Figure 2.

Definition of the inner core positions. (A–E) GST or peptides fused to GST as indicated were precoupled to glutathione–Sepharose and incubated with soluble rat brain extracts. Affinity-selected AP-2 (α-adaptin) was detected by Western blot. An aliquot of brain homogenate (starting material, SM) equal to one-tenth of that added to the fusion proteins was analyzed in parallel. The tetrapeptide cores of the motifs are underlined and bold amino acids indicate the substitutions introduced into the wild-type sequence. (A/D) Mutational analysis for V at position +1 and for Q at position +2 (B/E). (C) Sequence alignment of potential NECAP-like α-ear-binding motifs in proteins connected to vesicle trafficking. The conserved hydrophobic amino acids at the 0 and +3 positions are shaded black and similar residues are shaded gray. Small letters in superscript indicate distinct copies of the motif within the same protein. AAK1, adaptor-associated kinase 1; BIKe, BMP-2 inducible kinase; GAK, cyclin G-associated kinase; PACSIN, PKC and CK2 substrate in neurons; REPS1, RalBP1-associated Eps domain containing protein 1.