Dual Engagement Regulation of Protein Interactions with the AP-2 Adaptor α Appendage

Abstract

Clathrin-mediated endocytosis depends upon the coordinated assembly of a large number of discrete clathrin coat components to couple cargo selection with rapid internalization from the cell surface. Accordingly, the heterotetrameric AP-2 adaptor complex binds not only to clathrin and select cargo molecules, but also to an extensive family of endocytic accessory factors and alternate sorting adaptors. Physical associations between accessory proteins and AP-2 occur primarily through DP(F/W) or FXDXF motifs, which engage an interaction surface positioned on the C-terminal platform subdomain of the independently folded ␣ subunit appendage. Here, we find that the WXX(F/W)X(D/E) interaction motif found in several endocytic proteins, including synaptojanin 1, stonin 2, AAK1, GAK, and NE-CAP1, binds a second interaction site on the bilobal ␣ appendage, located on the N-terminal ␤ sandwich subdomain. Both ␣ appendage binding sites can be engaged synchronously, and our data reveal that varied assemblies of interaction motifs with different affinities for two sites upon the ␣ appendage can provide a mechanism for temporal ordering of endocytic accessory proteins during clathrin-mediated endocytosis.Clathrin-coated vesicles are a major portal of entry into eukaryotic cells, carrying macromolecular nutrients, ligands, select transmembrane proteins, and even viruses into the cell interior from the plasma membrane (1, 2). Cargo selectivity of these short-lived transport intermediates is often thought to be governed by a central triad of proteins, the cargo receptors, clathrin, and the heterotetrameric AP-2 adaptor complex. Cargo receptors contain cytosolic internalization sequences, such as the YXXØ motif (where X is any amino acid and Ø represents a residue with a bulky hydrophobic side chain), found within proteins like the receptor for the endocytosed iron transport protein transferrin. Several distinct internalization sequences or tags are known, each specifying internalization by promoting preferential incorporation into assembling clathrincoated vesicles (3). Clathrin functions as a trimer of heterodimers, composed of three 192-kDa heavy and three 20 -25-kDa light chains, that polymerize to form the characteristic polyhedral clathrin lattice (4,5). Assembled clathrin appears to act as a mechanical scaffold during the process of bud invagination. AP-2, the archetypical sorting adaptor, is composed of two large (ϳ100 kDa) subunits (␣ and ␤2), a 50-kDa medium 2 subunit, and a 17-kDa small 2 chain (2, 6). AP-2 binds physically to both clathrin, through the hinge and appendage domains of the ␤2 subunit (7), and to YXXØ-type internalization sequences, via the 2 subunit, in a phosphorylation-regulated manner (6, 8 -10). AP-2 is therefore a multifunctional protein that couples coat assembly with cargo selection. Surprisingly, after small interfering RNA silencing of either the AP-2 ␣ or 2 subunit mRNA in HeLa cells to deplete cellular AP-2 adaptor levels, certain transmembrane receptors, like the epidermal growth factor and low ...

α-AP-2 Directs Myosin VI-dependent Endocytosis of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in the Intestine

“…Protein and Peptide Preparation-Expression and purification of GST fusion proteins were performed as described (23,24). GST and the various GST fusion proteins produced in Escherichia coli BL21 cells were grown in 20 ml of LB Broth (Invitrogen) plus ampicillin (200 g) (Fisher) overnight at 37°C with constant shaking to create a starter culture.…”

Functional Dissection of an AP-2 β2 Appendage-binding Sequence within the Autosomal Recessive Hypercholesterolemia Protein

“…DNA Constructs-The recombinant proteins consisting of glutathione S-transferase (GST) fused to ARHC1 (human residues 180 -308), AP-2 ␣ C appendage (murine residues 701-938), ␣ C appendage (Q782A) point mutant, AP-2 ␤2 appendage (rat residues 714 -951), and the epsin 1 DPW domain (rat residues 229 -407) fusion proteins have been described previously (22,(33)(34)(35). GST-ARHM1 (human ARH residues 254 -269) was generated by ligation of complementary oligonucleotides, after annealing and digestion, into EcoRI/XhoI cleaved pGEX-4T-1.…”

“…The ITC data also reveal that there is a single binding site for the ARH peptide upon the ␤2 appendage. With a K d of 1-2 M, the affinity of ARH for the ␤2 appendage is ϳ5-fold higher than the best interaction motif for the AP-2 ␣ subunit appendage; the WXX(FW)X(DE) motif binds the ␣ appendage with a K d of ϳ10 M, albeit to a topologically distinct surface, located on the sandwich subdomain of the ␣ appendage (35). Mutation of key residues (Trp 841 and Tyr 888 ) that comprise the site of high hydrophobic potential, believed to be the major ligand binding surface on the ␤2 appendage platform subdomain (Fig.…”

Canonical Interaction of Cyclin G–associated Kinase with Adaptor Protein 1 Regulates Lysosomal Enzyme Sorting

“…Both sequences are contained within a relatively unstructured segment (designated clathrin-binding or CB) of the protein that also contains binding motifs for the clathrin terminal domain (i.e., DLL) and for the ear domains of the AP2-␣ and -␤2 subunits (i.e., DPF and WAAW; Mishra et al, 2004;Figure 1). GAK also has a serine/threonine-kinase domain homologous to that of the mammalian adaptor-associated kinase AAK1 and the yeast Ark/Prk proteins (Zeng and Cai, 1999;Watson et al, 2001), and tensin (also known as PTEN-homology) and J domains similar to those of auxilin (Ahle and Ungewickell, 1990; Figure 1).…”