Acute Regulation of Na/H Exchanger NHE3 by Adenosine A1 Receptors is Mediated by Calcineurin Homologous Protein

Francesca Di Sole, Robert Cerull, Victor Babich, Henry Quiñones, Serge M. Gisler, Jürg Biber, Heini Murer, Gerhard Burckhardt, Corinna Helmle-Kolb, Orson W. Moe

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Abstract

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, NHE3C terminus; prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3C terminus (amino acids 462-552 of opossum NHE3). The yeast data were confirmed in OK cells where truncated NHE3 (NHE3Δ552) still shows CPA-stimulated CHP interaction. Overexpression of the polypeptide from the CHP binding region (NHE3462-552) 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.

Original languageEnglish (US)
Pages (from-to)2962-2974
Number of pages13
JournalJournal of Biological Chemistry
Volume279
Issue number4
DOIs
StatePublished - Jan 23 2004

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Adenosine A1 Receptors
Sodium-Hydrogen Antiporter
Calcineurin
Proteins
Opossums
Kidney
Yeast
Adenosine
Protein Kinase C
Chemical activation
Autacoids
Amino Acids
Two-Hybrid System Techniques
Phosphorylation
Xenopus laevis
Type C Phospholipases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Acute Regulation of Na/H Exchanger NHE3 by Adenosine A1 Receptors is Mediated by Calcineurin Homologous Protein. / Di Sole, Francesca; Cerull, Robert; Babich, Victor; Quiñones, Henry; Gisler, Serge M.; Biber, Jürg; Murer, Heini; Burckhardt, Gerhard; Helmle-Kolb, Corinna; Moe, Orson W.

In: Journal of Biological Chemistry, Vol. 279, No. 4, 23.01.2004, p. 2962-2974.

Research output: Contribution to journalArticle

Di Sole, F, Cerull, R, Babich, V, Quiñones, H, Gisler, SM, Biber, J, Murer, H, Burckhardt, G, Helmle-Kolb, C & Moe, OW 2004, 'Acute Regulation of Na/H Exchanger NHE3 by Adenosine A1 Receptors is Mediated by Calcineurin Homologous Protein', Journal of Biological Chemistry, vol. 279, no. 4, pp. 2962-2974. https://doi.org/10.1074/jbc.M306838200
Di Sole, Francesca ; Cerull, Robert ; Babich, Victor ; Quiñones, Henry ; Gisler, Serge M. ; Biber, Jürg ; Murer, Heini ; Burckhardt, Gerhard ; Helmle-Kolb, Corinna ; Moe, Orson W. / Acute Regulation of Na/H Exchanger NHE3 by Adenosine A1 Receptors is Mediated by Calcineurin Homologous Protein. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 4. pp. 2962-2974.
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abstract = "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, NHE3C terminus; prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3C terminus (amino acids 462-552 of opossum NHE3). The yeast data were confirmed in OK cells where truncated NHE3 (NHE3Δ552) still shows CPA-stimulated CHP interaction. Overexpression of the polypeptide from the CHP binding region (NHE3462-552) 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.",
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AU - Cerull, Robert

AU - Babich, Victor

AU - Quiñones, Henry

AU - Gisler, Serge M.

AU - Biber, Jürg

AU - Murer, Heini

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