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

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Abstract
Many plasma membrane proteins destined for endocytosis are concentrated into clathrin‐coated pits through the recognition of a tyrosine‐based motif in their cytosolic domains by an adaptor (AP‐2) complex. The μ2 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 motif. AP‐2 is the first protein known (in any context) to interact with phosphatidylinositol 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.