(A) Muniscin–AP-2 interactions. T-Coffee (Notredame et al., 2000) generated multiple sequence alignment of the phylogenetically conserved linker region within muniscin members. Amino acid regions of Fcho1 from selected species: Homo sapiens (Hs; NP_001154829), Rattus norvegicus (Rn; XP_006252925), Python bivittatus (Pb; XP_007436694), Fcho2: Hs (NP_620137), Danio rerio (Dr; NP_001018617), Fcho2-like (Fcho2l): Aplysia californica (Ac; XP_005111676), the single FCHO member in Drosophila melanogaster (Dm; NP_001097723), and Daphnia pulex (Dp; EFX87825), as well as Sgip1: Mus musculus (Mm; NP_659155) and Dr (XP_005165952) are shown with appropriate residues numbers indicated. Identical residues are highlighted in magenta, highly similar residues in pale pink and conservatively substituted amino acids in yellow. The location of mass-spectrometry authenticated phosphosites (Hornbeck et al., 2012) are shown (red font). (B) Samples of 100 μg of GST, GST-FCHO1 (316–467) or GST-ARH (180–308) immobilized on glutathione-Sepharose were incubated with rat brain cytosol, washed and samples of each supernatant (S) and pellet (P) fraction separated by SDS-PAGE. Replicate gels were either stained with Coomassie blue (left) or immunoblotted with the designated antibodies (right). The positions of the molecular mass standards (in kDa) are indicated on the left. (C) Schematic illustration of the overall chain and domain composition of the AP-2 adaptor complex. (D) Samples of 100 μg of GST, GST-FCHO1 (316–467), GST-FCHO2 (314–444) or GST-Sgip1 (77–214) immobilized on glutathione-Sepharose were used in pull-down assays with the purified AP-2 heterameric core complex as in (B). The identity of the large subunit trunk polypeptides (α and β2 subunits) and the myc-tagged μ2 subunit is confirmed on immunoblots with mAb clone 8, mAb 100/1 and mAb clone 31, respectively. (E) Aliquots of 100 μg of GST or either 25 μg or 100 μg of GST-EPS15 (595–896) or GST-EPS15 (595–896/Δ617–636) immobilized on glutathione-Sepharose were incubated with soluble lysate from K562 cells. After washing, portions of the supernatant and pellet fractions were analyzed as in (B). FCHO1 antibody 1 is a mAb while antibody 2 is an affinity-purified antibody that also recognizes FCHO2 weakly. Non-specific cross-reactive bands are indicated with asterisks; reactivity of the GST-EPS15 fusion proteins with the anti-FCHO1 and anti-FCHO2 antibody preparations is also indicated with asterisks. Notice the decreased FCHO1 and FCHO2 binding upon deletion of the minimal μHD sequence within the EPS15 C-terminal region that correlates with reduced clathrin association.