the plasma membrane, and acts as a mechanoenzyme that tubulates the membrane. Phosphorylation of EPS15 and the CHC results in their translocation to the plasma membrane, causing nucleation of the CCP. By forming ringlike structures around the neck of CCPs, CCVs pinch off the plasma membrane. In addition, higher levels of PIP2 (Phosphatidylinositol 4,5-bisphosphate) and PIP3 (Phosphatidylinositol 3,4,5-trisphosphate) are generated at the site of EGFR activation. Dynamin, required for vesicle fission, binds to PIP3. Epsins (EPS-15 Interacting Protein), which tubulate the plasma membrane, bind to PIP2. Finally, activation of EGFR signaling results in efficient internalization by recruiting the endocytic machinery to the site of receptor activation (Ref.2, 4 & 5).

Once sorted to CCVs, the receptors enter the tubular vesicular early Endosomal compartment as the CCVs shed their Clathrin coat and progress into the EEs. Early Endosomal events comprise internalization of EGFRs, EE fusion and EE sorting. The EGFRs sorted into the EEs can either be quickly recycled to the plasma membrane from peripherally located EEs or be sorted in the Late Endosomal compartment, from where they are transported to Lysosomes for degradation. Most of the activated cell surface EGFRs are not recycled back to the plasma membrane but rather traffic to LEs (Late Endosomes), also referred to as MVBs (Multivesicular Bodies) composed of a limiting membrane with internal vesicles. The sorting of receptors from the limiting membrane into internal vesicles of MVBs shunts them to the Lysosomal degradation pathway. This step is controlled by interdependent mechanisms, including ubiquitination of the EGFR, recognition of the ubiquitinated receptor and invagination of the limiting membrane to form internal vesicles in the MVB. After being sorted to internal vesicles of the MVB, the receptors are finally transported to the Lysosomes for degradation (Ref.2, 5 & 6).

The endocytic machinery acts constitutively, where the activated cell surface receptors are routed to Lysosomes for degradation, and inactive receptors are constantly recycled back to the cell surface. Simultaneous to the endocytosis of the EGF-activated EGFRs, signal transduction pathways also operate inside the cell, through two effector cascades: the MAPK (Mitogen-Activated Protein Kinase) and the PI3K (Phosphoinositide-3-Kinase) cascades, which are involved in the regulation of cell survival, proliferation and differentiation, the outcomes of EGF signaling. The endocytosed EGF-EGFR complex continues to operate signal transduction even from the EEs and MVBs until its disassembly and degradation. Under physiological conditions, Clathrin-mediated processes are the main routes for internalization of EGFRs, but these receptors can also be internalized by Clathrin-independent processes that resemble Micropinocytosis, particularly in cells in which the receptors are overexpressed (Ref.2 & 6).