Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes

Many plasma membrane proteins destined for endocytosis are concentrated into clathrin-coated pits through the recognition of a tyrosine-based moth in their cytosolic domains by an adaptor (AP-2) complex. The mu 2 subunit of isolated AP-2 complexes binds specifically, but rather weakly, to proteins bearing the tyrosine-based signal. We now demonstrate, using peptides with a photoreactive probe, that this binding is strengthened significantly when the AP-2 complex is present in clathrin coats, indicating that there is cooperativity between receptor-AP-2 interactions and coat formation. Phosphoinositides with a phosphate at the D-3 position of the inositol ring, but not other isomers, also increase the affinity of the AP-2 complex for the tyrosine-based moth. AP-2 is the first protein known (in any context) to interact with phosphatidyl-inositol 3-phosphate. Our findings indicate that receptor recruitment can be coupled to clathrin coat assembly and suggest a mechanism for regulation of membrane traffic by lipid products of phosphoinositide 3-kinases.