AP-2-dependent internalization of potassium channel Kir2.3 is driven by a novel di-hydrophobic signal.
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 alpha 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 alpha 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 alphaC-sigma2 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.
Mason
Amanda K
AK
Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Jacobs
Brandiese E
BE
Welling
Paul A
PA
D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism*; D033965:Adaptor Protein Complex alpha Subunits / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D033922:Clathrin Heavy Chains / Q000235:genetics / Q000378:metabolism; D034281:Dynamins / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D005260:Female; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D016297:Mutagenesis, Site-Directed; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D015704:CD4 Antigens; C099489:KCNJ4 protein, human; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D033922:Clathrin Heavy Chains; D034281:Dynamins