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Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling

Nature Reviews Molecular Cell Biology volume 6, pages 112–126 (2005)Cite this article

Key Points

  • Cell membranes are arranged into various lipid assemblies, such as regions that are rich in a particular phosphoinositide and lipid-raft microdomains. The latter are assemblies of cholesterol and glycosphingolipids, and they can function as signalling platforms for a host of different membrane receptors and their downstream effectors.

  • From the cell surface, receptors can be internalized through clathrin-mediated endocytosis. In this pathway, the receptors move to different compartments depending on various sorting signals, such as peptide motifs or post-translational modifications, which are recognized by specific trafficking proteins. Alternatively, cargo molecules can be partitioned and internalized through lipid-raft-dependent pathways, which are clathrin-independent, cholesterol-sensitive and either dynamin-dependent or -independent.

  • The tyrosine-kinase epidermal-growth-factor receptor (EGFR) is the canonical example of a cell-membrane receptor that is internalized through clathrin-mediated endocytosis. However, it has also been detected in lipid-raft microdomains. From the cell surface, EGFR is directed towards early endosomes, where it can be sorted back to the cell surface or to the degradative pathway. The itinerary of EGFR is controlled by protein complexes and lipids, which include the E3 ubiquitin ligase Cbl, adaptor protein-2 (AP2), EGFR-pathway substrate-15 (EPS15), epsin, HRS (hepatocyte-growth-factor-regulated tyrosine-kinase substrate), TSG101 (tumour susceptibility gene-101), and phosphoinositides.

  • Members of the transmembrane serine/threonine kinase transforming-growth-factor-β-receptor family are the first examples of membrane receptors that are internalized not only through clathrin-mediated endocytosis (which results in signalling), but also through lipid-raft-dependent routes (which results in receptor downregulation). Other proteins, such as interleukin-2 receptor-β and autocrine-motility-factor receptor, are also internalized through lipid-raft-mediated endocytic routes.

  • A role for receptor endocytosis and trafficking in the regulation of Hedgehog and Wingless/Int-1 (Wnt) signalling has been proposed on the basis of studies that analysed the distribution of proteins that are implicated in the signalling pathways of these two morphogens.

Abstract

The internalization of various cargo proteins and lipids from the mammalian cell surface occurs through the clathrin and lipid-raft endocytic pathways. Protein–lipid and protein–protein interactions control the targeting of signalling molecules and their partners to various specialized membrane compartments in these pathways. This functions to control the activity of signalling cascades and the termination of signalling events, and therefore has a key role in defining how a cell responds to its environment.

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Figure 1: Epidermal-growth-factor-receptor trafficking.
Figure 2: Transforming-growth-factor-β-receptor internalization by clathrin- and lipid-raft-mediated endocytosis.
Figure 3: Internalization through lipid-raft-dependent endocytosis.

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Acknowledgements

The authors wish to thank L. Izzi for helpful advice and for her critical review of the manuscript. Work in J.L.W.'s laboratory is supported by grants from the NCIC (National Cancer Institute of Canada), CIHR (Canadian Institute of Health Research), NSERC (National Science and Engineering Research Council) and Genome Canada. J.L.W. is a CIHR Investigator and International Scholar of the HHMI (Howard Hughes Medical Institute). We apologize to the many researchers whose papers could not be cited here because of space limitations.

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Authors and Affiliations

  1. Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Room 1075, Mount Sinai Hospital, Toronto, M5G 1X5, Ontario, Canada

    Christine Le Roy & Jeffrey L. Wrana

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  1. Christine Le Roy
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Correspondence to Jeffrey L. Wrana.

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Supplementary information

Supplementary table S1

Examples of phosphoinositides and their binding partners. (PDF 22 kb)

Related links

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DATABASES

Flybase

Arrow

Dishevelled

Fz

Fz4

Hh

Ptc

Smo

Wg

Wnt5

Interpro

BAR

C2

ENTH

FYVE

HECT

PH

PX

SH2

SoHo

WW

Swiss-Prot

AMFR

CAP

caveolin-1

Cbl

Dickkopf1

EGF

EGFR

EPS15

EPS15R

H-Ras

HRS

IL2Rβ

Kremen-2

NEDD4

Rab11

Raf

SARA

Shh

SMAD2

SMURF1

SMURF2

TβRI

TβRII

TβRIII

TNFα

TNFR1

TSG101

Glossary

LIPID RAFTS

Rich in cholesterol, glycosphingolipids, glycosylphosphatidylinositol-anchored proteins and signalling molecules, these membrane microdomains, which are distinct from clathrin-coated pits, function as signalling platforms.

MORPHOGEN SYSTEM

In morphogen systems, signalling molecules are produced at a localized region and spread away from their source. In embryonic tissue, morphogens influence the movement and organization of cells by forming a concentration gradient.

PHOSPHATIDYLINOSITOL 3-KINASES

(PI3Ks). Type-I and -II PI3Ks phosphorylate the D-3 position of PtdIns4P and PtdIns(4,5)P2, respectively. Type III PI3Ks phosphorylate the D-3 position of PtdIns. Type-I PI3Ks are thought to be involved in numerous signal-transduction and membrane-trafficking systems.

PTEN

(phosphatase and tensin homologue). PTEN dephosphorylates PtdIns(3,4,5)P3 and PtdsIns(3,4)P2 at the D-3 position, and is a negative regulator of PI3K signalling.

GLYCOSPHINGOLIPIDS

Lipids that contain at least one monosaccharide residue and either a sphingolipid or a ceramide (N-acylated sphingoid).

PHAGOCYTOSIS

Phagocytosis is a process that is used by cells to internalize large particles such as debris, apoptotic cells and pathogens. The internalized particles can be stored or degraded by cells.

MACROPINOCYTOSIS

Macropinocytosis is a form of regulated endocytosis that involves the formation of large endocytic vesicles after the closure of cell-surface membrane ruffles.

TRANSFERRIN RECEPTOR

The transferrin receptor is the archetypical cargo for internalization through clathrin-mediated endocytosis. At the cell surface, it binds its ligand (ferrotransferrin) and is internalized into early endosomes, where it releases the iron and then recycles back to the plasma membrane.

(MONO)UBIQUITYLATION

A highly conserved 76-amino-acid protein is covalently attached to a lysine residue in the target protein. Ubiquitylation requires ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-protein ligase (E3) enzymes that are responsible for selecting targets for ubiquitin modification. Two families of E3s are the HECT-domain-containing enzymes (such as SMURFs and NEDD4) and RING-domain-containing enzymes (such as Cbl).

NEDD4

(neuronal-precursor-cell-expressed developmentally downregulated-4). The prototypical protein in a family of E3 ubiquitin ligases that contain a HECT (homologous to E6-AP C terminus) catalytic C-terminal domain and WW and C2 domains that are involved in substrate recognition and cellular localization.

UIM

(ubiquitin-interacting motif). A module of 20 amino acids that specifically recognizes ubiquitin. It is present in endocytic proteins (such as EPS15 and HRS) and in proteasomal components.

LYSOSOME

A type of organelle that is characterized by a low internal pH, contains hydrolytic enzymes and is involved in the post-translational maturation of proteins, the degradation of receptors and the extracellular release of active enzymes.

RAB PROTEINS

Ras-like small G-proteins that control trafficking, exocytosis, endocytosis and endosome fusion. They can be modified by geranylgeranyl groups, are tightly associated with membranes and can be specifically localized to different compartments (for example, Rab5 is localized to early endosomes).

MULTIUBIQUITYLATION

The addition of several monoubiquitin molecules to a target protein. (As opposed to polyubiquitylation, in which a single chain of several ubiquitin molecules is appended to the target protein.)

ESCRT

(endosomal sorting complex required for transport). The multiprotein ESCRT machinery (ESCRT-I, -II and -III) promotes inward vesiculation at the limiting membrane of the sorting endosome, and selects cargo proteins for delivery to the intralumenal vesicles of multivesicular bodies.

MULTIVESICULAR BODIES

Endocytic intermediate organelles in the lysosomal degradative pathway that contain small vesicles and are surrounded by a limiting membrane.

EEA1

(early endosome antigen-1). EEA1 is a membrane-bound, FYVE-domain-containing protein that binds PtdIns3P. It is an effector of the small GTPase Rab5 that controls early-endosome fusion dynamics.

ACYL CHAIN

An acyl chain is a carbonyl group with an alkyl group attached.

FLOTILLIN

Flotillins are integral membrane proteins and constituents of lipid rafts. Flotillin-1 and -2 were originally discovered in neuronal cells as Reggie-2 and -1, respectively, and they are thought to be involved in insulin-receptor and T-cell-receptor signalling.

ANNEXIN

A family of Ca2+- and phospholipid-binding proteins that are found in lipid rafts. They are involved in membrane-trafficking events and in the organization of membrane compartments and the plasma membrane.

CAVEOLIN

These integral membrane proteins have a central hydrophobic domain that forms a hairpin loop inside the membrane to leave both C- and N-terminal domains facing the cytosol.

SIMIAN VIRUS 40

A member of the papilloma-, polyoma- and vacuolating-virus family of non-enveloped DNA viruses. At the cell surface, the virus probably binds the major histocompatibility (MHC) class-I antigen receptor, but whether this mediates its endocytosis into caveolae is unknown.

FLUID-PHASE MARKERS

Markers of fluid-phase endocytosis, which presumably does not involve receptor-mediated trafficking. These markers include proteins such as horseradish peroxidase and dextran.

R-SMADS

(receptor-regulated-SMADs). R-SMADs are transcription factors, and they contain two domains (MAD homologue-1 and -2 (MH1 and MH2)) that are separated by a proline-rich linker. The MH1 domain mediates interactions with proteins and DNA, whereas the MH2 domain mediates protein–protein interactions.

β-ARRESTIN-2

Arrestins are an important family of proteins that are known to be negative regulators of G-protein-coupled receptor (GPCR) signalling. Arrestins bind to phosphorylated GPCRs and recruit clathrin and AP2, which results in receptor internalization and desensitization. These proteins have also been shown to regulate Wnt and TGFβR internalization.

DYNAMIN

An important component of the endocytic machinery that might function both as a regulatory GTPase (by recruiting various components into coated pits and inducing vesicular budding) and as a mechanochemical enzyme (that forms a collar-like structure at the necks of invaginated membranes and promotes fission of the buds).

CHOLERA-TOXIN B SUBUNIT

A type of bacterial toxin that contains an enzymatic A subunit and a binding B subunit. The uptake of cholera toxin is mediated by a glycolipid, the GM1 ganglioside.

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Le Roy, C., Wrana, J. Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling. Nat Rev Mol Cell Biol 6, 112–126 (2005). https://doi.org/10.1038/nrm1571

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