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 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 oligonudeotide 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 similar to 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.