A Phosphotyrosine Switch for Cargo Sequestration at Clathrin-coated Buds*

Background: The AP-2 adaptor is a central protein-protein interaction hub during clathrin-mediated endocytosis. Results: Introducing a specific phosphotyrosine mimetic on the large AP-2 β2 subunit selectively regulates protein interactions with the adaptor complex. Conclusion: A directed post-translational modification can locally rewire the AP-2 interaction network. Significance: This provides a plausible mechanism for regional cargo uncoupling at cell surface clathrin coats. The AP-2 clathrin adaptor complex oversees endocytic cargo selection in two parallel but independent manners. First, by physically engaging peptide-based endocytic sorting signals, a subset of clathrin-dependent transmembrane cargo is directly collected into assembling buds. Synchronously, by interacting with an assortment of clathrin-associated sorting proteins (CLASPs) that independently select different integral membrane cargo for inclusion within the incipient bud, AP-2 handles additional cargo capture indirectly. The distal platform subdomain of the AP-2 β2 subunit appendage is a privileged CLASP-binding surface that recognizes a cognate, short α-helical interaction motif. This signal, found in the CLASPs β-arrestin and the autosomal recessive hypercholesterolemia (ARH) protein, docks into an elongated groove on the β2 appendage platform. Tyr-888 is a critical constituent of this spatially confined β2 appendage contact interface and is phosphorylated in numerous high-throughput proteomic studies. We find that a phosphomimetic Y888E substitution does not interfere with incorporation of expressed β2-YFP subunit into AP-2 or alter AP-2 deposition at surface clathrin-coated structures. The Y888E mutation does not affect interactions involving the sandwich subdomain of the β2 appendage, indicating that the mutated appendage is folded and operational. However, the Y888E, but not Y888F, switch selectively uncouples interactions with ARH and β-arrestin. Phyogenetic conservation of Tyr-888 suggests that this residue can reversibly control occupancy of the β2 platform-binding site and, hence, cargo sorting.

The AP-2 clathrin adaptor complex oversees endocytic cargo selection in two parallel but independent manners. First, by physically engaging peptide-based endocytic sorting signals, a subset of clathrin-dependent transmembrane cargo is directly collected into assembling buds. Synchronously, by interacting with an assortment of clathrin-associated sorting proteins (CLASPs) that independently select different integral membrane cargo for inclusion within the incipient bud, AP-2 handles additional cargo capture indirectly. The distal platform subdomain of the AP-2 ␤2 subunit appendage is a privileged CLASP-binding surface that recognizes a cognate, short ␣-helical interaction motif. This signal, found in the CLASPs ␤-arrestin and the autosomal recessive hypercholesterolemia (ARH) protein, docks into an elongated groove on the ␤2 appendage platform. Tyr-888 is a critical constituent of this spatially confined ␤2 appendage contact interface and is phosphorylated in numerous high-throughput proteomic studies. We find that a phosphomimetic Y888E substitution does not interfere with incorporation of expressed ␤2-YFP subunit into AP-2 or alter AP-2 deposition at surface clathrin-coated structures. The Y888E mutation does not affect interactions involving the sandwich subdomain of the ␤2 appendage, indicating that the mutated appendage is folded and operational. However, the Y888E, but not Y888F, switch selectively uncouples interactions with ARH and ␤-arrestin. Phyogenetic conservation of Tyr-888 suggests that this residue can reversibly control occupancy of the ␤2 platform-binding site and, hence, cargo sorting.
Post-translational phosphorylation of Ser, Thr, and Tyr residues is a very prevalent modification across eukaryotic phyla, occurring in at least 30% of expressed proteins from the unicellular Saccharomyces cerevisiae to complex metazoans like primates (1)(2)(3)(4). There are estimates of 500,000 potential phosphor-ylation acceptor residues (5) and Ͼ100,000 phosphosites/cell (4); a data set of 68,379 phosphopeptides has been compiled from growth-arrested HeLa cell populations (6). Appropriately, it is widely accepted that phosphorylation regulates the activity of endocytosis. Perhaps the best established mechanism is when extracellular ligand binding elicits receptor phosphorylation upon cytosol-oriented residues, which then act either as physical internalization signals or initiate the display or generation of a controlled uptake signal (7,8). Internalization of G protein-coupled receptors (9) and the epidermal growth factor receptor (10) are emblematic of this mode of post-translationally activated clathrin-mediated endocytosis. The reciprocal is also known, where site-specific phosphorylation can cloak or incapacitate a constitutively internalization signal. For instance, in the ionotropic ␥-aminobutyric acid A (GABA A ) receptor, phosphorylation of flanking Tyr side chains within a YXYXXØ-type sorting signal (where Ø represents a bulky hydrophobic residue) on the ␥2 subunit prevents efficient clathrin-mediated endocytosis (11). Analogously, the prompt internalization directed by a YXXØ sorting signal in the co-receptor CTLA4 expressed on activated T lymphocytes is negatively regulated by Tyr phosphorylation (12).
In addition to trafficking transmembrane cargo, numerous soluble core components of the endocytic coat are also phosphorylated. Vertebrate clathrin coat proteins are dubbed "dephosphins" (13) because many of these factors are swiftly dephosphorylated in concert by calcineurin upon depolarization of the nerve terminal (14). This established rapid change in overall phosphostatus is not, however, accompanied by a clear mechanistic understanding of the precise regulatory consequences of phosphorylation-dephosphorylation cycles. It is known that there are global phosphorylation differences in the heterotetrameric clathrin adaptor AP-2 when exchanging between cytosolic and membrane-associated pools and that hyperphosphorylation impedes AP-2 interactions with clathrin triskelia (15). Also, phosphorylation of Thr-156 on the AP-2 2 subunit promotes recognition of YXXØ-type endocytic sorting signals (16 -18). The PhosphoSitePlus discovery mode database (19) currently identifies Thr(P)-156 in at least 35 independent mass spectrometry studies. Yet the crystal structure of the AP-2 heterotetramer core in the open, cargo-binding conformation fails to reveal precisely why this phosphorylation event biases cargo capture (20). More recently, phosphoproteomics has categorized numerous additional phosphorylated side chains in the AP-2 ␣, ␤2, 2, and 2 subunits from many different organisms, cell types, and tissues (19).
Proteins can be multiply, sequentially, and redundantly phosphorylated (19,21), often making categorical dissection of the precise physiological role of individual modifications challenging. In fact, the biological role of the vast majority of the phosphorylated residues catalogued in high-throughput mass spectrometry-based screens is presently elusive and unsettled, and only about 2% or less of mapped phosphosites are Tyr(P), the bulk being Ser(P) (4,5,22). The low relative frequency of Tyr(P) makes delineation of phosphoacceptor function more tractable. Also germane is that binding interfaces are predisposed to evolutionarily conserved phosphorylation (23), and the preponderance of phosphorylation sites map to intrinsically disordered regions of proteins (22,24). Nevertheless, overall, Tyr(P) tends to be enriched on folded domains and at structured contact interfaces (24). Here, we examine the physiological consequences of the most prevalent Tyr phosphosite modification, Tyr(P)-888, upon the independently folded appendage of the AP-2 adaptor ␤2 subunit (the product of the AP2B1 gene).

EXPERIMENTAL PROCEDURES
All the experiments described here were carried out at least three times independently, with comparable results obtained. Selected but representative data are shown for each set of experiments.
Protein Preparation-GST and various GST fusion proteins were expressed in Escherichia coli strain BL21 with a standard protocol (33,(37)(38)(39). In brief, protein expression was induced in 2ϫ TY medium by adding isopropyl-1-thio-␤-D-galactopyranoside (0.2 mM final concentration) to log phase bacterial cultures (A 600 ϳ0.7) at room temperature. Following the addition of isopropyl-1-thio-␤-D-galactopyranoside, cultures were allowed to grow for an additional 16 h with constant shaking. Bacteria were harvested by centrifugation at 15,000 ϫ g for 15 min at 4°C and stored at Ϫ80°C. Bacterial pellets were resuspended in 50 mM Tris-HCl, pH 7.5, 10 mM ␤-mercaptoethanol, 300 mM NaCl, and 0.2% (w/v) Triton X-100 before sonication on ice. Cellular debris was removed by centrifugation at 23,700 ϫ g for 30 min at 4°C, and GST fusion proteins were recovered on glutathione-Sepharose. After extensive washing with phosphate-buffered saline (PBS), GST fusion proteins were eluted with 25 mM Tris-HCl, pH 8.0, 5 mM DTT, 200 mM NaCl, and 10 mM glutathione on ice, dialyzed into PBS supplemented with 1 mM DTT, and stored at Ϫ80°C until further use.
Cell and Tissue Extract Preparation-Rat brain cytosol was prepared from frozen rat brains (PelFreez) essentially as described (33,(37)(38)(39). In brief, the cytosol was prepared by sequential differential centrifugation of homogenized rat brains in a buffer containing 25 mM HEPES-KOH, pH 7.2, 2 mM EDTA, 2 mM EGTA, 250 mM sucrose, supplemented with 1 mM phenylmethylsulfonyl fluoride and Complete protease inhibitor (Roche Applied Science). The supernatant fraction obtained after centrifugation at 105,000 ϫ g is designated the cytosol and was stored in small aliquots at Ϫ80°C until further use. Prior to the binding assays, rat brain cytosol aliquots were thawed and adjusted to 25 mM HEPES-KOH, pH 7.2, 125 mM potassium acetate, 5 mM magnesium acetate, 2 mM EDTA, 2 mM EGTA, and 1 mM DTT (1ϫ assay buffer) by dilution of a 10ϫ stock and then centrifuged at 245,000 ϫ g (TLA-100.4 FIGURE 1. Adenovirus-mediated re-expression of the AP-2 ␤2 subunit in ␤2 ؊/؊ iMEFs. A, serial dilutions of whole-cell lysates from either ␤2 ϩ/ϩ (lanes a-c and aЈ-cЈ) or ␤2 Ϫ/Ϫ iMEFs (lanes d-f and dЈ-fЈ) were resolved by SDS-PAGE. Duplicate gels were either stained with Coomassie Blue (left) or transferred to nitrocellulose (right) and probed with antibodies directed against the clathrin heavy chain (HC) mAb TD.1 and the anti-␤1/␤2 subunit antibody GD/1 or with an anti-AP-2 ␣ subunit mAb C-8 or affinity-purified anti-AP-1 ␥ subunit antibody AE/1 or affinity-purified anti-ARH or anti-Eps15 antibodies. The position of the molecular mass standards (in kDa) is shown on the left, and only the relevant portion of each blot is presented. Homozygous gene disruption of the Ap2b1 locus results in loss of the ϳ100-kDa ␤2 subunit product (identified by arrowheads in the ␤2 ϩ/ϩ lysates) and an approximate 2-fold up-regulation of the ␤1 subunit in compensation. B, control ␤2 ؊/؊ MEF cells (MOI ϭ 0, lane a) or iMEFs infected for 24 h with an adenovirus encoding a YFP-tagged full-length wild-type AP-2 ␤2 subunit (␤2-YFP) at the indicated viral MOIs (lanes b-d) were lysed and resolved by SDS-PAGE, and replicate immunoblots were probed with anti-␤1/␤2 subunit antibody GD/1 (top), polyclonal anti-GFP antibodies (middle), or mouse anti-tubulin mAb E7 (bottom). The appearance of a minor ␤2-YFP degradation product, migrating just below the native ␤1 subunit (arrowhead), is indicated. rotor) at 4°C for 20 min to remove any remaining insoluble debris.
For fibroblast lysates, control ␤2 Ϫ/Ϫ iMEFs, ␤2 Ϫ/Ϫ iMEFs transfected with the tdRFP-␤-arrestin 1 (IV 3 AA) mutant or ␤2 Ϫ/Ϫ iMEFs infected with adenovirus encoding either ␤2-YFP wild type or Y888E or Y888F mutant at multiplicities of infec-tion (MOI) of 500 for 24 h, were trypsinized, washed in PBS, and lysed on ice in 25 mM Hepes-KOH, pH 7.2, 250 mM sucrose, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, and Complete miniprotease inhibitor mixture (Roche Applied Science) for 30 min, followed by brief sonication on ice. Cell debris was removed by centrifugation at 18,000 ϫ g for 20 min at 4°C, and the super-natants were stored at Ϫ80°C until further use. Prior to use in binding assays, thawed lysates were adjusted to 1ϫ assay buffer by diluting a 10ϫ stock and then centrifuged at 18,000 ϫ g at 4°C for 20 min to remove any remaining insoluble debris. For experiments other than the binding assays, control ␤2 Ϫ/Ϫ iMEFs or those infected with adenovirus encoding ␤2WT-YFP, ␤2Y888E-YFP, or ␤2Y888F-YFP at various MOIs for 24 h or at an MOI of 500 for 24 -72 h were trypsinized, washed in PBS, and lysed in SDS-PAGE sample buffer using sonication and then heated at 95°C for 5 min.
Binding Assays-GST fusion protein pull-down assays were performed as described previously (33,(37)(38)(39). In brief, GST or GST fusion proteins (100 -200 g) were immobilized on ϳ25 l of packed glutathione-Sepharose beads at 4°C for 1 h. Beads were then collected by centrifugation (10,000 ϫ g, 1 min), washed, and resuspended in 50 l of assay buffer. Aliquots of clarified rat brain cytosol (250 l) or ␤2 Ϫ/Ϫ iMEF lysate (200 l) was added, and the tubes were incubated at 4°C for 2 h with continuous mixing. The glutathione-Sepharose beads were recovered by centrifugation (10,000 ϫ g, 1 min), and then an aliquot of 40 l of each supernatant was retained and adjusted to 100 l with SDS sample buffer (designated the supernatant). The pellets were washed thoroughly (four times with ϳ1.5 ml of ice-cold PBS/wash), and then each pellet was resuspended in SDS sample buffer up to ϳ80 l (designated the pellet fraction). Unless otherwise mentioned, 10-l aliquots, corresponding to ϳ2% (␤2 Ϫ/Ϫ iMEF lysate) or ϳ1.5% (rat brain cytosol) of each supernatant and ϳ12.5% of each pellet fraction, were analyzed using SDS-PAGE and immunoblotting.
Electrophoresis and Immunoblotting-Samples were resolved on freshly prepared 11% polyacrylamide gels formulated from a stock solution of acrylamide/bisacrylamide (30:0.4 ratio). After SDS-PAGE, proteins were either stained with Coomassie Blue or transferred to nitrocellulose membranes. Blots were blocked overnight or for 2 h in 5% skim milk in 10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 0.1% Tween 20 (TBST), and then portions of the blots were incubated with primary antibodies as indicated in the figure legends. After incubation with horseradish peroxidase (HRP)-conjugated anti-mouse and/or anti-rabbit IgG, immunoreactive protein bands were visualized with enhanced chemiluminescence. For the portions of the blots probed with anti-␤-arrestin antibody, the blots were stripped using 62 mM Tris-HCl, pH 6.8, 100 mM ␤-mercaptoethanol, 2% SDS, washed with TBST, blocked, and reprobed with anti-DsRed monoclonal antibody. Densitometric quantitation was carried out using ImageJ (National Institutes of Health) (40).
Recombinant Adenovirus Preparation and Infection-The recombinant YFP-tagged wild-type, Y888E mutant, or Y888F mutant AP-2 ␤2 subunit adenoviruses were generated (27) and purified as described (28,41). Viral titer was estimated by measuring A 260 of the final preparation and was typically 7-9 ϫ 10 12 particles/ml. Adenoviral infection was performed as described previously (28). In brief, AP-2 ␤2 Ϫ/Ϫ iMEFs were counted, and an equal number of cells were seeded onto tissue culture dishes in the presence or absence of glass coverslips and allowed to attach and grow for 24 h. After 24 h, cells were washed with PBS supplemented with 1 mM MgCl 2 (PBS-M) and incubated in fresh PBS-M for an additional 5 min at 37°C. Following incubation, PBS-M was removed, and adenovirus was added to cells (at various MOIs) in PBS-M and allowed to adsorb for several h before the addition of culture medium.
Immunofluorescence and Confocal Microscopy-AP-2 ␤2 Ϫ/Ϫ iMEFs seeded on round glass coverslips and infected with adenovirus encoding ␤2WT-YFP at a MOI of 500 for 24, 48, or 72 h were fixed with 2% paraformaldehyde, permeabilized, and immunolabeled with anti-AP-2 ␣ subunit mAb AP.6 and Alexa568-labeled goat anti-rabbit secondary antibody. AP-2 ␤2 Ϫ/Ϫ iMEFs infected with adenovirus encoding ␤2-YFP wild type, Y888E, or Y888F at a MOI of 500 for 24 h and transfected with tdRFP-␤-arrestin 1 (IV 3 AA) mutant were fixed with 2% paraformaldehyde and washed with PBS. The cells were then mounted with Cytoseal. Cell surface transferrin binding experiments were performed by switching AP-2 ␤2 Ϫ/Ϫ iMEFs infected with adenovirus encoding the various ␤2-YFP proteins at a MOI of 500 for 24 h to prewarmed DMEM supplemented with 25 mM Hepes, pH 7.2, and 0.5% BSA (starvation medium) and incubated at 37°C for 1 h. Following incubation, the cells were switched to ice-cold starvation medium for 1 h at 4°C and incubated for an additional 1 h at 4°C in ice-cold starvation medium containing 25 g/ml Alexa 568-labeled transferrin (Molecular Probes) and then washed with PBS, fixed, and mounted with Cytoseal.
Confocal images were acquired using Olympus Fluoview 1000 microscope with a PlanApo N (ϫ60/1.42 numerical aperture) oil objective. Sequential scanning was applied for acquiring individual emission channels when multiple fluorophores were involved. Data images were acquired using FV10-ASW software and exported in TIFF format. The TIFF files were then imported into Adobe Photoshop CS for making minor adjustments to brightness and/or contrast. The final figures were imported and assembled in Adobe Illustrator software.
Through-the-objective-type total internal reflection fluorescence microscopy (TIR-FM) was conducted as described (25,42), utilizing a ϫ100 CFI Apochromat series 1.49 numerical aperture TIR-FM objective mounted on a Nikon Eclipse Ti  lanes m-p). After incubation at 4°C for 2 h, the Sepharose beads were centrifuged and washed. Aliquots corresponding to ϳ2% of each supernatant (S) and 12.5% of each pellet (P) fraction were resolved by SDS-PAGE and either stained with Coomassie Blue or transferred to nitrocellulose. Replicate blots were probed with a mixture of anti-clathrin HC mAb TD.1 and the anti-␤1/␤2 subunit antibody GD/1 or with an anti-AP-2 ␣ subunit mAb. The position of the molecular mass standards (in kDa) is shown on the left, and only the relevant portion of each blot is presented. Note that when ␤2-YFP is expressed, this chain reduces the recovery of the endogenous ␤1 subunit in the pellet fractions containing AP-2 (lanes h, l, and p compared with lane d). B, ribbon representation of the structure of the ␤2 appendage domain (pale green) complexed with either the ARH (Protein Data Bank code 2G30; chartreuse) or ␤-arrestin 1 (Protein Data Bank code 2IV8; yellow) (D/E) n X 1-2 FXX(F/L)XXXR peptide. Selected Tyr residues on the ␤2 appendage (in a stick representation) are indicated (magenta circles). The primary sequence of each bound peptide is indicated, and the side chains on the bound ␣-helical peptides are shown in a stick representation with oxygen colored red and nitrogen blue.
inverted microscope equipped with a Perfect Focus System device, Agilent 4 color launch, and Elements AR software. Images were collected using an Andor Zyla 5.5 camera (Andor Technology USA, South Windsor, CT), and all colors were collected with a single quad pass (405, 488, 561, 633 nm) cube to ensure registration. Emission images were sorted with a high speed (20 ms) FLI (Finger Lakes Instrumentation, Lima, NY) filter wheel.
The reconstitution approach entails infecting the ␤2 Ϫ/Ϫ iMEFs with adenovirus encoding a full-length ␤2 subunit polypeptide tagged at the C terminus with YFP; numerous studies attest to the proper folding and operation of this ␤2-YFP subunit (25,36,55,58). Preliminary titration experiments show dose-dependent expression of the wild-type ␤2-YFP in the ␤2 Ϫ/Ϫ iMEFs (Fig. 1B, lanes b-d compared with lane a). On immunoblots, anti-␤1/␤2 antibodies reveal that adding adenovirus at a MOI of 500 results in production of the ␤2-YFP chain in excess of the endogenous ␤1 subunit at 24 h postinfection (Fig. 1C, lane a) and that the level of the virus-encoded protein drops substantially by 72 h postinfection (Fig. 1C, lane c). This reconstitution procedure therefore approximates the normal ␤1/␤2 expression ratios (Fig. 1A, lanes aЈ-cЈ) 1 day following infection. Also, the ␤2-YFP fluorescent signal is located primarily in heterogeneously sized puncta distributed irregularly over the ventral surface of infected iMEFs at 24 h postinfection (Fig.  1D), with a high degree of coincidence with the endogenous AP-2 ␣ subunit. By 72 h postinfection, the intensity of the ␤2-YFP drops, and a diffuse, featureless pool of ␣ subunit immunoreactivity is increasingly evident, suggesting that the ectopic ␤2 chain is becoming limiting.
The proper incorporation of the virally transduced ␤2-YFP into heterotetrameric AP-2 complexes is verified biochemically by pull-down assays using cell lysates from uninfected or infected iMEFs (Fig. 2A). In uninfected ␤2 Ϫ/Ϫ iMEF lysates, only the ϳ105-kDa ␤1 subunit is present (25), and this chain is recovered in the pellet fraction, along with the AP-2 ␣ subunit ( Fig. 2A, lane d), which binds physically to the tandem Asp-Pro-Trp (DPW) repeats positioned within a GST fusion of the central region of the endocytic protein epsin 1 (33,59). Because this tract from epsin 1 also contains a clathrin box sequence, a fraction of the cytosolic clathrin heavy chain associates independently with the immobilized GST fusion protein (33,59,60). Neither AP-1/2 subunits nor soluble clathrin trimers interact appreciably with GST alone (Fig. 2A, lane b). Similar analysis of the lysates from virus-infected iMEFs shows the incorporation of the ectopic ϳ140-kDa ␤2-YFP chain into GST-epsin 1-bound AP-2 at the expense of the native ␤1 subunit (Fig. 2A,  lane h, arrowhead). However, the expressed larger ␤2-YFP chain remains entirely in the supernatant, along with the endogenous ␣ and ␤1 subunits, when incubated with the immobilized GST control ( Fig. 2A, lanes e and f, arrowhead).
Post-translational Modification at a Secondary Cargo Selection Site on the ␤2 Appendage-The bilobal ␣ and ␤2 subunit appendages of AP-2 both operate as protein-protein interaction hubs, each with two spatially separate binding sites and numerous shared binding partners (7,(61)(62)(63). However, despite this structural homology, we have proposed that the distal platform subdomain of the AP-2 ␤2 appendage is a privileged docking site (25,37,38). This interaction surface is used primarily by the CLASPs ARH and ␤-arrestins to accomplish prompt cargo packaging in the face of ongoing uptake of differ-  (37) is shown with side chains in a stick representation. Element coloring is as follows: red, oxygen; blue, nitrogen; orange, phosphorus. A Tyr(P) modification is modeled on Tyr-888 (orange broken line on the left or the molecular surface of Tyr(P)-888 demarcated with spheres on the right). Obvious steric discordance with the ARH main chain ␣ helix is indicated (orange arrow). B, aliquots of ϳ100 g of GST (lanes a and b); the wild-type GST-␤2 appendage (lanes c and d); or mutant GST-␤2 (Y888E) (lanes e and f), GST-␤2 (Y888F) (lanes g and h), or GST-␤2 (Y815A) (lanes i and j) mutant appendage immobilized on glutathione-Sepharose were incubated with precleared rat brain cytosol. After incubation at 4°C for 2 h, the Sepharose beads were recovered by centrifugation and washed. Aliquots corresponding to ϳ1.5% of each supernatant (S) and 12.

AP-2 ␤2 Appendage Phosphorylation and Cargo Loading
ent cargo (Fig. 2B) (37,62,64). Dissimilar to the extended interaction motifs that engage the three other binding surfaces on the ␣ and ␤2 appendages, the (D/E) n X 1-2 FXX(F/L)XXXR motif found in ␤-arrestins and ARH contacts the AP-2 ␤2 platform subdomain structured as a three-turn ␣ helix, packing the protruding anchor aromatic/aliphatic anchor side chains located on one face of the helix into the complementary ␤2 surface groove (Fig. 2B) (37,62). Main chain hydrogen bonding of the ␣-helical scaffold reinforces the hydrophobic nature of the protein-protein interaction surface, analogous to a transmembrane ␣ helix. This altered binding form accounts for ␤2-selective interactions. A vital residue on the cognate AP-2 ␤2 platform surface is Tyr-888, which contributes structurally to the hydrophobic acceptor pockets for both the proximal Phe and the trailing Phe/Leu motif side chains (Fig. 2B). A Y888A (65) or a Y888V (37, 62, 66) substitution eliminates ARH and ␤-arrestin binding, and Tyr-888 is virtually phylogenetically invariant from unicellular invertebrates to mammals. Intriguingly, natively phosphorylated Tyr-888 (Tyr(P)-888) has been identified at least 50 times in discovery mode high-throughput mass spectrometry investigations (see the PhosphoSitePlus Web site) (19,67,68). This is thus a natural post-translational modification and, because of its location, suggests that Tyr-888 could be a key phosphoregulatory side chain that could determine occupancy of the ␤2 platform (Fig. 3A), a hypothesis we explored using, in part, the ␤2-null iMEF reconstitution system.
First, in pull-down assays utilizing the AP-2 ␤2 appendage domain (residues 714 -951) fused to GST, the general consequence of selected mutation of Tyr-888 within the platform site illustrates that either a conservative Y888F or a phosphomimetic Y888E change is well tolerated (Fig. 3B, lanes e-h compared with lanes c and d). These results rule out gross misfolding or serious structural perturbations in these Tyr-888 mutants. The binding of the endocytic partner Eps15 to the mutants is very similar to the wild-type appendage (Fig. 3B,  lanes f and h compared with lane d) and distinct from the inhibitory consequence of a Y815A mutation (Fig. 3B, lane j) within the proximal sandwich site on the adjacent ␤2 appendage subdomain (see Fig. 2B) (37,62). By contrast, engagement of the platform site partner epsin 1 (37) is diminished weakly by the Y888E but not by the Y888F or Y815A mutations (Fig. 3B, lane f); some epsin 1 is still present in the supernatant fraction (Fig.  3B, lane e). The fact that the Y888F substitution has no appreciable effect on ␤2-platform binding indicates that the Tyr hydroxyl group is not essential for establishing or maintaining these interactions. Indeed, the crystal structures of either the ARH (37) or ␤-arrestin (62) (D/E) n X 1-2 FXX(F/L)XXXR peptide complexed with the ␤2 appendage reveal that Tyr-888 engages in aromatic stacking interactions without hydrogen bond donation through the hydroxyl group. Our interpretation of these findings is that the strict selection/retention of Tyr at this position over millions of years of evolution (with the exception of slime molds, where a Phe is instead present; Fig. 3C) is consis-tent with a primary (reversible) regulatory role for the hydroxyl group.
In addition, the full-length wild-type, Y888F-, and Y888Eencoding cDNAs transduced transiently into ␤2 Ϫ/Ϫ nullizygous iMEFs are equally expressed and equivalently incorporated into heterotetrameric AP-2 complexes at ϳ25 h postinfection ( Fig. 2A). Again, in each case, co-assembly of the ectopic ␤2-YFP chain reduces the amount of endogenous ␤1 subunit incorporated into the AP-2 population ( Fig. 2A, lanes h,  i, and p compared with lane d). After infection, the ␤2-YFP wild type represents on average 85.9% of the total affinity-purified AP-2-associated ␤ chain and 80.0 and 78.3% in the ␤2-YFP (Y888E) and ␤2-YFP (Y888F) mutants, respectively. Also, on the immunoblots, there is no evidence of extensive or major degradation products within the cell extracts from cells expressing the mutant forms of the ␤2-YFP protein. The three forms of ␤2-YFP each display random punctate distributions (69) on the ventral surface of infected iMEFs (Fig. 3D). These structures colocalize well with ligand-labeled surface transferrin receptors irrespective of whether ␤2 residue 888 is Tyr, Phe, or Glu, identifying the spots as plasma membrane clathrincoated structures. We conclude that all three ␤2-YFP polypeptides are similarly incorporated into functional AP-2 heterotetramers in ␤2 Ϫ/Ϫ iMEFs.
To investigate a selective effect of modification Tyr-888 on ␤2 appendage platform binding partners, we used a constitutively active form of ␤-arrestin 1, the so-called IV 3 AA mutant (25,64). In the closed basal state, a tract of the C-terminal ␤2 platform-binding (D/E) n X 1-2 FXX(F/L)XXXR sequence of the ␤-arrestins is bound intramolecularly to the globular core of the protein (37, 70 -72). By perturbing this intermolecular contact, the vicinal di-Ala substitution of Ile-386 and Val-387 shifts the equilibrium of ␤-arrestin 1 to the open, endocytically competent form (25,64,73) (see Fig. 7), which now binds robustly in pull-down assays. Compared with lysates from untransfected cells (Fig. 4A, lane f), a ϳ110 kDa band is visible on Coomassie Blue-stained gels in the pellet fractions from incubations of the GST-␤2 appendage with lysates of ectopic tdRFP-␤-arrestin 1 (IV 3 AA)-producing cells (Fig. 4A, lane h). The identity of this band is substantiated with antibodies against both ␤-arrestin 1 and the DsRed RFP, and the distinctive stained band is not seen in parallel pellet fractions using immobilized control GST (Fig.  4A, lane d) and remains fully in the supernatant fraction (Fig.  4A, lane c). Nor is this immunoreactivity detected in lysates not transfected with the mutant ␤-arrestin 1 (Fig. 4A, lanes a, b, e, f,  i, and j). In the same experiment, the GST-␤2 appendage containing a Y888E substitution behaves quite differently; a dra-  -d), the wild-type GST-␤2 appendage (lanes e-h), or the GST-␤2 (Y888E) mutant appendage (lanes i-l) immobilized on glutathione-Sepharose were incubated with precleared lysates prepared from either control ␤2 Ϫ/Ϫ iMEFs or from iMEFs ectopically expressing tdRFP-␤-arrestin 1 (IV 3 AA). Following incubation with the lysates at 4°C for 2 h, the Sepharose beads were recovered by centrifugation and washed. Aliquots corresponding to ϳ1.5% of each supernatant (S; lanes a, c,  e, g, i, and k) and 12.5% of each pellet (P; lanes b, d, f, h, j, and l) fraction were resolved by SDS-PAGE and either stained with Coomassie Blue (top) or transferred to nitrocellulose. Replicate blots were probed with anti-␤-arrestin polyclonal antibody or an anti-DsRed mAb. The position of the molecular mass standards (in kDa) on the Coomassie Blue-stained gel is shown at the left, and only the relevant portion of each blot is presented. A nonspecific band on immunoblots probed with the anti-␤-arrestin antibody is indicated (arrowheads) and is not detected with anti-DsRed antibody; the apparent mass of this band (ϳ95 kDa) rules out the possibility that it represents the endogenous cytosolic ␤-arrestin (ϳ50 kDa), which also binds the ␤2 appendage very poorly (37). B, aliquots of ϳ100 g of GST (lanes a and b), the wild-type GST-␤2 appendage (lanes c and d), or GST-␤2 (Y888E) (lanes e and f) or GST-␤2 (Y888F) (lanes g and h) mutant appendages immobilized on glutathione-Sepharose were incubated with precleared lysates obtained from ␤2 ؊/؊ iMEFs transfected with the tdRFP-␤-arrestin 1 (IV 3 AA) mutant. Gels and blots were prepared as in A and immunoblotted with antibodies directed against ␤-arrestin 1, DsRed, Eps15, or epsin 1. Note that the tdRFP-␤-arrestin 1 (IV 3 AA) mutant protein remains almost completely in the supernatant fraction after incubation with the Y888E ␤2 appendage mutant. matic decrease in tdRFP-␤-arrestin 1 binding is seen, and no equivalent stained band is evident in the pellet (Fig. 4, A (lane l) and B (lane f)). The majority of the RFP-tagged ␤-arrestin is recovered in the supernatant fraction (Fig. 4, A (lane k) and B (lane e)). However, with the Y888F switch, the particular Coomassie-stained 110-kDa ␤-arrestin band is present in the pellet, and the GST fusion protein affinity-isolates immunoreactive tdRFP-␤-arrestin 1 (Fig. 4B, lane h). Across phyla, only slime molds display a Phe at the Tyr-888 equivalent position (Fig. 3B), but these results restate that for platform binding, Tyr and Phe are functionally interchangeable at this location. Additional immunoblots confirm that interactions of Eps15 and epsin 1 with the wild-type, Y888E, and Y888F GST-␤2 proteins are similar (Fig. 4B, lanes d, f, and h). Our interpretation of these results is that the Y888E mutant is selectively defective in interactions with (D/E) n X 1-2 FXX(F/L)XXXR-containing binding partners. The disparity between ␤-arrestin and epsin 1 in engaging the ␤2 appendage Y888E mutation stems from the fact that although epsin 1 contains a (D/E) n X 1-2 FXX(F/ L)XXXR sequence, the K D for the ␤2 platform surface is at least 10-fold weaker than ARH or ␤-arrestins (37); therefore, epsin 1 utilizes a molecularly distinct manner to productively engage the ␤2 appendage.
Last, infecting the ␤2 Ϫ/Ϫ iMEFs with adenovirus encoding a ␤2-YFP subunit reconstitutes cellular AP-2 and the recruitment of RFP-tagged ␤-arrestin 1 to surface clathrin puncta in the absence of any acute G protein-coupled receptor activation. The fluorescently-tagged ␤-arrestin 1 (IV 3 AA) reporter is distributed together with the random array of ␤2-YFP-containing clathrin puncta (Fig. 5A). Parallel infections with a ␤2-YFP (Y888E) mutant, while still being incorporated into AP-2 and targeted to bud sites, result in strongly diminished RFP-␤-arrestin 1 deposition at AP-2-positive coated structures and hence loss of close proximity of the clustered signals (Fig. 5B).
The deposition and concentration of mutated ␤-arrestin 1 at ␤2-YFP puncta are better visualized with TIR-FM (25) (Figs. 5D and 6). Again, the extent of tdRFP-␤-arrestin 1 (IV 3 AA) colocalizing with the Y888E mutant ␤2-YFP is clearly reduced, but not completely abolished, relative to the transduced wild-type ␤2 chain (Figs. 5C and 6). Following a 24-h transduction with ␤2-YFP-encoding adenovirus, the cellular AP-2 pool in the ␤2 Ϫ/Ϫ iMEFs still contains some endogenous ␤1 subunit (Fig.  2). The reason for incomplete loss of tdRFP-␤-arrestin 1 (IV 3 AA) clustering at ␤2-YFP-positive patches, therefore, may be due to imperfect reconstitution of AP-2 with the Y888E mutant, because native AP-2 has a biological half-life of ϳ35 h (49), or due to the capability of the exposed clathrin box motif to still engage assembled clathrin in the absence of ␤2 platform binding, or both. Parallel TIR-FM experiments with a tdRFP-␤arrestin 1 (IV 3 AA ϩ LIEL 3 AAEA) compound mutant show that disrupting the LIELD clathrin box motif diminishes further the residual coincidence between the tagged ␤-arrestin 1 and the ␤2-YFP (Y888E) surface puncta (Figs. 5E and 6). However, corroborating the biochemical data, AP-2 reconstituted with the Y888F mutant operates analogously to the wild-type ␤2-YFP (Fig. 5F), with tdRFP-␤-arrestin 1 (IV 3 AA) clustered at ␤2-YFP-positive structures at the cell surface. This assessment is supported by quantitative line scan analysis of the TIR-FM data (Fig. 6). A ␤-arrestin 1 variant (IV 3 AA), which displays constitutive physical interaction with AP-2 through the wild-type ␤2 platform contact site, thus interacts only poorly with plasma membrane-tethered AP-2 when a single structural modification, the phosphomimicking Y888E, is present in the ␤2 chain.
Perturbation of the ␤2⅐(D/E) n X 1-2 FXX(F/L)XXXR Motif Interaction Interface-Although strongly suggestive, a legitimate concern with the studies described above is that although Ser/Thr 3 Asp/Glu substitutions are well accepted functional phosphomimetics (21,74,75), the Y888E mutation loses the aromatic character of the side chain. Active phosphomimetic Tyr 3 Glu mutations are reported in the literature (76 -87), but this is clearly a suboptimal substitution in certain instances (82, 88 -91). To further demonstrate that Tyr(P)-888 modification interferes with motif accommodation, we also tested whether complex formation can be disrupted by artificially altering the reciprocal binding partner. The rationale is to probe whether the physical bulk of an additional aromatic ring or the hydration shell of an attached hydroxyl or carboxyl group impedes the close apposition of interfacial hydrophobic surfaces (Fig. 7, A and C).
Using immobilized GST-␤-arrestin 1 CT (residues 331-418) in pull-down assays with brain cytosol, both F391Y and F391W substitutions at the F/L anchor site interfere with the binding of soluble AP-2 without similarly affecting clathrin association (Fig. 7B, lanes f and h compared with lane d), which is dictated by the separate adjacent sequence (92,93). Neither clathrin nor AP-2 interacts with GST alone (Fig. 7B, lane b), and these soluble coat components remain completely in the supernatant (Fig. 7B, lane a).
Analogous but stronger inhibitory effects are seen with L262Y or L262E mutations in GST-ARH C1 (residues 180 -308) (Fig. 7D, lanes h and j), whereas a L262F change does not perturb AP-2 binding substantially, as expected (Fig. 7D, lane f). Cytosolic AP-2 remains fully within the supernatant fraction in pull-down assays with GST-ARH C1 (L262Y) or GST-ARH C1 (L262E) (Fig. 7D, lanes g and i), indicating that the lack of AP-2 recovery in the pellets is not due to the substitution altering the off rate and causing the adaptor to be lost during washing. The different substitutions to ARH Leu-262 have comparably minor effects on clathrin binding through the independent interaction motif (35,94). Although the C-terminal regions of both the ␤-arrestins and ARH each contain a single (D/E) n X 1-2 FXX(F/ L)XXXR interaction motif and a clathrin-binding motif, we have previously documented that ARH engages AP-2 and clathrin more avidly in pull-down assays (38) (Fig. 7, B and D). This may be related to the relative spacing between the two interaction motifs, which is much shorter in ␤-arrestins (Fig. 7A), or perhaps due to inhibitory residues in ␤-arrestin 1 (72). Nevertheless, the impact of the ␤-arrestin 1 F391Y and ARH L262Y mutations is significant; introducing only a para-hydroxyl group has an unmistakable inhibitory effect on productive AP-2 binding. Most important, the highly disruptive ARH L262E substitution does not replace the aliphatic character of the natural leucine at this position, unlike in the case of the ␤2 Y888E switch. We conclude that engineered bulky or polar alterations to the ␤2 platform interaction interface can directly obstruct proper CLASP engagement. The electrostatic perturbation of a new dianionic charge, in addition to the physical bulk of the added phosphate group on Tyr-888 (Fig. 3A), will function similarly in vivo.

DISCUSSION
At a rudimentary level, reversible Tyr phosphorylation refashions the local binding features of protein interaction partners. Although this often operates in a positive manner, like the engagement of Tyr(P) motifs by Src homology 2, phosphotyrosine binding (and structurally related FERM), and C2 domains (95)(96)(97)(98), obstruction of protein-protein contacts is also known to accompany Tyr(P) modification (11,12,99,100). Parsing the phosphoproteome (19), we noticed that a functionally critical, solvent-exposed Tyr side chain upon the ␤2 subunit platform interface is repeatedly catalogued in the database. The vast majority of Tyr(P)-888 is reported from leukocyte cell extracts. Tantalizingly, the LDL receptor, which in leukocytes depends on ARH (and hence on the AP-2 ␤2 appendage) for packaging into clathrin coats, is not concentrated at surface AP-2-positive clathrin puncta in lymphocytes (101), quite dissimilar to fibroblasts (102) and other cultured cells (55,103). Our structure-guided argument is that the positioning, charge, and large ionic hydration shell of the affixed phosphate will change the chemical properties of the ␤1/2 platform interaction surface, interfering sterically with successful docking and engagement of the (D/E) n X 1-2 FXX(F/L)XXXR interaction motif (Fig. 3A). Our results attest that indeed there is a spatially confined environment upon the ␤2 platform for productive ␣-helical peptide encounters. It seems a remote possibility that the Tyr(P)-888 modification serves as a signal for the binding of a Tyr(P)-selective modular fold to AP-2. First, the phosphoty- The background fluorescence intensities for the red and green channels (colored brick red and forest green, respectively) are shown for each image analyzed. Note that the amplitudes of ␤2-YFP intensity are similar in the four reconstituted iMEF populations, supporting the conclusion that all three products of the YFP-tagged ␤2 transgene are incorporated into functional AP-2 that is deposited at maturing clathrin assemblies at the cell surface. However, unlike the wild-type and Y888F ␤2-YFP (light blue bars), the Y888E mutant does not have strongly colocalized peaks of red tdRFP-␤arrestin 1 fluorescence, and, in general, the maximal peak amplitude above cytosolic baseline fluorescence for this mutant is considerably smaller than that for wild-type or Y888F ␤2-YFP-expressing cells.
rosine binding domain typically recognizes Tyr(P) in the context of a conserved sequence tract ((F/Y)XNPXpY) and a structurally defined orientation (a tight ␤-turn) (104), which is unrelated to the flanking ␤2 primary sequence and impossible for Tyr-888 to assume in the context of a residue projecting off of a laterally hydrogen-bonded antiparallel ␤-strand within the ␤2 appendage platform subdomain (66). Second, similar structural considerations indicate that Tyr(P)-888 is too buried and inaccessible and lacks flexible adjacent residues to engage an Src homology 2 domain in the accepted fashion (97). But even AP-2 ␤2 Appendage Phosphorylation and Cargo Loading JUNE 20, 2014 • VOLUME 289 • NUMBER 25 were this to occur, the functional consequence would be to compete with the engagement of ARH or ␤-arrestins through an overlapping site upon the ␤2 platform, so the Tyr(P) modification would still be molecularly translated into an inhibitory input on regular ␤2 platform interactions.
Besides operating at the cell surface for receptor internalization, both the ␤-arrestins and ARH have been localized to endosomal compartments under various conditions (105)(106)(107)(108)(109)(110)(111)(112). This is dissimilar to many other AP-2 appendage-binding CLASPs and accessory proteins, which cycle strictly between plasma membrane assemblages and a reserve cytosolic pool. In this regard, it is significant that the high degree of similarity between the AP-1 ␤1 and AP-2 ␤2 chains is also seen in common post-translational modifications. The positionally equivalent side chain to Tyr-888 in the ␤1 chain is Tyr-897, and Tyr(P)-897 is also represented in the phosphoproteome database (113). Actually, Tyr(P)-897 is currently the most frequently detected phosphorylation event on the ␤1 subunit (50 reports), like the Tyr(P)-888 in ␤2, and is present in numerous other cell types besides leukocytes. A phosphopeptide encompassing the Tyr-888 analogous residue in the D. melanogaster ␤-adaptin (Tyr-871) has also been detected in Kc167 cells (114). Thus, because Tyr-888 is structurally and functionally conserved in the AP-1 ␤1 appendage platform subdomain (66), phosphorylation probably regulates engagement of this surface as well. This reversible modification may underlie the variable assembly of ARH and ␤-arrestins on AP-1-positive endosomal structures (112).
It is instructive to compare the representation of Tyr(P)-888 with the frequency of other phosphosites currently mapped within the ␤2 chain (19). In the basal AP-2 heterotetramer conformation, the first occurring Tyr at the ␤2 subunit N terminus (Tyr-6) along with adjacent Phe-7 occlude the binding site for the (D/E)XXXL(L/I/M)-type dileucine sorting signal on the ␣⅐2 hemicomplex (115). Phosphorylation of Tyr-6 is linked to dileucine signal recognition (116) and may dislodge the N-terminal region of ␤2 from the ␣⅐2 molecular surface (115) when AP-2 attaches to the inner leaflet of the plasma membrane to permit transmembrane cargo capture. However, Tyr(P)-6 is only reported once in low throughput (116) and currently only once in high throughput experiments and is recorded a single time in the ␤1 chain (19). On the C-terminal ␤2 appendage, Tyr-737 within the sandwich subdomain is connected to the regulation of ␤-arrestin interactions through phosphorylation by the non-receptor Tyr kinase Src (58,117,118). Curiously, Tyr(P)-737 is remote from the platform site utilized by ␤-arrestins (and ARH) (see Fig. 2B) and has been observed nine times by high throughput detection, compared with 53 instances for Tyr(P)-888.
The widespread occurrence of phosphoregulation through eukaryotic evolution is argued to be due, in part, to the fact that the fundamental chemical properties of phosphoamino acids are unlike any natural side chains within proteins (119). Even so, there is evidence that phosphosites can evolve to replace acidic residues by Ser/Thr/Tyr substitution to impose conditional regulation (120). In the Raf-1 Ser/Thr kinase, residues Tyr 340 -Tyr 341 within the catalytic domain require phosphorylation for kinase activation (76,77). These residues correspond to vicinal Asp 448 -Asp 449 in the enzymatically more active B-Raf (76), so Asp/Glu can functionally substitute for Tyr(P) under at least some conditions, and in invertebrate Raf kinases, this vicinal pair is composed of Asp and/or Glu residues (120). Furthermore, there is a computational argument that a Glu-for-Tyr(P) substitution has occurred in the CMGC kinase group (120). Taken together, it appears that Asp/Glu can mimic Tyr(P) in certain biological contexts. Still, because of our lingering reservation with the Y888E substitution on the molecular surface of the ␤2 platform subdomain, we mutated the core F/L position side chain in ARH and ␤-arrestin 1 (D/E) n X 1-2 FXX(F/L)XXXR motifs with similar inhibitory outcomes.
The identity of the protein kinase(s) that reversibly phosphorylates Tyr-888 on the ␤2 appendage and Tyr-897 on the ␤1 chain remains to be established. Because both AP-1 and AP-2 are modified at this position, it is possible that a non-receptor Tyr kinase catalyzes this post-translational modification as opposed to a receptor Tyr kinase activated at the cell surface. An interesting aspect of the recognition of this particular substrate is that the acceptor side chain is not located within a flexible loop or a largely unstructured polypeptide region housing an identifiable phosphorylation motif (121,122). Instead, Tyr-888 projects out from strand ␤11 of the five-stranded antiparallel ␤-sheet that forms the base of the ␤2 platform subdomain (37,62,66) (Fig. 2B) and displays a computed 6.87% of the surface area as accessible (similar to the 4.95% surface accessibility for Tyr-737) (123). This suggests that conformational recognition as opposed to a linear peptide-based identification motif depending on specificity-determining flanking residues is necessary for the Tyr(P)-888 modification of this FIGURE 7. Complementary mutagenesis of the ␤2-platform binding partner interface. A, primary sequence alignment of the ␤-arrestin 1 and ARH (D/E) n X 1-2 FXX(F/L)XXXR-containing regions. The location and selected substitutions for residues mutated are indicated, as are LIELD clathrin box 1 (blue highlight) and the IV residues (red) mutated to Ala in the IV 3 AA mutation of ␤-arrestin 1. In ARH, the functionally analogous LLDLE clathrin box motif is positioned 35 residues proximal to the ␤2 platform binding sequence shown. B, aliquots of ϳ100 g of GST (lanes a and b), the wild-type GST-␤-arrestin 1 CT (residues 331-418) (lanes c and d), or the GST-␤-arrestin 1 CT (F391Y) (lanes e and f) or GST-␤-arrestin 1 CT (F391W) (lanes g and h) single point mutants each immobilized on glutathione-Sepharose were incubated with precleared rat brain cytosol. After incubation at 4°C for 2 h, the Sepharose beads were recovered by centrifugation and washed. Aliquots corresponding to ϳ1.5% of each supernatant (S; lanes a, c, e, and g) and ϳ12.5% of each pellet (P; lanes b, d, f, and h) were resolved by SDS-PAGE and either stained with Coomassie Blue (top) or transferred to nitrocellulose. Replicate blots were probed with a mixture of anti-clathrin HC mAb TD.1 and affinity-purified anti-␤1/␤2 antibody GD/1 or anti-AP-2 2 subunit antibodies. The position of the molecular mass standards (in kDa) is shown at the left, and only the relevant portion of each blot is presented. C, combined ribbon and space-filling surface representation of the ␤2 appendage platform subdomain bound to the ARH DEAFSRLAQSRT interaction peptide (Protein Data Bank code 2G30) (37) shown in a stick representation with oxygen colored red and nitrogen blue. Compared with the native structure, models of allowed rotomers of the L262F, L262Y, and L262E substitutions (shown in a purple stick representation) are illustrated. D, aliquots of ϳ100 g of GST (lanes a and b); wild-type GST-ARH C1 (residues 180 -308) (lanes c and d); or GST-ARH C1 (L262F) (lanes e and f), GST-ARH C1 (L262Y) (lanes g and h), or GST-ARH C1 (L262E) (lanes i and j) single point mutants each immobilized on glutathione-Sepharose were incubated with precleared rat brain cytosol. After incubation at 4°C for 2 h, the Sepharose beads were recovered by centrifugation and washed. Aliquots corresponding to ϳ1.5% of each supernatant (S) and ϳ12.5% of each pellet (P) were resolved by SDS-PAGE and analyzed as in B with antibodies against the clathrin HC, ␤1/␤2, and 2 adaptor subunits.

AP-2 ␤2 Appendage Phosphorylation and Cargo
Loading solvent-exposed side chain. The relevant protein Tyr phosphatase that reverses this modification is also not yet known.
Finally, an attractive emerging concept is the existence of compositionally distinct clathrin-coated structures that can relay distinct populations of cargo from the surface into the cell interior in parallel (124 -130). On one level, this idea seems generally improbable because the majority of inchoate clathrincoated structures at the surface show a similar sequence of pioneer protein arrival (131), and at steady state, clathrin assembly zones are populated with mostly the same adaptors, CLASPs, and accessory factors (55,(131)(132)(133)(134). Our results illustrate that specialized or cargo-biased clathrin-coated vesicles are not necessarily characterized by different sets of co-assembled adaptors and CLASPs but could possibly arise through different local, switchable binding capabilities of these cargo-sorting components at individual clathrin internalization zones. The ultimate effectiveness and precision of this type of toggle mechanism will depend upon the rate and extent of post-translational modification and the half-life of the covalent adduct and also upon the degree of functional redundancy in sorting. In several cell types, Tyr(P)-888 will be more effective at uncoupling G protein-coupled receptor internalization than LDL receptor family uptake because alternate CLASPs like Dab2 (55,103) and possibly Ced-6 (135) can also recognize the (F/Y)XNPX(Y/F) sorting signal decoded by ARH, and these proteins do not engage the AP-2 ␤2 subunit platform surface.