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Author Notes:

Address for reprint requests and other correspondence: G. Chen, WMRB Rm. 338, 1639 Pierce Dr., Atlanta, GA 30322 (e-mail: gchen3@emory.edu).

Subject:

Research Funding:

This work was supported by American Heart Association Beginning Grant-In-Aid 0765202B and National Institutes of Health (NIH) Grant R21-DK-080431 to G. Chen, and NIH Grants P01-DK-61521 and R01-DK-41707 to J. M. Sands.

Keywords:

  • membrane protein
  • urea transport
  • endocytosis

Internalization of UT-A1 urea transporter is dynamin dependent and mediated by both caveolae- and clathrin-coated pit pathways

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Journal Title:

American Journal of Physiology - Renal Physiology

Volume:

Volume 299, Number 6

Publisher:

, Pages F1389-F1395

Type of Work:

Article | Post-print: After Peer Review

Abstract:

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 NH2 and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH2 and 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.

Copyright information:

© 2010 the American Physiological Society

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