A third specificity-determining site in μ2 adaptin for sequences upstream of YxxΦ sorting motifs

Internalization signals of the Yxx Phi type (Phi = bulky hydrophobic side chain) interact with the mu2 chain of AP-2 adaptors. Internalization activity is intolerant of non-conservative substitution of either the tyrosine or the Phi side chains, which bind to hydrophobic pockets in mu2 adaptin in a conformation described as 'a two pinned plug into a socket'. P-selectin, a type I transmembrane protein, contains the Yxx Phi -like sequence YGVF in its cytoplasmic domain, but substitution of either the tyrosine or phenylalanine with alanine in the full-length protein causes only small changes in the rate of endocytosis. It is shown here that the sequence YGVF contained within a peptide corresponding to the 17 COOH-terminal amino acids of P-selectin binds to mu2 adaptin in the same fashion previously seen for other Yxx Phi motifs. In addition, the P-selectin peptide binds to a third hydrophobic pocket in mu2 adaptin through a leucine at position Y-3 in the peptide. This structure suggests that some sequences can function as a 'three pinned plug', in which internalization activity is not critically dependent on any one of the three interacting side chains.