title: Depolymerization of cortical actin inhibits UT-A1 urea transporter endocytosis but promotes forskolin-stimulated membrane trafficking.
abstract: The cytoskeleton participates in many aspects of transporter protein regulation. In this study, by using yeast two-hybrid screening, we identified the cytoskeletal protein actin as a binding partner with the UT-A1 urea transporter. This suggests that actin plays a role in regulating UT-A1 activity. Actin specifically binds to the carboxyl terminus of UT-A1. A serial mutation study shows that actin binding to UT-A1's carboxyl terminus was abolished when serine 918 was mutated to alanine. In polarized UT-A1-MDCK cells, cortical filamentous (F) actin colocalizes with UT-A1 at the apical membrane and the subapical cytoplasm. In the cell surface, both actin and UT-A1 are distributed in the lipid raft microdomains. Disruption of the F-actin cytoskeleton by latrunculin B resulted in UT-A1 accumulation in the cell membrane as measured by biotinylation. This effect was mainly due to inhibition of UT-A1 endocytosis in both clathrin and caveolin-mediated endocytic pathways. In contrast, actin depolymerization facilitated forskolin-stimulated UT-A1 trafficking to the cell surface. Functionally, depolymerization of actin by latrunculin B significantly increased UT-A1 urea transport activity in an oocyte expression system. Our study shows that cortical F-actin not only serves as a structural protein, but directly interacts with UT-A1 and plays an important role in controlling UT-A1 cell surface expression by affecting both endocytosis and trafficking, therefore regulating UT-A1 bioactivity.
authors.affiliation: Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Xu
authors.forename: Hua
authors.initials: H
authors.lastname: Su
authors.forename: Conner B
authors.initials: CB
authors.lastname: Carter
authors.forename: Otto
authors.initials: O
authors.lastname: Fröhlich
authors.forename: Guangping
authors.initials: G
authors.lastname: Chen
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology*; D003593:Cytoplasm / Q000378:metabolism / Q000502:physiology; D004285:Dogs; D004705:Endocytosis; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D053778:Thiazolidines / Q000494:pharmacology; D014982:Xenopus laevis; D015003:Yeasts / Q000378:metabolism; D000096982:Urea Transporters
chemical_list: D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D051242:Caveolin 1; D002966:Clathrin; D005576:Colforsin; D026901:Membrane Transport Proteins; D053778:Thiazolidines; D000096982:Urea Transporters; C037068:latrunculin B