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 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.
Weinman
E J
EJ
Department of Medicine, University of California, Los Angeles School of Medicine, USA.
Steplock
D
D
Wang
Y
Y
Shenolikar
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015870:Gene Expression; D007668:Kidney / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009928:Organ Specificity; D010750:Phosphoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D015345:Oligonucleotide Probes; D010750:Phosphoproteins; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D017868:Cyclic AMP-Dependent Protein Kinases