Figure 1.

NHERF1 and NHERF2 bind to an internal region of the NHE3 C terminus (aa 590–610). A, the interactions between GST-NHERF1 (panel a) or GST-NHERF2 (panel b) and ³⁵S-labeled (panel c) NHE3 C-terminal His₆-tagged truncations (C refers to C terminus) C589 (aa 475–589), C610 (aa 475–610), C660 (aa 475–660), C689 (aa 475–689), C711(aa 475–711), and C832(aa 475–832) were determined with pulldown assays (see “Experimental Procedures”). 6 μl of ³⁵S-labeled NHE3 C-terminal truncations and 3 μg of recombinant proteins (GST-NHERF1 or GST-NHERF2) were loaded for 10% SDS-PAGE. B, putative Class I (-⁵⁹²SAV-) and Class II (-⁵⁹⁵CLDM-) PBMs in NHE3 C terminus (aa 590–610): amino acid sequence alignment of conserved amino acids for NHERF2-binding sites in internal sequences of the mammalian NHE gene family 1–5 cytoplasmic domains. The putative internal Class I PBM (-⁵⁹²SAV-) is boxed (dotted line), and critical amino acids for Class I PBM are underlined. The putative internal Class II PBM (-⁵⁹⁵CLDM-) is boxed (solid line), and the conserved hydrophobic amino acids are underlined. Accession numbers for the sequences, top to bottom, are P26432 (NHE3_Rabbit), NP_004165 (NHE3_Human), NP_036786 (NHE3_Rat), NP_620213.1 (NHE5_Norway rat), AAI42672 (NHE5_Human), XP_005008798.1 (NHE5_G. Pig (domestic guinea pig)), P23791 (NHE1_Rabbit), NP_003038 (NHE1_Human), NP_036784 (NHE1_Norway rat), P50482 (NHE2_Rabbit), P48763 (NHE2_Rat), and NP_775121 (NHE4_Rat). Sequences were aligned with ClustalW. An asterisk indicates the hydrophobic amino acids of the class II PBM.