Recruitment of AP-2 complex and clathrin

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Reaction [binding]
Homo sapiens
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Recruitment of early acting proteins such as the FCHo and ITSN proteins stabilizes the transient AP-2:clathrin complex at the plasma membrane and is rapidly followed by incorporation of many more molecules of AP-2 and clathrin. AP-2 binding to the plasma-membrane enriched PI(4,5)P2 is reinforced early in the formation of a CCP by the interaction of AP-2 with PIP5K1C, which synthesizes PI(4)P to PI(4,5)P2 (Krauss et al, 2006; Bairstow et al, 2006; Thieman et al, 2009).

AP-2 recruitment is also promoted by conformational changes upon lipid and protein binding. AP-2 is a heterotetramer consisting of two large subunits (alpha and beta1 adaptin), a medium mu2 subunit and a small sigma2 subunit, and exists in a closed conformation when not part of a clathrin-coated pit (Jackson et al, 2010).
Interactions between the AP-2 mu2 subunit and PIP2 within the lipid bilayer stabilize the 'open' conformation of AP-2, exposing binding sites for cargo proteins. The open conformation is also promoted by interaction of AP-2 with early CCP proteins such as SGIP and FCHo2 (Hollopeter et al, 2014). Recruitment of clathrin stimulates the activity of AAK1, an AP-2 kinase that phosphorylates the mu2 subunit of the adaptor complex at Thr156, further stabilizing the open conformation and promoting cargo recruitment (Olusanya et al, 2001; Ricotta et al, 2002; Conner et al, 2002; Conner et al, 2003).

NECAP1 and 2 may also aid in the assembly of an emergent clathrin-coated pit. NECAP proteins have a WxxF motif at the C-terminus that binds with high affinity to the alpha-ear sandwich domain of AP-2 and an N-terminal PH ear domain that interacts both with AP-2 and a wide range of endocytic accessory proteins containing FxDxF motifs (Ritter et al, 2003; Wasiak et al, 2002; Ritter et al, 2013). Clathrin and the NECAP PH ear domain appear to compete for an AP-2 binding site. Clathrin-mediated displacement of the NECAP PH ear domain from its lower affinity AP-2 site may allow release this domain, allowing it to transition to a role in recruiting endocytic accessory proteins and cargo (Ritter et al, 2007; Ritter et al, 2013; reviewed in McMahon and Boucrot, 2011).

Finally, studies have highlighted a role for ARF6 and its GTPase activating protein ARFGAP1 in CCP formation, although the details remain to be established.
ARFGAP1 and ARF6 appear to contribute to the recruitment of some cargo, but may also play a more generalized role in CCP formation (Moravec et al, 2012; Bai et al, 2011). ARFGAP1 binds directly to AP-2 and its GAP activity is required for CME. Consistent with this, silencing of ARFGAP1 impairs CME (Schmid et al, 2006; Rawet et al 2010; Bai et al 2011). ARFGAP1 has activity towards several ARFs, including ARF6 which is found is some CCPs and is known to regulate CME under some circumstances (Moravec et al, 2003; Palacios et al, 2002; Paleotti et al, 2005; Kraus et al, 2003). ARF6 is thought to contribute to the recruitment of AP-2 and clathrin to the plasma membrane, possibly in part by affecting the lipid composition (Paleotti et al, 2002; Krauss et al, 2003).

Literature References
PubMed ID Title Journal Year
21499258 ARFGAP1 promotes AP-2-dependent endocytosis

Fu, L, Gad, H, Collawn, JF, Luo, R, Nie, Z, Li, J, Cocucci, E, Bai, M, Yang, JS, Beznoussenko, GV, Kirchhausen, T, Turacchio, G, Luini, A, Hsu, VW

Nat. Cell Biol. 2011
1847920 Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2

Beck, KA, Keen, JH

J. Biol. Chem. 1991
15802264 The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes

Chardin, P, Macia, E, Klein, S, Kirchhausen, T, Partisani, M, Franco, M, Luton, F, Paleotti, O

J. Biol. Chem. 2005
21779028 Molecular mechanism and physiological functions of clathrin-mediated endocytosis

Boucrot, E, McMahon, HT

Nat. Rev. Mol. Cell Biol. 2011
20211604 ArfGAP1 interacts with coat proteins through tryptophan-based motifs

Szafer-Glusman, E, Levi-Tal, S, Rawet, M, Cassel, D, Parnis, A

Biochem. Biophys. Res. Commun. 2010
14555962 Identification of a family of endocytic proteins that define a new alpha-adaptin ear-binding motif

Blondeau, F, Tung, EC, Philie, J, Ritter, B, McPherson, PS, Girard, M

EMBO Rep. 2003
12847086 ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma

Kinuta, M, Krauss, M, Haucke, V, Wenk, MR, Takei, K, De Camilli, P

J Cell Biol 2003
22863004 The first five seconds in the life of a clathrin-coated pit

Kirchhausen, T, Aguet, F, Cocucci, E, Boulant, S

Cell 2012
19287005 Clathrin regulates the association of PIPKIgamma661 with the AP-2 adaptor beta2 appendage

Ling, K, Mishra, SK, Thieman, JR, Traub, LM, Anderson, RA, Doray, B

J. Biol. Chem. 2009
26403691 Forty Years of Clathrin-coated Vesicles

Robinson, MS

Traffic 2015
11877461 Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis

Conner, SD, Schmid, SL

J. Cell Biol. 2002
12447393 ARF6-GTP recruits Nm23-H1 to facilitate dynamin-mediated endocytosis during adherens junctions disassembly

D'Souza-Schorey, C, Boshans, RL, Schweitzer, JK, Palacios, F

Nat. Cell Biol. 2002
16903783 Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly

McMahon, HT, Ford, MG, Praefcke, GJ, Benmerah, A, Burtey, A, Peak-Chew, SY, Schmid, EM, Mills, IG

PLoS Biol. 2006
24130457 NECAP 1 regulates AP-2 interactions to control vesicle size, number, and cargo during clathrin-mediated endocytosis

Murphy, S, Gudheti, MV, Kozlov, G, Dokainish, H, Philie, J, Ritter, B, Gehring, K, McPherson, PS, Halin, M, Girard, M, Jorgensen, EM

PLoS Biol. 2013
16880396 Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2mu-cargo complexes

Kukhtina, V, Haucke, V, Pechstein, A, Krauss, M

Proc. Natl. Acad. Sci. U.S.A. 2006
11877457 Phosphorylation of the AP2 mu subunit by AAK1 mediates high affinity binding to membrane protein sorting signals

Höning, S, Ricotta, D, von Figura, K, Conner, SD, Schmid, SL

J. Cell Biol. 2002
11516654 Phosphorylation of threonine 156 of the mu2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo

Smythe, E, Andrews, PD, Swedlow, JR, Olusanya, O

Curr. Biol. 2001
14581451 Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor micro2 kinase

Smythe, C, Jackson, AP, Wettey, FR, Smythe, E, Hufton, L, Flett, A

J. Cell Biol. 2003
17762867 The NECAP PHear domain increases clathrin accessory protein binding potential

Philie, J, Ritter, B, Zylbergold, P, Allaire, PD, Legendre-Guillemin, V, Denisov, AY, Gehring, K, McPherson, PS

EMBO J. 2007
12213833 Enthoprotin: a novel clathrin-associated protein identified through subcellular proteomics

Blondeau, F, Wasiak, S, Puertollano, R, Bell, AW, Legendre-Guillemin, V, Bonifacino, JS, de Heuvel, E, Boismenu, D, McPherson, PS, Girard, M

J. Cell Biol. 2002
16707488 Type Igamma661 phosphatidylinositol phosphate kinase directly interacts with AP2 and regulates endocytosis

Ling, K, Carbonara, C, Firestone, AJ, Bairstow, SF, Anderson, RA, Su, X

J. Biol. Chem. 2006
22815487 BRAG2/GEP100/IQSec1 interacts with clathrin and regulates ?5?1 integrin endocytosis through activation of ADP ribosylation factor 5 (Arf5)

Casanova, JE, Moravec, R, Allison, AB, D'Souza, R, Conger, KK

J. Biol. Chem. 2012
14617351 AAK1-mediated micro2 phosphorylation is stimulated by assembled clathrin

Conner, SD, Schmid, SL, Schröter, T

Traffic 2003
15916959 Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2

Höning, S, Haucke, V, Robinson, C, Owen, DJ, Robinson, M, Ricotta, D, Motley, A, Späte, K, Krauss, M, Spolaore, B

Mol. Cell 2005
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