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 YXXΦ, where Φ represents a bulky hydrophobic residue. This motif interacts with the medium chain (μ) subunits of adaptor complexes that link the cytosolic domains of integral membrane proteins to the clathrin coat involved in vesicle formation. The YXXΦ 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 YXXΦ motifs and the μ2 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 tyrosine-binding pocket in μ2 provides a structural explanation for A23 being able to inhibit the interaction between YXXΦ motifs and μ2. Furthermore, we show that A23 inhibited the internalization of 125I-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 YXXΦ motif/μ2 interaction, did not inhibit endocytosis. These data are consistent with A23 inhibition of the YXXΦ motif/μ2 interaction in intact cells and with the possibility that different tyrphostins may be used to inhibit specific membrane trafficking events in eukaryotic cells.