Molecular approaches to examine the phosphorylation state of the C type natriuretic peptide receptor.
The intracellular domain of the C type natriuretic peptide receptor (NPRC) contains one threonine and several serine residues where phosphorylation is thought to occur. Several phosphorylation consensus sequences for various kinases have been identified within the intracellular domain of NPRC, but the exact residues that are phosphorylated and the specific kinases responsible for their phosphorylation have not been thoroughly defined. Here we introduce a recombinant GST fusion protein and a rat gastric mucosa (RGM1) cell line as molecular tools to study the phosphorylation state of NPRC in vitro and in vivo, respectively. We utilize a previously characterized polyclonal antibody against NPRC to probe for total NPRC protein and various phosphospecific and substrate motif antibodies to probe for phosphorylation of NPRC. Phosphoprotein staining reagents were used with a phosphoprotein control set to detect phosphorylation of NPRC at serine and threonine residues. Recombinant GST-NPRC fusion protein was phosphorylated in vitro by RGM1 lysate in the presence of adenosine-5'-triphosphate (ATP). Western blot analysis using a monoclonal phospho-Thr antibody, which exclusively detects phosphorylated threonine residues, and does not cross-react with phosphorylated serine residues revealed NPRC immunoprecipitated from RGM1 lysate is phosphorylated on a threonine residue. Global analysis of the entire rat NPRC sequence using a protein kinase A (PKA) prediction algorithm, identified five putative PKA phosphorylation sites containing a serine residue and one containing a threonine residue, Thr 505. Taken together, the data presented here suggest that rat NPRC is a substrate for PKA and Thr 505 located within the intracellular domain of NPRC is a likely candidate site for the phosphorylation.
Alli
Abdel A
AA
Research Service, James A. Haley Veterans Hospital, Tampa, Florida 33612, USA.
Gower
William R
WR
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005753:Gastric Mucosa / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D011993:Recombinant Fusion Proteins; D017461:Receptors, Atrial Natriuretic Factor; C073616:atrial natriuretic factor receptor C