A FUNCTIONAL PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE PHOSPHOINOSITIDE BINDING DOMAIN IN THE CLATHRIN ADAPTER AP-2 ALPHA-SUBUNIT - IMPLICATIONS FOR THE ENDOCYTIC PATHWAY/

Clathrin-coated pits are sites of concentration of ligand-bound signal ing receptors. Several such receptors are known to recruit, bind, and activate the heterodimeric phosphatidylinositol-3-kinase, resulting in the generation of phosphatidylinositol 3,4,5-trisphosphate. We report here that dioctanoyl-phosphatidylinositol-3,4,5-P-3 binds specificall y and saturably to soluble AP-2 and with greater affinity to AP-2 with in assembled coat structures. Soluble D-myo-inositol hexakisphosphate shows converse behavior. Binding to bovine brain clathrin-coated vesic les is evident, only after detergent extraction. These observations an d evidence for recognition of the diacylglyceryl backbone as well as t he inositol phosphate headgroup are consistent with AP-2 interaction w ith membrane phosphoinositides in coated vesicles and with soluble ino sitol phosphates in cytoplasm. A discrete binding domain is identified near the N terminus of the AP-2 alpha subunit, and an expressed fusio n protein containing this sequence exhibits specific, high affinity bi nding that is virtually identical to the parent protein. This region o f the AP-2 alpha sequence also shows the greatest conservation between a Caenorhabditis elegans homolog and mammalian alpha, consistent with a function in recognition of an evolutionarily unchanging low molecul ar weight ligand. Binding of phosphatidylinositol 3,4,5-trisphosphate to AP-2 inhibits the protein's clathrin binding and assembly activitie s. These findings are discussed in the context of the potential roles of phosphoinositides and AP-2 in the internalization and trafficking o f cell surface receptors.