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

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...

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Bibliographic Details
Published in:The Journal of clinical investigation 1995-05, Vol.95 (5), p.2143-2149
Main Authors: Weinman, E J, Steplock, D, Wang, Y, Shenolikar, S
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Base Sequence
Blotting, Northern
Cloning, Molecular
Cyclic AMP-Dependent Protein Kinases - metabolism
Gene Expression
Kidney - metabolism
Kidney Tubules, Proximal - metabolism
Microvilli - metabolism
Molecular Sequence Data
Oligonucleotide Probes
Organ Specificity
Phosphoproteins - biosynthesis
Phosphoproteins - chemistry
Phosphoproteins - metabolism
Polymerase Chain Reaction
Protein Conformation
Rabbits
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Sequence Homology, Amino Acid
Sodium-Hydrogen Exchangers - metabolism
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Summary: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.
ISSN:0021-9738
DOI:10.1172/JCI117903