Volume 6, Issue 6, Pages 1425-1436 (December 2000) Direct Binding of the Na–H Exchanger NHE1 to ERM Proteins Regulates the Cortical Cytoskeleton and Cell Shape Independently of H+ Translocation  Sheryl P Denker, Derek C Huang, John Orlowski, Heinz Furthmayr, Diane L Barber  Molecular Cell  Volume 6, Issue 6, Pages 1425-1436 (December 2000) DOI: 10.1016/S1097-2765(00)00139-8

Figure 1 Regulation of H+ Efflux, Focal Adhesions, and Cell Shape by NHE1 (A and B) Expression (A) and localization (B) of HA-tagged wild-type NHE1 and translocation-defective NHE1-E266I. The band migrating above 66 kDa is a nonspecific band detected with the 12CA5 antibody. (C) Effect of pHi on H+ efflux in parental and transfected cell lines. In a HEPES buffer, a pHi-dependent H+ efflux was observed in CCL39 (closed squares) and PSN1 (open circles) cells but not in PS120 (open squares) or PSN1-E266I (closed circles) cells. In a HCO3−-containing buffer, the steady-state pHi of cells maintained in continuous serum (inset) shows that CCL39 and PSN1 cells with NHE1 activity have a more alkaline pHi than PS120 and PSN1-E266I cells without NHE1 activity. (D) Effect of NHE1 activity on cell proliferation (cell number). NHE1 activity contributed to a faster proliferation rate in CCL39 (closed square) and PSN1 (open square) cells compared to PS120 (closed circle), PSneo (closed diamond), and PSN1-E266I (open circle) cells. Pooled data from three separate experiments are shown as mean ± SEM. The asterisk indicates different from PSN1 cells (p < 0.05) by Student's t test. (E) Integrin-induced focal adhesion organization, determined by the distribution of paxillin in cells plated on fibronectin, was induced in cells with NHE1 (CCL39, PSN1, and PSN1-E266I), but not in cells without NHE1 (PS120 and PSneo). CCL39, PSN1, and PSN1-E266I cells are pyramidal, whereas PS120 and PSneo cells are fusiform. Cells are representative of more than 85% of the total population of each cell type. Images were acquired using Nomarski optics. Scale bars: top panel, 10 μm; bottom panel, 60 μm. Molecular Cell 2000 6, 1425-1436DOI: (10.1016/S1097-2765(00)00139-8)

Figure 2 Amino Acid Sequence Alignment of Transmembrane 4.1 Binding Proteins and NHE1 In Vitro Binding Assays (A) The sequences of the juxtamembrane regions (shaded boxes) of the cytoplasmic domain of AE1, CD44, and NHE proteins are shown; the approximate location of the E266I (rat sequence numbering) mutation in NHE1 is indicated. Proximal and distal motifs for 4.1 binding are indicated by single and double underlines, respectively. Accession numbers for the sequences, top to bottom, are P02730, JH0417, P19634, P26431, Q61165, P48763, P48764, P26434, and AAD16413. (B) Autoradiograph of in vitro translated amino-terminal (N), carboxy-terminal (C), and full-length (FL) ERM proteins. (C) Autoradiograph of in vitro binding assay demonstrating that the cytoplasmic domain of NHE1 bound to the amino-terminal (N), but not carboxy-terminal (C) or full-length (FL) ERM proteins. (D) GST fusion proteins (SDS–PAGE gel, top panel) consisting of deletion constructs of NHE1 were used in binding assays (autoradiograph, bottom panel) to determine that the first 170 amino acids (503–670) of the NHE1 cytoplasmic domain were sufficient for N-moesin binding. Identical results were obtained with N-ezrin and N-radixin. (E) GST-fusion proteins (SDS–PAGE gel, amino acids 506–576, top panel) that contain alanine substitutions in the 4.1 binding motif were used in binding assays (autoradiograph, bottom panel) to determine that mutation of the distal (lane 3), but not proximal (lane 2), cluster of charged amino acids abolishes N-radixin binding to wild-type NHE1 (lane 1). Binding is abolished by mutations in both clusters (lane 4). Molecular Cell 2000 6, 1425-1436DOI: (10.1016/S1097-2765(00)00139-8)

Figure 3 Localization and Association of NHE1, Ezrin, and Radixin in Fibroblasts (A–D) HA-tagged NHE1 (A and C) colocalizes with both ezrin (B) and radixin (D) in lamellipodia (arrowheads). NHE1 ([A], arrows) and ezrin ([B], arrows) have an almost identical labeling pattern along the membrane, whereas NHE1 ([C], arrows) appears to have a broader distribution than radixin (D) along the membrane. This labeling pattern is representative of more than 70% of the PSN1 cells. Scale bar, 5 μm. (E) Ezrin coimmunoprecipitates with stably expressed, HA-tagged NHE1 in PSN1 cells but not in NHE1-deficient PSneo cells. Immune complexes were separated by 8% SDS–PAGE and transferred to PVDF membranes. Duplicate aliquots were probed with ezrin (left panel) or 12CA5 followed by the appropriate HRP-conjugated secondary antibodies. The position of ezrin is indicated on the left, that of NHE1 on the right. (F) Ezrin, detected by immunoblotting, specifically coimmunoprecipitates with endogenous NHE1 in both CCL39 and Ltk− cells, but not in NHE1-deficient PS120 or LAP-1 cells. The position of ezrin is indicted on the left. The band at 55 kDa is IgG heavy chain. Molecular Cell 2000 6, 1425-1436DOI: (10.1016/S1097-2765(00)00139-8)

Figure 4 Localization and Distribution of NHE1 and Ezrin in Fibroblasts Expressing Wild-Type and Mutant NHE1 Proteins (A) Equal amounts of NHE1 are expressed in PSN1, PSN1-E266I, and PSN1-KR/A cells. (B) Colabeling experiments demonstrate that NHE1 and ezrin labeling were reduced in the lamellipodia of PSN1-KR/A cells. In PSN1 and PSN1-E266I cells, NHE1 and ezrin labeling was predominantly localized in lamellipodia (arrowheads). In PSN1-KR/A cells, NHE1 and ezrin labeling was less intense in the lamellipodia (arrowheads) and more intense along the smooth edge of the plasma membrane. The distribution of ezrin labeling in PSN1-KR/A cells was similar to that in PSneo cells. Images are representative of more than 70% of cells in the total population of each cell type. Scale bar, 10 μm. (C) The abundance of ezrin in total membrane fractions was similar in all cell lines. Equal amounts of protein in postnuclear supernatants (lanes 1) were centrifuged at 400,000 × g to obtain total membrane (lanes 2) and soluble (lanes 3) fractions. (D) Compared to PSN1 and PSN1-E266I cells, the abundance of ezrin, as determined by immunoblotting, in the TX-insoluble membrane fraction (lanes 2) was reduced in PSneo and PSN1-KR/A cells. Postnuclear supernatants (lanes 1) were used to prepare membrane fractions as in (C). Membranes were solubilized in 1% TX and recentrifuged at 100,000 × g to obtain TX-insoluble (lanes 2) and -soluble (lanes 3) fractions. Molecular Cell 2000 6, 1425-1436DOI: (10.1016/S1097-2765(00)00139-8)

Figure 5 Effects of Disrupting the NHE1–Ezrin Interaction on Focal Adhesions, Cell Shape, and Cell Growth (A) Ezrin distribution in cells plated on fibronectin for 3 hr. Compared to PSN1 and PSN1-E266I cells, ezrin labeling in PSneo and PSN1-KR/A cells was less intense in lamellipodia and redistributed in a punctate pattern along the sides of the membrane. Scale bar, 5 μm. (B) Paxillin distribution in cells. Compared to PSN1 cells, less intense labeling of paxillin in focal adhesions (arrowheads) was observed in NHE1-KR/A cells, whereas diffuse cytoplasmic labeling was more intense. Cells are representative of more than 85% of the total population. Scale, bar, 5 μm. (C) Paxillin expression is similar in PSN1 and PSN1-KR/A cells. (D) The morphology of PSN1-KR/A cells was elongated and fusiform, unlike the pyramidal shape of PSN1 cells. Scale bar, 10 μm. (E) The growth rate of PSN1-KR/A cells was similar to that of PSN1 cells. Molecular Cell 2000 6, 1425-1436DOI: (10.1016/S1097-2765(00)00139-8)

Figure 6 NHE1–Ezrin Binding Regulates the Formation of Actin Stress Fibers Phalloidin labeling of quiescent cells (left panel) and cells treated with LPA (200 ng/ml, 10 min; middle panel). The LPA-induced increase in stress fiber formation observed in PSN1 and PSN1-E266I cells was markedly attenuated in PSneo and PSN1-KR/A cells. High magnification of the peripheral edge of membrane protrusions (arrows) reveals that actin stress fibers extend to the membrane in PSN1 and PSN1-E266I cells, but not in PSneo and PSN1-KR/A cells. Cells are representative of more than 85% of the total population of each cell type. Scale bars in left and middle panels, 5 μm; in right panel, 50 μm. Molecular Cell 2000 6, 1425-1436DOI: (10.1016/S1097-2765(00)00139-8)

Figure 7 Proposed Model of the Anchoring Function of NHE1 Activation of NHE1 and ERM proteins facilitates the binding of ERM proteins to the cytoplasmic domain of NHE1. Rho-dependent signaling, possibly mediated by ROCK, regulates the association of this complex with F-actin. The amino termini (N) of ERM proteins bind to NHE1; the carboxyl termini (C) bind to F-actin. Molecular Cell 2000 6, 1425-1436DOI: (10.1016/S1097-2765(00)00139-8)