Cargo recognition during clathrin-mediated endocytosis: a team effort
Introduction
By facilitating fast and selective internalization of receptors, channels, transporters and other integral membrane proteins, clathrin-mediated endocytosis (CME) regulates the cell-surface expression and function of these proteins. The cycle of coated vesicle formation consists of several steps 1., 2.. First, clathrin triskelia assemble into polyhedral lattices on the cytoplasmic surface of the plasma membrane. Binding of clathrin to the membrane is mediated by ‘adaptor’ proteins that possess phospholipid-interacting motifs and may also bind transmembrane proteins. The major clathrin adaptor protein functioning at the plasma membrane is the AP-2 complex, although several other proteins are capable of linking clathrin to the membrane bilayer. The clathrin lattice serves as a binding scaffold for several stoichiometrically minor components that assist or regulate coated vesicle budding. For example, at the early stages, generation of membrane curvature and formation of a coated pit requires proteins such as epsin, while later events are thought to be monitored by endophilin and amphiphysin, which associate with the clathrin lattice and can produce and/or sense deformation of the lipid bilayer.
The selective recruitment of transmembrane proteins (cargo) into coated pits occurs by binding of specific sequences in the cytoplasmic domains of cargo proteins to AP-2 and possibly-other coat-associated proteins. Several lines of evidence suggest that coated pits can form independently of AP-2 interaction with cargo 3., 4. and that cargo is recruited into pre-existing coated pits 5., 6.. However, it is also possible that recruitment of some cargo proteins is coordinated with and necessary for the efficient coat assembly.
The process of coat invagination continues during the third stage, leading to the formation of deeply invaginated pits and the scission of a nascent endocytic vesicle. The latter step requires the cytoplasmic GTPase dynamin, which is proposed to function either directly as a GTP-dependent ‘pinchase’ or as a regulator of a distinct mechanochemical enzyme. Finally, uncoating of the vesicle is accomplished with the participation of the coat-associated 5-phosphoinositide phosphatase synaptojanin 1 and a chaperone complex consisting of auxilin and Hsc70.
It is generally thought that the late stages of endocytosis — formation of curved coats and vesicle budding — can occur independently of what type of cargo is present in the coated pit. Thus, cargo recruitment to the clathrin-containing lattice structure is the key sorting step defining the specificity of the internalization process. Hence, this review will focus on progress in understanding the cargo recognition phase of CME in mammalian cells.
Section snippets
Recruitment of cargo containing tyrosine-based and di-leucine motifs
The best-understood mechanism of cargo recruitment is the direct interaction of AP-2 with a YxxΘ motif (where Θ is a bulky hydrophobic residue and x is a variable residue) present in the cytoplasmic tail of the cargo protein (Figure 1). AP-2 is a stable heterotetramer consisting of four subunits: α and β2 adaptins, μ2 and σ2 [7]. High-resolution atomic structures of several parts of AP-2 and, more recently, of an entire ‘core’ domain of AP-2 have been solved 7., 8.••. The structure of the
Recruitment of signaling receptors into coated pits during ligand-induced endocytosis
CME is a principal pathway for endocytosis of many receptors that trigger signal transduction events, for example receptors for growth factors and G-protein-coupled receptors (GPCR). Tyrosine-based and di-leucine motifs have been identified in several signaling receptors, and in a limited number of studies the interaction of signaling receptors with AP-2 was reported 30., 31., 32., 33.. However, mutational analysis revealed that neither these motifs nor AP interactions played an important role
RNAi attack
The development of cell-biologist-friendly methodologies allowing efficient depletion of proteins in cultured mammalian cells by siRNA-mediated RNA interference (RNAi) led to a plethora of studies utilizing this technique to test the function of coated-pit proteins including AP-2 and cargo-specific adaptors (see Table 1). These studies confirmed an essential role for AP-2 in the internalization of YxxΘ-containing cargo such as transferrin receptor 19.•, 61., 62. and for ‘intermediate’ adaptor
Conclusions and future directions
During past two to three years many mechanistic aspects of AP-2 function in coat assembly, recruitment of accessory proteins and cargo recognition have been elucidated. The current views of AP-2 function are, however, rather static. Much needs to be done to understand how the many functions of AP-2 are regulated and coordinated. It also has become clear that cargo sorting to coated pits is a collective effort of AP-2 and other adaptor proteins, which are linked to AP-2 or can even function in
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
- •
of special interest
- ••
of outstanding interest
Acknowledgements
I am indebted to B Wendland, M Marsh and M von Zastrow for critical reading of the manuscript, and to P McPherson for communicating unpublished data. This work is supported by grants from NCI, NIDA, ACS and NSF.
References (69)
- et al.
Phosphorylation of threonine 156 of the mu2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo
Curr Biol
(2001) - et al.
Recruitment of activated G-protein-coupled receptors to pre-existing clathrin-coated pits in living cells
J Biol Chem
(2002) - et al.
Molecular architecture and functional model of the endocytic AP2 complex
Cell
(2002) - et al.
AAK1-mediated μ2 phosphorylation is stimulated by assembled clathrin
Traffic
(2003) - et al.
The μ2 subunit of the clathrin adaptor AP-2 binds to FDNPVY and YppO sorting signals at distinct sites
Traffic
(2002) Sorting it out: AP-2 and alternate clathrin adaptors in endocytic cargo selection
J Cell Biol
(2003)- et al.
Distinct saturable pathways for the endocytosis of different tyrosine motifs
J Biol Chem
(1998) - et al.
YXXM motifs in the PDGF-β receptor serve dual roles as phosphoinositide 3-kinase binding motifs and tyrosine-based endocytic sorting signals
J Biol Chem
(2003) - et al.
β-arrestin acts as a clathrin adaptor in endocytosis of the β2- adrenergic receptor
Nature
(1996) - et al.
Role of β-arrestin in mediating agonist-promoted G protein-coupled receptor internalization
Science
(1996)
Endocytosis of G-protein-coupled receptors: roles of G-protein-coupled receptor kinases and β-arrestin proteins
Prog Neurobiol
β-arrestin 2 mediates endocytosis of type III TGF-β receptor and down-regulation of its signaling
Science
Association of epidermal growth factor receptors with coated pit adaptins via a tyrosine-phosphorylation-regulated mechanism
J Biol Chem
Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis
J Biol Chem
Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation
Cell
Cbl–CIN85–endophilin complex mediates ligand-induced downregulation of EGF receptors
Nature
The endophilin–CIN85–Cbl complex mediates ligand-dependent downregulation of c-Met
Nature
Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1
Mol Cell
RING finger mutations that abolish c-Cbl-directed polyubiquitination and downregulation of the EGF receptor are insufficient for cell transformation
Mol Cell
Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins
Annu Rev Cell Dev Biol
The yeast Epsin Ent1 is recruited to membranes through multiple independent interactions
J Biol Chem
Trafficking patterns of β-arrestin and G-protein-coupled receptors determined by the kinetics of β-arrestin deubiquitination
J Biol Chem
Effect of clathrin heavy chain- and α-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells
J Biol Chem
Desensitization, internalization, and signaling functions of β-arrestins demonstrated by RNA interference
Proc Natl Acad Sci USA
Tandem mass spectrometry analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling
Proc Natl Acad Sci USA
Regulated endocytosis of G-protein-coupled receptors by a biochemically and functionally distinct subpopulation of clathrin-coated pits
J Biol Chem
The adaptor protein β-arrestin2 enhances endocytosis of the low density lipoprotein receptor
J Biol Chem
Biological basket weaving: formation and function of clathrin-coated vesicles
Annu Rev Cell Dev Biol
Clathrin-dependent endocytosis
Biochem J
Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis
EMBO J
G protein-coupled receptor/arrestin3 modulation of the endocytic machinery
J Cell Biol
Linking endocytic cargo to clathrin: structural and functional insights into coated vesicle formation
Biochem Soc Trans
A structural explanation for the recognition of tyrosine-based endocytic signals
Science
Phosphorylation of the AP2 μ subunit by AAK1 mediates high affinity binding to membrane protein sorting signals
J Cell Biol
Cited by (174)
Numerical simulation of the viral entry into a cell driven by receptor diffusion
2021, Computers and Mathematics with ApplicationsLive-cell imaging of early coat protein dynamics during clathrin-mediated endocytosis
2018, Biochimica et Biophysica Acta - Molecular Cell ResearchMechanisms of canalicular transporter endocytosis in the cholestatic rat liver
2018, Biochimica et Biophysica Acta - Molecular Basis of DiseaseGanglioside GM2 catabolism is inhibited by storage compounds of mucopolysaccharidoses and by cationic amphiphilic drugs
2019, Molecular Genetics and Metabolism