Signal-binding Specificity of the μ4 Subunit of the Adaptor Protein Complex AP-4

Abstract

The medium (μ) chains of the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of tyrosine-based (Y<i>XX</i>Ø) sorting signals found within the cytoplasmic domains of integral membrane proteins. Here, we describe the signal-binding specificity and affinity of the medium subunit μ4 of the recently described adaptor protein complex AP-4. To elucidate the determinants of specificity, we screened a two-hybrid combinatorial peptide library using μ4 as a selector protein. Statistical analyses of the results revealed that μ4 prefers aspartic acid at position Y+1, proline or arginine at Y+2, and phenylalanine at Y−1 and Y+3 (Ø). In addition, we examined the interaction of μ4 with naturally occurring Y<i>XX</i>Ø signals by both two-hybrid and <i>in vitro</i> binding analyses. These experiments showed that μ4 recognized the tyrosine signal from the human lysosomal protein LAMP-2, HTGYEQF. Using surface plasmon resonance measurements, we determined the apparent dissociation constant for the μ4-Y<i>XX</i>Ø interaction to be in the micromolar range. To gain insight into a possible role of AP-4 in intracellular trafficking, we constructed a Tac chimera bearing a μ4-specific Y<i>XX</i>Ø signal. This chimera was targeted to the endosomal-lysosomal system without being internalized from the plasma membrane.