Clathrin-dependent endocytosis

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Almost forty years ago, Roth and Porter identified clathrin-coated pits as specialized plasma membrane domains responsible for the selective recruitment of cargo molecules [29]. Extensive work then provided critical insight into clathrin-dependent endocytosis, a major pathway responsible for the efficient uptake of various proteins from the cell surface [30]. Clathrin coats at the plasma membrane give rise to endosome-targeted vesicles, by means of a two-step process: membrane budding, and subsequent fission of the resulting vesicle. The major structural components of clathrin coats are two protein complexes, clathrin, and clathrin adaptor proteins. Clathrin consists of three 192-KDa heavy chains, each bound to one light chain of approximately 30 KDa [31]. This complex is called a triskelion and may polymerize in vitro to form a polygonal lattice [32]. In vivo, triskelia polymerize to form rounded baskets that coat invaginated pits and vesicles. The heterotetrameric adaptors (APs) are responsible both for recognizing signals in the cargoes, and for bridging the clathrin lattices to the membrane [33]. AP2 is involved in internalization at the plasma membrane. Clathrin is also involved in other trafficking steps, together with other APs, such as AP1, in particular, which is involved in Golgi-to-endosome trafficking. Like the other APs, AP2 consists of two large subunits (a2 and b2), one medium (m2), and one small subunit (s2). The m2 and b2 subunits interact with sorting signals in the cytoplasmic domains of transmembrane proteins, notably Tyr-based and di-Leu-based motifs, respectively. The b2 subunit also interacts with clathrin heavy chains via a specific motif, the clathrin box, also found in other clathrin partners (e.g. epsins, amphiphysin). The a2 and b2 subunits both contain a 30 kDa "appendage" domain, which is joined to the rest of the protein via a flexible linker. Resolution of the crystal structure of the flexible domain of a2 subunit revealed a single binding site for its ligands, which include amphiphysin, Eps15, epsin, and possibly dynamin [34]. A single site for the binding of multiple ligands would facilitate temporal and spatial regulation in the recruitment of components of the endocytic machinery. Eps15 was identified as a partner of AP2, capable of binding the a2 subunit via the DPF motifs in its C-terminal domain [35]. The N-terminal region of this protein contains three repeated sequences, conserved throughout evolution, which define a family of proteins [36]. These EH (Eps15 homology) domains of Eps15 have been demonstrated to bind several proteins, including epsin [37]. The C-terminal domain of Eps15 also contains also two UIMs (ubiquitin interacting motif). Eps15 has been demonstrated to be crucial for clathrin-dependent endocytosis, probably in the recruitment of AP2 to clathrin-coated pits. It was recently suggested that epsin, a protein that interacts with Eps15 via an NPF motif and may also bind AP2, plays an important role in internalization. Epsin contains a highly conserved amino-terminal region [38], the ENTH domain (epsin N-terminal homology). This domain binds phosphoinositol-(4, 5)-bisphosphate and it was recently shown that such binding modifies membrane curvature in conjunction with clathrin polymerization [39]. Epsin also contains UIM domains. Following initial invagination of the membrane, the clathrin-pit recruits dynamin, a GTPase which plays a key role in constriction, and endophilin, which may play a direct role in the fission reaction by virtue of its lipid modifying activity [40] before the pinching off of vesicles from the plasma membrane [41]. Another AP2 partner, that also binds clathrin and dynamin, amphiphysin, was demonstrated to induce curvature of lipid bilayers in vitro, a feature also displayed by epsin, endophilin and dynamin, indicating that roles in invagination or fission may not be mutually exclusive [42]. AP2 is not the only adaptor molecule involved in clathrin-dependent endocytosis. For some receptors, such as the b-adrenergic receptor, the role of adaptor in clathrin-mediated endocytosis is played by b-arrestins, which link activated receptors to both AP2 and clathrin [43].

Models ordering the successive steps and actors involved in membrane recruitment, invagination, constriction and fission have been raised, in which invaginating clathrin-coated pits were traditionally thought to be covered by AP2. Recent investigations performed in living cells expressing fluorescent-tagged proteins have challenged old models. The use of total internal reflection fluorescence microscopy (TIR-FM) has enabled the tracking of individual clathrin coated pits and vesicles and the direct observation of events occuring within a restricted area adjacent to plasma membrane. Experiments revealing that AP2 is absent from disappearing clathrin spots have suggested that contrary to predictions, AP2 complexes may form stable plateforms from which clathrin coated pits lacking AP2 would laterally emerge [42].

New insights on clathrin-mediated endocytosis were also obtained with the use of small interfering RNA to knock down AP-2 subunits and clathrin heavy chain to undetectable levels. Receptor-mediated endocytosis of several receptors including transferrin receptor (TfR), or Epidermal Growth Factor receptor (EGFR) was severely inhibited in clathrin depleted cells. Strikingly, however, if internalization of TfR was inhibited in AP2-depleted cells, internalization of EGFR was as efficient in these cells as in control cells. AP-2 is thus only one of several endocytic adaptors required for clathrin-dependent the uptake of certain cargo proteins [44].