The AP‐2 adaptin α/σ2 hemicomplex binds to a di‐isoleucine motif to mediate Kir2.3 endocytosis

Abstract

We have previously shown that a prototype 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 endocytic signal. Present evidence indicates these signals sequentially interact with intracellular sorting machinery and Lin‐7/CASK to drive basolateral‐directed traffic in the biosynthetic and endocytotic pathways. Although channel endocytosis is dependent on the AP‐2 adaptor, it is unknown if and how the di‐isoleucine signal is recognized by the adaptin. To explore these questions, the subunits of AP‐1 and AP‐2 were expressed either individually or as hemicomplex forms and tested for interaction with Kir2.3 in a GST pull down assay. We found Kir2.3C binds strongly and preferentially to the AP‐2 α/σ 2 hemicomplex and modestly binds to the AP‐1γ/σ 1 hemicomplex. Kir2.3 did not recognize the β2/μ2 (AP‐2) hemicomplex. Replacement of the di‐isoleucine motif with di‐alanine significantly reduced the interaction. By contrast, the canonical dileucine motif binds preferentially to the γ/σ 1 (AP‐1) hemicomplex, with a weaker binding to α/σ 2 (AP‐2). In conclusion, the di‐isoleucine signal in Kir2.3 differs from canonical di‐leucine signals and may act purely as an endocytic signal, providing new insights into the trafficking mechanism of di‐hydrophobic sorting signals. Research supported by NIH.