Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (mu 1-mu 4) that recognizes YXXO-sequences (O is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in m subunits mediates recognition of YXXO-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXO-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the mu 4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of mu 4 on the recognition of the non-canonical YXXO-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXO-signal. This structure showed no significant difference compared to that of wild-type mu 4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered mu 4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of mu 4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXO-signal of APP is limited to the non-canonical site of mu 4.