AP2 Interactions with Cargo

One of the best understood interactions in endocytosis is the recognition of cargo membrane proteins bearing Yxx0-based (where x is any amino acid and 0 is a bulky hydrophobic residue) endocytic sorting motifs within their cytoplasmic domains by

Site within AP-2

AP-2 subunit/

AP-2 binding partners domain involved


LDLR, megalin)

CD4, MPR46)

Membrane cargo (i.e., |_l2 GluR2, GABAaR-ß3, synaptotagmin 1)

Clathrin ß2-hinge

Stonin2 a-ear

Synaptojaninl(170) |_l2 AAK1 NECAP1/2



Amphiphysinl a-ear API 80

Synaptojaninl (170)


Motif in AP-2 binding partner Yxx(|)


Basic cluster

ß-propeller (N-terminal domain) Wxx[FW]ac



Subdomain A (F174, W421, R423) Unknown site within |_l2 alpha/ sigma2 interface within AP-2 core Subdomain B

Platform domain (i.e., W840 of a-ear)

Approximate Kd

120 |iM ND

Platform domain (W840)

Platform domain (Y888) Sandwich domain (Y815)

K341,K343,K345,K354,K356 Sandwich domain (F740, G742) subdomain A (F174, W421, R423)

Shown are also the consensus motifs for interaction with AP-2 and their approximate binding constants (Kd). Gh/R2, a subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate-receptors; GABAaR- 5, |33 subunit of type A y-aminobutyric acid receptors; TfR, transferrin receptor; EGFR, epidermal growth factor receptor; CD4, cluster of differentiation antigen 4; LDLR, low-density lipoprotein receptor; AAK1, adaptor-associated kinase 1; Dab2, disabled-2; AP180, adaptor protein of 180 kDa; ARH, autosomal recessive hypercholesterolemia protein; NECAPl/2, adaptin-ear-binding coat-associated proteins 1 and 2; epsl5, epidermal growth factor receptor protein substrate 15

AP-2|| (Fig. 1, no. 5). As described in detail above, peptides with this motif fit into a hydrophobic pocket on the surface of subdomain A of C-|2 (Owen et al. 1999). A good example is represented by the sequence 20YTRF within the cytosolic domain of the transferrin receptor, which functions as an autonomous and trans-plantable signal for internalization (Traub 2003).

AP-2 has also been postulated to recognize FxNPxY motifs present in the cyto-plasmic tail of low-density-lipoprotein (LDL) receptor family members (Boll et al. 2002). It does not require the Yxx0 binding pocket of |2, but depends on other, unidentified residues. Given that specialized endocytic adaptors for FxNPxY motifs, such as Dab2 and ARH, have been identified, the physiological significance of this interaction is uncertain. Dileucine-based motifs [DE]xxxL[LI] (Bonifacino and Lippincott-Schwartz 2003) serve as internalization signals in many single and multi-spanning transmembrane proteins (Bonifacino and Traub 2003). Again, conflicting data exist regarding the exact site of interaction within AP-2, but recent results suggest that the alpha/sigma2 hemicomplex of the AP-2 core is involved. Association with AP-2 is dependent on LL or LI pairs preceded by acidic residues at the -4 and -5 position with respect to the first leucine (Fig. 1, no. 4). So far, attempts to co-crystallize AP-2 with [DE]xxxL[LI] peptides have failed (Collins et al. 2002).

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors (AMPARs) are the main class of receptors involved in rapid excitatory neurotransmission in the mammalian CNS (Derkach et al. 2007). AMPA receptors undergo constitutive and ligand-induced internalization that requires dynamin and the AP-2 complex. Recently, it was reported that an atypical basic motif within the cytoplasmic tails of AMPA-type glutamate receptors directly associates with nanomolar affinity with | 2-adaptin. Recognition by AP-2| involves a basic motif within the cytolasmic tails of AMPA receptor subunits GluR1-3, GABAa receptors, and in the C2 domain of synaptotagmin 1 (Kastning et al. 2007) (Fig. 1, no. 6). Unlike constitutively internalized cargo proteins, these synaptic membrane proteins interact with subdomain B of C-|2. Why such non-conventional endocytosis signals are used is unknown, but may be related to the fact that pre- and postsynaptic membrane proteins must undergo sorting to a specific endosomal subpopulation (Lee et al. 2004) that may be capable of segregating synaptic from constitutively internalized proteins. This is similar to what has been reported recently for other ligand-activated receptors (Lakadamyali et al. 2006).

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