Multiple C-terminal motifs of the 46-kDa mannose 6-phosphate receptor tail contribute to efficient binding of medium chains of AP-2 and AP-3.
The interaction of adaptor protein (AP) complexes with signal structures in the cytoplasmic domains of membrane proteins is required for intracellular sorting. Tyrosine- or dileucine-based motifs have been reported to bind to medium chain subunits (mu) of AP-1, AP-2, or AP-3. In the present study, we have examined the interaction of the entire 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR46-CT) containing tyrosine- as well as dileucine-based motifs with mu2 and mu3A chains using the yeast two-hybrid system. Both mu2 and mu3A bind specifically to the MPR46-CT. In contrast, mu3A fails to bind to the cytoplasmic domain of the 300-kDa mannose 6-phosphate receptor. Mutational analysis of the MPR46-CT revealed that the tyrosine-based motif and distal sequences rich in acidic amino acid residues are sufficient for effective binding to mu2. However, the dileucine motif was found to be one part of a consecutive complex C-terminal structure comprising tyrosine and dileucine motifs as well as clusters of acidic residues necessary for efficient binding of mu3A. Alanine substitution of 2 or 4 acidic amino acid residues of this cluster reduces the binding to mu3A much more than to mu2. The data suggest that the MPR46 is capable of interacting with different AP complexes using multiple partially overlapping sorting signals, which might depend on posttranslational modifications or subcellular localization of the receptor.
Storch
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S
Department of Biochemistry, Children's Hospital, University of Hamburg, D-20246 Hamburg, Germany.
Braulke
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T
D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033965:Adaptor Protein Complex alpha Subunits; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D034041:Monomeric Clathrin Assembly Proteins; D011485:Protein Binding; D017527:Receptor, IGF Type 2 / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033965:Adaptor Protein Complex alpha Subunits; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D017931:DNA Primers; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D034041:Monomeric Clathrin Assembly Proteins; D017527:Receptor, IGF Type 2; D014157:Transcription Factors; C499065:adaptor protein complex 1, mu 2 subunit; C438886:adaptor protein complex 2, mu 2 subunit; C439023:clathrin assembly protein AP180; C060759:enhancer-binding protein AP-3