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.
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 YXXPhi, where Phi represents a bulky hydrophobic residue. This motif interacts with the medium chain (mu) subunits of adaptor complexes that link the cytosolic domains of integral membrane proteins to the clathrin coat involved in vesicle formation. The YXXPhi 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 YXXPhi motifs and the mu2 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 mu2 provides a structural explanation for A23 being able to inhibit the interaction between YXXPhi motifs and mu2. 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 YXXPhi motif/mu2 interaction, did not inhibit endocytosis. These data are consistent with A23 inhibition of the YXXPhi motif/mu2 interaction in intact cells and with the possibility that different tyrphostins may be used to inhibit specific membrane trafficking events in eukaryotic cells.
Banbury
David N
DN
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
Oakley
Jacqueline D
JD
Sessions
Richard B
RB
Banting
George
G
D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D003911:Dextrans / Q000493:pharmacokinetics; D004705:Endocytosis / Q000187:drug effects*; D011992:Endosomes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008958:Models, Molecular; D009930:Organic Chemicals; D017434:Protein Structure, Tertiary; D011990:Receptors, Transferrin / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D020032:Tyrphostins / Q000737:chemistry / Q000494:pharmacology*; D021601:trans-Golgi Network / Q000378:metabolism
D033962:Adaptor Protein Complex 2; C417664:Alexa594; D003911:Dextrans; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D009930:Organic Chemicals; D011990:Receptors, Transferrin; D020032:Tyrphostins; D014443:Tyrosine; C060641:tyrphostin A23