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An extended di‐isoleucine motif mediates Kir2.3 endocytosis via direct binding to the AP‐2 adaptin α/σ2 subunits
Author(s) -
Ortega Bernardo,
Mason Amanda K,
Welling Paul A
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.608.5
Subject(s) - endocytic cycle , endocytosis , binding site , clathrin , microbiology and biotechnology , isoleucine , amino acid , chemistry , signal transducing adaptor protein , biochemistry , plasma protein binding , biology , leucine , signal transduction , receptor
A prototypical basolateral membrane potassium channel, Kir2.3, contains a C‐terminal trafficking cassette comprised of a biosynthetic sorting signal, a PDZ binding site, and a novel di‐isoleucine (di‐Ile) endocytic signal that orchestrates polarized trafficking in biosynthetic and endocytotic pathways. Here, we explore how the endocytotic signal is recognized by the AP‐2 clathrin adaptor. Similar to canonical di‐Leu signals, such as in the HIV protein Nef, we found that Kir2.3 signal directly interacts with AP‐2 complex, binding to α/σ2 subunits. Mutation of a key residue in the di‐Leu binding pocket of σ2 abrogated binding to both Kir2.3 and Nef fusion proteins, confirming a common binding site in AP‐2. Extensive mutagenesis analysis of Kir2.3 revealed that additional amino‐acid residues downstream from the di‐Ile motif contribute to the overall binding. Given that Nef motif does not tolerate di‐Leu to di‐Ile substitution, the additional binding sites in Kir2.3 motif may be required to compensate for the sub‐optimal binding provided by the Ile residues. Our data illustrate how diverse endocytic motifs in Kir2.3 and Nef bind to a common hydrophobic pocket in AP‐2, but then require specific amino‐acid residues to confer additional specificity to their bindings. Research supported by NIH & NKF‐MD