Abstract
Select plasma membrane proteins can be marked as cargo for inclusion into clathrin-coated pits by common internalization signals (e.g. YXXΦ, dileucine motifs, NPXY) that serve as universal recognition sites for the AP-2 adaptor complex or other clathrin-associated sorting proteins. However, some surface proteins, such as the Kir2.3 potassium channel, lack canonical signals but are still targeted for clathrin-dependent endocytosis. Here, we explore the mechanism. We found an unusual endocytic signal in Kir2.3 that is based on two consecutive pairs of hydrophobic residues. Characterized by the sequence ΦΦXΦΦ (a tandem di-hydrophobic (TDH) motif, where Φ is a hydrophobic amino acid), the signal shows no resemblance to other endocytic motifs, yet it directly interacts with AP-2 to target the Kir2.3 potassium channel into the endocytic pathway. We found that the tandem di-hydrophobic motif directly binds to the ασ2 subunits of AP-2, interacting within a large hydrophobic cleft that encompasses part of the docking site for di-Leu signals, but includes additional structures. These observations expand the repertoire of clathrin-dependent internalization signals and the ways in which AP-2 can coordinate endocytosis of cargo proteins.
Highlights
Some membrane proteins, such as Kir2.3, are internalized by a clathrin-dependent mechanism even though they lack canonical endocytic signals
Select plasma membrane proteins can be marked as cargo for inclusion into clathrin-coated pits by common internalization signals (e.g. YXX⌽, dileucine motifs, NPXY) that serve as universal recognition sites for the AP-2 adaptor complex or other clathrin-associated sorting proteins
Because expression of the AP-2 complex as heterodimers (␣2 and 22) generates appropriately folded hemicomplexes that have exposed binding sites for di-Leu or YXX⌽ motifs [23], we felt that this approach might be optimal to map the recognition site for the unique endocytic motif in Kir2.3
Summary
Some membrane proteins, such as Kir2.3, are internalized by a clathrin-dependent mechanism even though they lack canonical endocytic signals. We found that the tandem di-hydrophobic motif directly binds to the ␣2 subunits of AP-2, interacting within a large hydrophobic cleft that encompasses part of the docking site for di-Leu signals, but includes additional structures. These observations expand the repertoire of clathrin-dependent internalization signals and the ways in which AP-2 can coordinate endocytosis of cargo proteins. Many clathrin-mediated endocytosis-targeted proteins contain a common endocytic signal (i.e. either YXX⌽ or di-Leu ((DE)XXXL(LIM)) [1,2,3]) that directly interacts with distinct binding pockets in AP-2. We used a similar approach to fully characterize the endocytic motif in Kir2.3 and map its binding site in AP-2
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