Binding of AP2 to sorting signals is modulated by AP2 phosphorylation.
The two clathrin-associated adaptor complexes AP1 and AP2 are known to participate in the formation of clathrin-coated vesicles at the trans-Golgi network and at the plasma membrane. During this process adaptors are involved in the sequestration of vesicle cargo by binding to the sorting signals within the cytoplasmic domains of the cargo proteins and in the recruitment of the clathrin coat. After budding of the clathrin-coated vesicles, the clathrin and adaptors dissociate from the vesicles. Here we show that in vitro binding of AP2 to sorting signals, which is one of the initial steps in receptor-mediated endocytosis, is modulated by adaptor phosphorylation. AP2 was phosphorylated by incubating purified AP2 in the presence of ATP and dephosphorylated by incubation with alkaline phosphatase. Affinity for tyrosine-, leucine-based and noncanonical sorting motifs was 15-33 times higher for phosphorylated than for dephosphorylated AP2. Also the binding of AP2 to membranes was regulated by adaptor phosphorylation/dephosphorylation and was about 8-fold higher for phosphorylated than for dephosphorylated AP2. Moreover, AP2 isolated from cytosol is higher phosphorylated than membrane-extracted and exhibits a 5-fold higher binding affinity than AP2 extracted from membranes. Taken together these data point to a cycle of phosphorylation/dephosphorylation as a mechanism for regulating the reversible association of AP2 with membranes and sorting signals during the process of receptor-mediated endocytosis.
Fingerhut
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Institute for Biochemistry II, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany.
von Figura
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Honing
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D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D018699:Coated Vesicles / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D021382:Protein Sorting Signals; D021381:Protein Transport; D021601:trans-Golgi Network / Q000378:metabolism
D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D008565:Membrane Proteins; D021382:Protein Sorting Signals