beta-Arrestins bind and decrease cell-surface abundance of the Na+/H+ exchanger NHE5 isoform.
The neuronal Na(+)/H(+) exchanger NHE5 isoform not only resides in the plasma membrane but also accumulates in recycling vesicles by means of clathrin-mediated endocytosis. To further investigate the underlying molecular mechanisms, a human brain cDNA library was screened for proteins that interact with the cytoplasmic C-terminal region of NHE5 by using yeast two-hybrid methodology. One candidate cDNA identified by this procedure encoded beta-arrestin2, a specialized adaptor/scaffolding protein required for internalization and signaling of members of the G protein-coupled receptor superfamily. Direct interaction between the two proteins was demonstrated in vitro by GST fusion protein pull-down assays. Sequences within the N-terminal receptor activation-recognition domain and the C-terminal secondary receptor-binding domain of beta-arrestin2 conferred strong binding to the C terminus of NHE5. Full-length NHE5 and beta-arrestin2 also associated in intact cells, as revealed by their coimmunoprecipitation from extracts of transfected CHO cells. Moreover, ectopic expression of both proteins caused a redistribution of beta-arrestin2 from the cytoplasm to vesicles containing NHE5, and significantly decreased the abundance of the transporter at the cell surface. Comparable results were also obtained for the beta-arrestin1 isoform. These data reveal a broader role for arrestins in the trafficking of integral plasma membrane proteins than previously recognized.
Szabó
Elöd Z
EZ
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Numata
Masayuki
M
Lukashova
Viktoria
V
Iannuzzi
Pietro
P
Orlowski
John
J
D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D008565:Membrane Proteins; D010766:Phosphorylation; D020033:Protein Isoforms; D021381:Protein Transport; D017923:Sodium-Hydrogen Exchangers / Q000032:analysis / Q000378:metabolism*; D014162:Transfection; D000071557:beta-Arrestins
D019390:Arrestins; D008565:Membrane Proteins; D020033:Protein Isoforms; D017923:Sodium-Hydrogen Exchangers; D000071557:beta-Arrestins; C117561:sodium-hydrogen exchanger 5