FIGURE 10.

Phosphorylation of the C-terminal acidic Ser/Thr cluster is important for β-arrestin2-mediated internalization of NHE5. A, AP-1 cells in 10-cm dishes were transiently cotransfected (24 h) with 10 μg of cDNA containing a fixed amount (2 μg) of WT or mutant (⁷⁰²ST/AA⁷¹⁴ or ⁶⁹⁷IL/AA + ⁷⁰²ST/AA⁷¹⁴ + ⁷²²II/AA) NHE5_HA3 and increasing amounts (0–8 μg) of β-Arr2_FLAG. The total DNA/transfection was held constant at 10 μg/dish by adjusting with empty parental vector pCMV. Cell surface NHE5 protein was isolated using a cell surface biotinylation assay (see “Experimental Procedures”) and compared with total cellular levels of NHE5 (10% of lysate). The fractions were resolved by SDS-PAGE and immunoblotted using antibodies to the respective epitope tags, and the intensities of the bands were quantified by densitometry. B, for comparative purposes, the ratio of surface/total NHE5 for cells expressing NHE5_HA alone (control) was normalized to a value of 1. Values are the means ± S.E. (error bars) of at least four independent experiments (*, p < 0.05).