β-Arrestins bind and decrease cell-surface abundance of the Na~+/H~+ exchanger NHE5 isoform

摘要

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 β-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 β-arrestin2 conferred strong binding to the C terminus of NHE5. Full-length NHE5 and β-arrestin2 also associated in intact cells, as revealed by their coimmunoprecipitation from extracts of trans-fected CHO cells. Moreover, ectopic expression of both proteins caused a redistribution of β-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 β-arrestin1 isoform. These data reveal a broader role for arrestins in the trafficking of integral plasma membrane proteins than previously recognized.