Stabilization of the transient binding of AP-2 and clathrin at the plasma membrane is effected by the recruitment of a number of early acting proteins, including FCHo (F-BAR domain-containing Fer/Cip4 homology domain-only) proteins 1 and 2, intersectins (ITSNs), EPS15, EPS15L1, REPS1 and SGIP1 among others (Henne et al, 2010; Stimpson et al, 2009; Reider et al, 2009; Dergai et al, 2010; Antonescu et al, 2011; reviewed in McMahon and Boucrot, 2011).
FCHo proteins interact with the plasma membrane-enriched PI(4,5)P2 through the F-BAR domain, which recognizes curvature in the membrane (Henne et al, 2010; Henne et al, 2007; Shimada et al, 2007; Umasankar et al, 2012). Other F-BAR proteins, such as FNBP1 and FNBP1L may join the nascent clathrin-coated pit at a slightly later stage (Shimada et al, 2007). Recruitment of EPS15 and ITSN1 and 2 appears coincident with binding of FCHo2 and depends on direct interaction with the AP2 mu homology domain of FCHo2 (Henne et al, 2010).
SGIP1 (Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1) interacts with numerous endocytic proteins including AP-2, ITSN1, REPS1, EPS15, endophilin and amphiphsyin1 and is thought to play a role in clathrin-mediated endocytosis (Trevaskis et al, 2005; Dergai et al, 2010; Uezu et al, 2007). SGIP1 is related to the FCHo proteins and is co-immunoprecipitated in a tripartite complex containing ITSN1 and REPS1 (Dergai et al, 2010). The exact function of SGIP1 in clathrin-mediated endocytosis remains to be elucidated, however recent work suggests SGIP1 and FCHo proteins may contribute to allosteric changes in AP-2 that promote membrane binding and cargo recognition (Hollopeter et al, 2014).
The recruitment of this group of early CCP proteins is rapidly followed by the incorporation of many AP-2 and clathrin molecules, stimulated in part by the FCHo- and SGIP-dependent stabilization of the open, membrane binding conformation of AP-2 (Hollopeter et al, 2014). Alternately, a proportion of the nascent CCPs may undergo abortive initiation (Loerke et al, 2009; Aguet et al, 2013; Antonescu et al, 2011). This is prompted in part through the early recruitment of the 170 kDa isoform of synaptojanin 1 (SYNJ1-170, not shown in this reaction). SYNJ1 catalyzes the hydrolysis of PI(4,5)P2 to PI(4)P and destabilizes the interaction of many early CCP components with the plasma membrane (Perera et al, 2006).