Internalization of UT-A1 urea transporter is dynamin dependent and mediated by both caveolae- and clathrin-coated pit pathways.
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 NH(2) and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH(2) 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.
Huang
Haidong
H
Department of Medicine, Renal Division, School of Medicine, Emory University, Atlanta, Georgia, USA.
Feng
Xiuyan
X
Zhuang
Jieqiu
J
Fröhlich
Otto
O
Klein
Janet D
JD
Cai
Hui
H
Sands
Jeff M
JM
Chen
Guangping
G
D021941:Caveolae / Q000502:physiology*; D002966:Clathrin / Q000502:physiology; D003034:Coated Pits, Cell-Membrane / Q000502:physiology*; D034281:Dynamins / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D034622:RNA Interference; D047392:beta-Cyclodextrins / Q000494:pharmacology; D000096982:Urea Transporters
D002966:Clathrin; D034281:Dynamins; D026901:Membrane Transport Proteins; D047392:beta-Cyclodextrins; D000096982:Urea Transporters; C108732:methyl-beta-cyclodextrin