AP-2-dependent Internalization of Potassium Channel Kir2.3 Is Driven by a Novel Di-hydrophobic Signal

Abstract

The localization and density of Kir2.3 channels are influenced by the balance between PDZ protein interaction at the cell surface and routing into the endocytic pathway. Here, we explore mechanisms by which the Kir2.3 channel is directed into the endocytic pathway. We found that Kir2.3 channels are constitutively internalized from the cell surface in a dynamin-dependent manner, indicative of vesicle-mediated endocytosis. The rate of Kir2.3 endocytosis was dramatically attenuated following RNA interference-mediated knockdown of either ␣ adaptin (AP-2 clathrin adaptor) or clathrin heavy chain, revealing that Kir2.3 is internalized by an AP-2 clathrin-dependent mechanism. Structure-rationalized mutagenesis studies of a number of different potential AP-2 interaction motifs indicate that internalization of Kir2.3 is largely dependent on a non-canonical di-isoleucine motif (II413) embedded within the C terminus. Internalization assays using CD4-Kir2.3 chimeras demonstrate that the di-isoleucine signal acts in an autonomous and transplantable manner. Kir2.3 co-immunoprecipitates with ␣ adaptin, and disruption of the di-isoleucine motif decreased interaction of the channel with AP-2. Replacement of the di-isoleucine motif with a canonical di-leucine internalization signal actually blocked Kir2.3 endocytosis. Moreover, in yeast three-hybrid studies, the Kir2.3 di-isoleucine motif does not bind the AP-2 ␣C-2 hemicomplex in the way that has been recently observed for canonical di-leucine signals. Altogether, the results indicate that Kir2.3 channels are marked for clathrin-dependent internalization from the plasma membrane by a novel AP-2-dependent signal.

WNK4 Diverts the Thiazide-sensitive NaCl Cotransporter to the Lysosome and Stimulates AP-3 Interaction

“…Cells were assayed 48 h post-transfection. After 2 washes with Ringer's solution (17), biotinylatable residues at the cell surface were blocked with 1.5 mg/ml Sulfo-NHS acetate (Pierce) in Ringer's solution at 4°C for 1 h. The cells were then washed, switched to complete Dulbecco's modified Eagle's medium/F-12 medium supplemented with 15 mM HEPES, and incubated at 20 degrees for 2 h to block biosynthetic traffic at the level of the TGN (18,19). Cells were then released from TGN block by transferring them to 37°C.…”

“…For samples subjected to SDS-PAGE, lysates were denatured in Laemmli buffer, heated for 90°C ϫ 5 min, and separated on 6 or 7.5% polyacrylamide gels. Dot blots and immunoblotting of samples fractionated by SDS-PAGE were performed as described previously (16,17).…”

Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway

“…A less well-characterized mechanism of control of NKCC1 activity involves its regulated endocytic retrieval from the basolateral membrane (37). Regulation of transport activity via endocytosis has been reported for a number of ion transporters, including the glutamate transporter (18), the Na ϩ -Cl Ϫ cotransporter (25), the Na ϩ -HCO 3 Ϫ cotransporter (42), and ion channels (3,32). Mechanistic details in many of these instances are lacking.…”

CK2 Phosphorylation of an Acidic Ser/Thr Di-isoleucine Motif in the Na+/H+ Exchanger NHE5 Isoform Promotes Association with β-Arrestin2 and Endocytosis

“…Aside from dileucine signals, di-isoleucine motifs (i.e. II) have also been found to serve as unique signals for endocytosis (88,89). Thus, our data suggest that phosphorylation of Ser/Thr clusters recognized by ␤-arrestins and acidic dihydrophobic signals, operating either independently or jointly, are involved in the endocytosis of NHE5.…”