Sequence requirements for the recognition of tyrosine-based endocytic signals by clathrin AP-2 complexes

We recently determined that fusion proteins containing tyrosine-based endocytic signals bind to the mu 2 subunit of AP-2, the complex that drives clathrin coat formation and mediates endocytosis from the plasma membrane, Here we analyze the selectivity of peptide recognition by mu 2 and by AP-2 using combinatorial selection methods and surface plasmon resonance, Both mu 2 and AP-2 are shown to interact with various sequences of the form tyrosine-polar-polar-hydrophobic (YppO) found on receptors that follow the clathrin pathway. The optimal sequence for interaction with mu 2 and with AP-2 has tyrosine as an anchor and prefers arginine at position Y+2 and leucine at position Y+3. In contrast, no preferred sequence is detected surrounding the YppO signal, indicating that recognition of the YppO endocytic signal does not require a prefolded structure. We conclude that sorting into the endocytic pathway is governed by a surprisingly simple interaction between the mu 2 chain and a tyrosine-containing tetrapeptide sequence.