Internalization of UT-A1 urea transporter is dynamin dependent and mediated by both caveolae- and clathrin-coated pit pathways
| Autor: | Hui Cai, Guangping Chen, Haidong Huang, Jieqiu Zhuang, Xiuyan Feng, Jeff M. Sands, Janet D. Klein, Otto Fröhlich |
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| Rok vydání: | 2010 |
| Předmět: |
Dynamins
Physiology media_common.quotation_subject Coated Pit GTPase Caveolae Endocytosis Clathrin Membrane Microdomains Humans Internalization media_common Dynamin biology beta-Cyclodextrins Membrane Transport Proteins Coated Pits Cell-Membrane Articles Cell biology HEK293 Cells Urea transport Gene Knockdown Techniques biology.protein RNA Interference |
| Zdroj: | American Journal of Physiology-Renal Physiology. 299:F1389-F1395 |
| ISSN: | 1522-1466 1931-857X |
| DOI: | 10.1152/ajprenal.00718.2009 |
| Popis: | Dynamin is a large GTPase involved in several distinct modes of cell endocytosis. In this study, we examined the possible role of dynamin in UT-A1 internalization. The direct relationship of UT-A1 and dynamin was identified by coimmunoprecipitation. UT-A1 has cytosolic NH2and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH2and COOH termini. In cell surface biotinylation experiments, coexpression of dynamin and UT-A1 in HEK293 cells resulted in a decrease of UT-A1 cell surface expression. Conversely, cells expressing dynamin mutant K44A, which is deficient in GTP binding, showed an increased accumulation of UT-A1 protein on the cell surface. Cell plasma membrane lipid raft fractionation experiments revealed that blocking endocytosis with dynamin K44A causes UT-A1 protein accumulation in both the lipid raft and nonlipid raft pools, suggesting that both caveolae- and clathrin-mediated mechanisms may be involved in the internalization of UT-A1. This was further supported by 1) small interfering RNA to knock down either caveolin-1 or μ2 reduced UT-A1 internalization in HEK293 cells and 2) inhibition of either the caveolae pathway by methyl-β-cyclodextrin or the clathrin pathway by concanavalin A caused UT-A1 cell membrane accumulation. Functionally, overexpression of dynamin, caveolin, or μ2 decreased UT-A1 urea transport activity and decreased UT-A1 cell surface expression. We conclude that UT-A1 endocytosis is dynamin-dependent and mediated by both caveolae- and clathrin-coated pit pathways. |
| Databáze: | OpenAIRE |
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