THE ROLE OF NHERF AND E3KARP IN THE CAMP-MEDIATED INHIBITION OF NHE3

NHE3 is the apically located Na+/H+ exchanger in the gut and in the re nal proximal tubule, Acute inhibition of this transporter by cAMP requ ires the presence of either of two NHE3-associated proteins, NHERF or E3KARP, It has been suggested that these proteins either directly regu late MHE3 activity after being phosphorylated by protein kinase A (PKA ) or that they may serve as adapters that localize PKA near NHE3, We s tudied the role of NHERF and E3KARP in opossum kidney cells, which end ogenously express NHE3, NHERF, and ezrin and display cAMP-dependent in hibition of NHE3, In vivo phosphorylation studies showed that NHERF is a phosphoprotein under basal conditions, but does not change its phos phorylation state after 8-bromo-cAMP treatment, and that E3KARP is not phosphorylated at all. Co-immunoprecipitation showed that NHERF and E 3KARP bind both NHE3 and ezrin, Using cAMP analogs it was demonstrated that NHE3 activity, measured as sodium-dependent recovery of the intr acellular pH after intracellular acidification, is inhibited by PKA ty pe II. Because others have shown that ezrin binds PKA type II and that NHE3 is phosphorylated by PKA we suggest that NHERF and E3KARP are ad apters that link NHE3 to ezrin, thereby localizing PKA near NHE3 to al low NHE3 phosphorylation.