Cargo recognition during clathrin-mediated endocytosis: a team effort

doi:10.1016/j.ceb.2004.06.001

Current Opinion in Cell Biology

Volume 16, Issue 4, August 2004, Pages 392-399

https://doi.org/10.1016/j.ceb.2004.06.001 Get rights and content

Abstract

Transmembrane proteins destined to endosomes are selectively accumulated in clathrin-coated pits at the plasma membrane and rapidly internalized in clathrin-coated vesicles. The recognition of specific sequence motifs in transmembrane cargo by coated-pit proteins confers specificity on the endocytic process. Interaction of membrane cargo with the clathrin adaptor protein complex AP-2 is the major mechanism of cargo sorting into coated pits in mammalian cells. Recent studies have revealed a variety of alternative mechanisms of cargo recruitment involving additional adaptor proteins. These alternative mechanisms appear to be particularly important during clathrin-mediated endocytosis of signaling receptors.

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:

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of special interest
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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.

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Cargo recognition during clathrin-mediated endocytosis: a team effort

Abstract

Introduction

Section snippets

Recruitment of cargo containing tyrosine-based and di-leucine motifs

Recruitment of signaling receptors into coated pits during ligand-induced endocytosis

RNAi attack

Conclusions and future directions

References and recommended reading

Acknowledgements

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