Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1995 May;95(5):2143–2149. doi: 10.1172/JCI117903

Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na(+)-H+ exchanger.

E J Weinman 1, D Steplock 1, Y Wang 1, S Shenolikar 1
PMCID: PMC295815  PMID: 7738182

Abstract

Activation of cAMP-dependent protein kinase A inhibits the renal proximal tubule brush border membrane Na(+)-H+ exchanger by a process involving participation of a regulatory cofactor (NHE-RF) that is distinct from the transporter itself. Recent studies from this laboratory reported a partial amino acid sequence of this putative cofactor (Weinman, E. J., D. H. Steplock, and S. Shenolikar. 1993. J. Clin. Invest. 92:1781-1786). The present experiments detail the structure of the NHE-RF protein as determined from molecular cloning studies. A codon-biased oligonucleotide probe to a portion of the amino acid sequence of the putative cofactor was used to isolate a 1.9-kb cDNA from a rabbit renal library. The encoded protein is 358 amino acids in length and is rich in proline residues. Search of existing data bases indicates that NHE-RF is a unique protein. Using a reticulocyte lysate, the cDNA translated a product of approximately 44 kD, which was recognized by an affinity-purified polyclonal antibody to NHE-RF. Potential phosphorylation sites for protein kinase A are present. The mRNA for the protein is expressed in kidney, proximal small intestine, and liver. Reverse transcription/PCR studies in the kidney indicate the presence of mRNA for NHE-RF in several distinct nephron segments including the proximal tubule.

Full text

PDF
2143

Images in this article

2144Image
on p.2144
2146Image
on p.2146
2146Image
on p.2146
2146Image
on p.2146
2146Image
on p.2146

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams J. A., Taylor S. S. Phosphorylation of peptide substrates for the catalytic subunit of cAMP-dependent protein kinase. J Biol Chem. 1993 Apr 15;268(11):7747–7752. [PubMed] [Google Scholar]
  2. Chen D., Magnuson V. L., Steffensen B., Klebe R. J. Use of stock solutions to simplify mRNA quantitation by reverse transcription--PCR assays. PCR Methods Appl. 1993 May;2(4):351–353. doi: 10.1101/gr.2.4.351. [DOI] [PubMed] [Google Scholar]
  3. Chen D., Magnuson V., Hill S., Arnaud C., Steffensen B., Klebe R. J. Regulation of integrin gene expression by substrate adherence. J Biol Chem. 1992 Nov 25;267(33):23502–23506. [PubMed] [Google Scholar]
  4. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  5. Dolson G. M., Hise M. K., Weinman E. J. Relationship among parathyroid hormone, cAMP, and calcium on proximal tubule sodium transport. Am J Physiol. 1985 Sep;249(3 Pt 2):F409–F416. doi: 10.1152/ajprenal.1985.249.3.F409. [DOI] [PubMed] [Google Scholar]
  6. Kahn A. M., Dolson G. M., Hise M. K., Bennett S. C., Weinman E. J. Parathyroid hormone and dibutyryl cAMP inhibit Na+/H+ exchange in renal brush border vesicles. Am J Physiol. 1985 Feb;248(2 Pt 2):F212–F218. doi: 10.1152/ajprenal.1985.248.2.F212. [DOI] [PubMed] [Google Scholar]
  7. Levine S. A., Montrose M. H., Tse C. M., Donowitz M. Kinetics and regulation of three cloned mammalian Na+/H+ exchangers stably expressed in a fibroblast cell line. J Biol Chem. 1993 Dec 5;268(34):25527–25535. [PubMed] [Google Scholar]
  8. Morell G., Steplock D., Shenolikar S., Weinman E. J. Identification of a putative Na(+)-H+ exchanger regulatory cofactor in rabbit renal BBM. Am J Physiol. 1990 Dec;259(6 Pt 2):F867–F871. doi: 10.1152/ajprenal.1990.259.6.F867. [DOI] [PubMed] [Google Scholar]
  9. Preston G. M., Jung J. S., Guggino W. B., Agre P. The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel. J Biol Chem. 1993 Jan 5;268(1):17–20. [PubMed] [Google Scholar]
  10. Weinman E. J., Dubinsky W. P., Dinh Q., Steplock D., Shenolikar S. Effect of limited trypsin digestion on the renal Na+-H+ exchanger and its regulation by cAMP-dependent protein kinase. J Membr Biol. 1989 Aug;109(3):233–241. doi: 10.1007/BF01870280. [DOI] [PubMed] [Google Scholar]
  11. Weinman E. J., Dubinsky W. P., Shenolikar S. Reconstitution of cAMP-dependent protein kinase regulated renal Na+-H+ exchanger. J Membr Biol. 1988;101(1):11–18. doi: 10.1007/BF01872815. [DOI] [PubMed] [Google Scholar]
  12. Weinman E. J., Dubinsky W., Shenolikar S. Regulation of the renal Na+-H+ exchanger by protein phosphorylation. Kidney Int. 1989 Oct;36(4):519–525. doi: 10.1038/ki.1989.226. [DOI] [PubMed] [Google Scholar]
  13. Weinman E. J., Shenolikar S., Kahn A. M. cAMP-associated inhibition of Na+-H+ exchanger in rabbit kidney brush-border membranes. Am J Physiol. 1987 Jan;252(1 Pt 2):F19–F25. doi: 10.1152/ajprenal.1987.252.1.F19. [DOI] [PubMed] [Google Scholar]
  14. Weinman E. J., Shenolikar S. Regulation of the renal brush border membrane Na(+)-H+ exchanger. Annu Rev Physiol. 1993;55:289–304. doi: 10.1146/annurev.ph.55.030193.001445. [DOI] [PubMed] [Google Scholar]
  15. Weinman E. J., Steplock D., Bui G., Yuan N., Shenolikar S. Regulation of renal Na(+)-H+ exchanger by cAMP-dependent protein kinase. Am J Physiol. 1990 May;258(5 Pt 2):F1254–F1258. doi: 10.1152/ajprenal.1990.258.5.F1254. [DOI] [PubMed] [Google Scholar]
  16. Weinman E. J., Steplock D., Shenolikar S. CAMP-mediated inhibition of the renal brush border membrane Na+-H+ exchanger requires a dissociable phosphoprotein cofactor. J Clin Invest. 1993 Oct;92(4):1781–1786. doi: 10.1172/JCI116767. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES