Binding of AP2 to sorting signals is modulated by AP2 phosphorylation

The two clathrin-associated adaptor complexes API and AP2 are known to part icipate in the formation of clathrin-coated vesicles at the trans-Golgi net work and at the plasma membrane. During this process adaptors are involved in the sequestration of vesicle cargo by binding to the sorting signals wit hin the cytoplasmic domains of the cargo proteins and in the recruitment of the clathrin coat. After budding of the clathrin-coated vesicles, the clat hrin 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 rec eptor-mediated endocytosis, is modulated by adaptor phosphorylation. AP2 wa s phosphorylated by incubating purified AP2 in the presence of ATP and deph osphorylated 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 m embranes was regulated by adaptor phosphorylation/dephosphorylation and was about 8-fold higher for phosphorylated than for dephosphorylated AP2. More over, AP2 isolated from cytosol is higher phosphorylated than membrane-extr acted and exhibits a B-fold higher binding affinity than AP2 extracted from membranes. Taken together these data point to a cycle of phosphorylation/d ephosphorylation as a mechanism for regulating the reversible association o f AP2 with membranes and sorting signals during the process of recptor-medi ated endocytosis.