Signal-binding specificity of the mu4 subunit of the adaptor protein complex AP-4.
The medium (mu) chains of the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of tyrosine-based (YXXphi) sorting signals found within the cytoplasmic domains of integral membrane proteins. Here, we describe the signal-binding specificity and affinity of the medium subunit mu4 of the recently described adaptor protein complex AP-4. To elucidate the determinants of specificity, we screened a two-hybrid combinatorial peptide library using mu4 as a selector protein. Statistical analyses of the results revealed that mu4 prefers aspartic acid at position Y+1, proline or arginine at Y+2, and phenylalanine at Y-1 and Y+3 (phi). In addition, we examined the interaction of mu4 with naturally occurring YXXphi signals by both two-hybrid and in vitro binding analyses. These experiments showed that mu4 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 mu4-YXXphi 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 mu4-specific YXXphi signal. This chimera was targeted to the endosomal-lysosomal system without being internalized from the plasma membrane.
Aguilar
R C
RC
Cell Biology and Metabolism Branch and the Laboratory of Molecular Genetics, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Boehm
M
M
Gorshkova
I
I
Crouch
R J
RJ
Tomita
K
K
Saito
T
T
Ohno
H
H
Bonifacino
J S
JS
D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D006367:HeLa Cells; D006801:Humans; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D034041:Monomeric Clathrin Assembly Proteins; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction; D014162:Transfection
D033942:Adaptor Proteins, Vesicular Transport; D015703:Antigens, CD; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D019151:Peptide Library; D010750:Phosphoproteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C439023:clathrin assembly protein AP180