Signal-binding specificity of the mu 4 subunit of the adaptor protein complex AP-4

The medium (p) chains of the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of tyrosine-based (YXX phi) sorting signal s found within the cytoplasmic domains of integral membrane proteins. Here, we describe the signal-binding specificity and affinity of the medium subu nit mu4 of the recently described adaptor protein complex AP-4. To elucidat e the determinants of specificity, we screened a two-hybrid combinatorial p eptide library using mu4 as a selector protein. Statistical analyses of the results revealed that mu4 prefers aspartic acid at position Y+1, proline o r arginine at Y+2, and phenylalanine at Y-1 and Y+3 (phi). In addition, we examined the interaction of mu4 with naturally occurring YXX phi signals by both two-hybrid and in vitro binding analyses. These experiments showed th at mu4 recognized the tyrosine signal from the human lysosomal protein LAMP -2, HTGYEQF. Using surface plasmon resonance measurements, we determined th e apparent dissociation constant for the mu4-YXX phi interaction to be in t he micromolar range. To gain insight into a possible role of AP-4 in intrac ellular trafficking, we constructed a Tac chimera bearing a mu4-specific YX X phi signal. This chimera was targeted to the endosomal-lysosomal system w ithout being internalized from the plasma membrane.