Ezrin binding domain-deficient NHERF attenuates cAMP-mediated inhibition of Na+/H+ exchange in OK cells

Na+/H+ exchanger regulatory factor (NHERF), an essential protein cofactor i n cAMP-mediated inhibition of Na+/H+ exchange transporter 3 (NHE3), facilit ates the formation of a signal complex of proteins that includes NHE3, NHER F, and ezrin. This model for NHE3 regulation was developed in fibroblasts a nd its applicability to epithelial cells remains to be established. Opossum kidney (OK) cells were transfected with either empty vector (control), ful l-length mouse (m) NHERF( 1-355), or a truncated mNHERF(1-325) that lacked ezrin binding and had been demonstrated in fibroblasts to bind NHE3 but not mediate its cAMP-associated inhibition. 8-Bromoadenosine 3', 5'-cyclic mon ophosphate (8-BrcAMP) at 10(-4) M inhibited Na+/H+ exchange activity in con trol and OK cells expressing wild-type mNHERF( 1-355) by >60% but by <10% i n cells expressing mNHERF(1-325). NHE3 coimmunoprecipitated with mNHERF( 1- 325), but cAMP phosphorylation of NHE3 was impaired in cells expressing mNH ERF(1-325). The inhibitory effect of hyperosmolality on NHE3 activity and t he uptake of 3-O-methyl-D-glucose was the same in all three cell lines. Cel l surface expression of NHE3 was not changed by cAMP in any of the cells li nes. These data indicate that disruption of the NHERF-ezrin signal complex attenuates the inhibitory effect of cAMP on NHE3 activity in OK cells and p rovides evidence supporting the proposed model of protein kinase A regulati on of NHE3 in epithelial cells.