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. 1998 May 15;101(10):2199–2206. doi: 10.1172/JCI204

Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF).

E J Weinman 1, D Steplock 1, K Tate 1, R A Hall 1, R F Spurney 1, S Shenolikar 1
PMCID: PMC508807  PMID: 9593775

Abstract

Inhibition of the renal brush border membrane (BBM) Na/H exchanger by cAMP-dependent protein kinase, PKA, requires participation of a recently cloned regulatory cofactor, Na/H exchanger-regulatory factor (NHE-RF). As deduced from the cDNA of this 358-amino acid protein, amino acids 11-101 and amino acids 150-241 of the NHE-RF protein share 74% overall homology suggesting duplication of these PDZ containing domains. The serine residues at amino acid position 289 and 340 are considered to be the most likely sites for PKA mediated phosphorylation. To study the structure- function relation between NHE-RF and PKA mediated inhibition of the rabbit BBM Na/H exchanger, the effect of recombinant proteins representing full-length NHE-RF as well as truncated and mutant forms of NHE-RF were determined using a reconstitution assay. The reconstitution assay employed a fraction of rabbit BBM proteins that contains Na/H exchanger activity that is not regulated by PKA. NHE-RF in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a significant left shift in the dose-response relation such that 10(-12) M NHE-RF inhibited Na/H exchange transport by 30% in the presence but not in the absence of PKA. A recombinant polypeptide representing amino acids 1-151 (Domain I) did not affect Na/H exchange transport in the presence or absence of PKA. A polypeptide representing amino acids 149-358 (Domain II) in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a left shift in the dose-response relation. 10(-12) M of Domain II polypeptide inhibited transport by 18% in the presence but not in the absence of PKA. Mutation of serine residues 287, 289, and 290 to alanine did not affect the inhibitory effect in the absence of PKA but abolished the left shift in the dose-response relation elicited by PKA. Mutation of serine residues 339 and 340 to alanine were without effect on PKA dependent regulation of Na/H exchange transport. These studies indicate that NHE-RF inhibits basal rabbit renal BBM Na/H exchange activity-an effect which is augmented by PKA. The amino acid sequences in the polypeptide containing only the NH2-terminal PDZ domain of NHE-RF have no intrinsic activity as an inhibitor but appears to be required for the full-length NHE-RF to express its full inhibitory effect on the BBM Na/H exchanger. One or more of the serine residues at positions 287, 289, and/or 290 represent the critical PKA phosphorylation site(s) on the NHE-RF protein that mediates the physiologic effect of cAMP on the renal BBM Na/H exchanger.

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Selected References

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

  1. 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]
  2. Hall R. A., Premont R. T., Chow C. W., Blitzer J. T., Pitcher J. A., Claing A., Stoffel R. H., Barak L. S., Shenolikar S., Weinman E. J. The beta2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange. Nature. 1998 Apr 9;392(6676):626–630. doi: 10.1038/33458. [DOI] [PubMed] [Google Scholar]
  3. Harper J. F. Peritz' F test: basic program of a robust multiple comparison test for statistical analysis of all differences among group means. Comput Biol Med. 1984;14(4):437–445. doi: 10.1016/0010-4825(84)90044-1. [DOI] [PubMed] [Google Scholar]
  4. Hoogerwerf W. A., Tsao S. C., Devuyst O., Levine S. A., Yun C. H., Yip J. W., Cohen M. E., Wilson P. D., Lazenby A. J., Tse C. M. NHE2 and NHE3 are human and rabbit intestinal brush-border proteins. Am J Physiol. 1996 Jan;270(1 Pt 1):G29–G41. doi: 10.1152/ajpgi.1996.270.1.G29. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Kurashima K., Yu F. H., Cabado A. G., Szabó E. Z., Grinstein S., Orlowski J. Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. phosphorylation-dependent and -independent mechanisms. J Biol Chem. 1997 Nov 7;272(45):28672–28679. doi: 10.1074/jbc.272.45.28672. [DOI] [PubMed] [Google Scholar]
  7. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  8. Moe O. W., Amemiya M., Yamaji Y. Activation of protein kinase A acutely inhibits and phosphorylates Na/H exchanger NHE-3. J Clin Invest. 1995 Nov;96(5):2187–2194. doi: 10.1172/JCI118273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Weinman E. J., Bennett S. C., Brady R. C., Harper J. F., Hise M. K., Kahn A. M. Effect of cAMP and calmodulin inhibitors on water absorption in rat proximal tubule. Proc Soc Exp Biol Med. 1984 Jul;176(3):322–326. doi: 10.3181/00379727-176-41878. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Weinman E. J., Shenolikar S., Cragoe E. J., Jr, Dubinsky W. P. Solubilization and reconstitution of renal brush border Na+-H+ exchanger. J Membr Biol. 1988;101(1):1–9. doi: 10.1007/BF01872814. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Weinman E. J., Shenolikar S. The Na-H exchanger regulatory factor. Exp Nephrol. 1997 Nov-Dec;5(6):449–452. [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Weinman E. J., Steplock D., Wang Y., Shenolikar S. Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na(+)-H+ exchanger. J Clin Invest. 1995 May;95(5):2143–2149. doi: 10.1172/JCI117903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yun C. H., Oh S., Zizak M., Steplock D., Tsao S., Tse C. M., Weinman E. J., Donowitz M. cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):3010–3015. doi: 10.1073/pnas.94.7.3010. [DOI] [PMC free article] [PubMed] [Google Scholar]

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