Tyrphostin A23 Inhibits Internalization of the Transferrin Receptor by Perturbing the Interaction between Tyrosine Motifs and the Medium Chain Subunit of the AP-2 Adaptor Complex

Abstract

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs within the cytosolic domains of integral membrane proteins. Many such motifs conform to the consensus YXX⌽, where ⌽ represents a bulky hydrophobic residue. This motif interacts with the medium chain ( ) subunits of adaptor complexes that link the cytosolic domains of integral membrane proteins to the clathrin coat involved in vesicle formation. The YXX⌽ motif is similar to motifs in which the tyrosine residue is phosphorylated by tyrosine kinases. Tyrphostins (structural analogs of tyrosine) are inhibitors of tyrosine kinases and function by binding to the active sites of the enzymes. We previously showed that, in vitro and in yeast two-hybrid interaction assays, some tyrphostins can inhibit the interaction between YXX⌽ motifs and the 2 subunit of the AP-2 adaptor complex (Crump, C., Williams, J. L., Stephens, D. J., and Banting, G. (1998) J. Biol. Chem. 273, 28073-28077). A23 is such a tyrphostin. We now show that molecular modeling of tyrphostin A23 into the tyrosinebinding pocket in 2 provides a structural explanation for A23 being able to inhibit the interaction between YXX⌽ motifs and 2. Furthermore, we show that A23 inhibited the internalization of 125 I-transferrin in Heb7a cells without having any discernible effect on the morphology of compartments of the endocytic pathway. Control tyrphostins, active as inhibitors of tyrosine kinase activity, but incapable of inhibiting the YXX⌽ motif/ 2 interaction, did not inhibit endocytosis. These data are consistent with A23 inhibition of the YXX⌽ motif/ 2 interaction in intact cells and with the possibility that different tyrphostins may be used to inhibit specific membrane trafficking events in eukaryotic cells.The trafficking and intracellular targeting of many transmembrane proteins depend on motifs found within their cytosolic domains. One such motif contains a critical tyrosine residue within the sequence YXX⌽, where ⌽ represents a bulky hydrophobic residue (1, 2). Tyrosine-based motifs conforming to this consensus sequence can interact directly with the medium chain ( ) subunits of heterotetrameric adaptor complexes involved in several different intracellular trafficking pathways. The AP-2 adaptor complex facilitates incorporation of transmembrane proteins containing YXX⌽ motifs into clathrincoated vesicles at the cell surface (1, 3-9), whereas AP-1, AP-3, and AP-4 adaptor complexes are involved in other intracellular vesicular transport steps (reviewed in Refs. 10 -13). The subunits from all four adaptor complexes have been shown to interact with the YXX⌽ motif, with the precise sequence and context of this motif determining the specificity of the interaction (14, 15). Mutation of residues within and around the YXX⌽ motif affect interaction with subunits; but, in all cases, mutation of the tyrosine residue in the motif (e.g. to alanine) blocks interaction with the subunit (4, 6, 8). Structural studies on peptides containing these motifs have generated data that hav...

Hitching a Ride on Vesicles: Cauliflower Mosaic Virus Movement Protein Trafficking in the Endomembrane System

“…Based on this result, and on the evidence that the localization of MP to endosomes is hampered upon treatment with the inhibitor of endocytosis tyrphostin A23, we conclude that CaMV MP traffics in the endocytic pathway. In animal systems, tyrphostin A23 interferes specifically with the m2 recognition of Tyr signals (Banbury et al, 2003). The evidence that tyrphostin A23 suppresses the internalization of MP and that this is dependent on functional YXXF cargo signals suggests strongly that interaction with the m-subunit of a plant AP-2 complex is required for the internalization of this protein, lending support to the hypothesis invoking a direct link between endocytosis and the clathrin machinery in plants.…”

“…Tyrphostin A23 inhibits endocytosis by interfering with the m2 recognition of Tyr signals (Banbury et al, 2003;Ortiz-Zapater et al, 2006;Dhonukshe et al, 2007). In transfected protoplasts, treatment with tyrphostin A23 led to the reorganization of GFP-MP distribution, which concentrated almost exclusively at the cell surface and no longer in foci ( Fig.…”

Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization

“…S11 A and B). In addition, we found that treatment with tyrphostin (tyr) A23, a specific inhibitor of clathrin-dependent endocytosis (26), can reduce the internalization of AMT1;3 spots and resulted in AMT1;3-EGFP coalescing into larger particles with an increased spot size and fluorescence intensity both under N-sufficient conditions (Fig. 4 C, I, and J and Movie S4) and high-ammonium treatment ( mutant.…”

Functional and structural requirements for the internalization of distinct BCR‐ligand complexes

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