1. Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, UK
2. Institute of Biochemistry I and Center for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany
3. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK
4. These authors contributed equally to this work.
5. Present address: Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany.

Correspondence to: David J. Owen¹ Correspondence and requests for materials should be addressed to D.J.O. (Email: djo30@cam.ac.uk).

Abstract

Most transmembrane proteins are selected as transport-vesicle cargo through the recognition of short, linear amino-acid motifs in their cytoplasmic portions by vesicle coat proteins. For clathrin-coated vesicles, the motifs are recognized by clathrin adaptors. The AP2 adaptor complex (subunits , 2, 2 and 2) recognizes both major endocytic motifs: Yxx motifs¹ (where can be F, I, L, M or V) and [ED]xxxL[LI] acidic dileucine motifs. Here we describe the binding of AP2 to the endocytic dileucine motif from CD4 (ref. 2). The major recognition events are the two leucine residues binding in hydrophobic pockets on 2. The hydrophilic residue four residues upstream from the first leucine sits on a positively charged patch made from residues on the 2 and subunits. Mutations in key residues inhibit the binding of AP2 to 'acidic dileucine' motifs displayed in liposomes containing phosphatidylinositol-4,5-bisphosphate, but do not affect binding to Yxx motifs through 2. In the 'inactive' AP2 core structure³ both motif-binding sites are blocked by different parts of the 2 subunit. To allow a dileucine motif to bind, the 2 amino terminus is displaced and becomes disordered; however, in this structure the Yxx-binding site on 2 remains blocked.

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