Acute regulation of Na/H exchanger NHE3 by adenosine A1 receptors is mediated by calcineurin homologous protein

Adenosine is an autacoid that regulates renal Na+ transport. Activation of adenosine A(1) receptor (A(1)R) by N-6-cyclopentidyladenosine (CPA) inhibits the Na+/H+ exchanger 3 (NHE3) via phospholipase C/Ca2+/protein kinase C (PKC) signaling pathway. Mutation of PKC phosphorylation sites on NHE3 does not affected regulation of NHE3 by CPA, but amino acid residues 462 and 552 are essential for A(1)R-dependent control of NHE3 activity. One binding partner of the NHE family is calcineurin homologous protein (CHP). We tested the role of NHE3-CHP interaction in mediating CPA-induced inhibition of NHE3 in opossum kidney (OK) and Xenopus laevis uroepithelial (A6) cells. Both native and transfected NHE3 and CHP are present in the same immunocomplex by co-immunoprecipitation. CPA (10(-6) M) increases CHP-NHE3 interaction by 30-60% (native and transfected proteins). Direct CHP-NHE3 interaction is evident by yeast two-hybrid assay (bait, NHE3(C terminus); prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3(C terminus) (amino acids 462-552 of opossum NHE3). The yeast data were confirmed in OK cells where truncated NHE3 (NHE3(Delta552)) still shows CPA-stimulated CHP interaction. Overexpression of the polypeptide from the CHP binding region (NHE3(462-552)) interferes with the ability of CPA to inhibit NHE3 activity and to increase CHP-NHE3(Full-length) interaction. Reduction of native CHP expression by small interference RNA abolishes the ability of CPA to inhibit NHE3 activity. We conclude that CHP-NHE3 interaction is regulated by A(1)R activation and this interaction is a necessary and integral part of the signaling pathway between adenosine and NHE3.