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 A₁ receptor (A₁R) 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 A₁R-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^Δ552) 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 A₁R activation and this interaction is a necessary and integral part of the signaling pathway between adenosine and NHE3.