title: Phosphorylation of the kinase homology domain is essential for activation of the A-type natriuretic peptide receptor.
abstract: Natriuretic peptide receptor A (NPR-A) is the biological receptor for atrial natriuretic peptide (ANP). Activation of the NPR-A guanylyl cyclase requires ANP binding to the extracellular domain and ATP binding to a putative site within its cytoplasmic region. The allosteric interaction of ATP with the intracellular kinase homology domain (KHD) is hypothesized to derepress the carboxyl-terminal guanylyl cyclase catalytic domain, resulting in the synthesis of the second messenger, cyclic GMP. Here, we show that phosphorylation of the KHD is essential for receptor activation. Using a combination of phosphopeptide mapping techniques, we have identified six residues within the ATP-binding domain (S497, T500, S502, S506, S510, and T513) which are phosphorylated when NPR-A is expressed in HEK 293 cells. Mutation of any one of these Ser or Thr residues to Ala caused reductions in the receptor phosphorylation state, the number and pattern of phosphopeptides observed in tryptic maps, and ANP-dependent guanylyl cyclase activity. The reductions were not explained by decreases in NPR-A protein levels, as indicated by immunoblot analysis and determinations of cyclase activity in the presence of detergent. Conversion of Ser-497 to Ala resulted in the most dramatic decrease in cyclase activity (approximately 20% of wild-type activity), but conversion to an acidic residue (Glu), which mimics the charge of the phosphoserine moiety, had no effect. Simultaneous mutation of five of the phosphorylation sites to Ala resulted in a dephosphorylated receptor which was unresponsive to hormone and had potent dominant negative inhibitory activity. We conclude that phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A.
authors.affiliation: Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA. lpotter@aim.salk.edu
authors.forename: L R
authors.initials: LR
authors.lastname: Potter
authors.forename: T
authors.initials: T
authors.lastname: Hunter
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000378:metabolism; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017830:Octoxynol / Q000494:pharmacology; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D017461:Receptors, Atrial Natriuretic Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014162:Transfection; D000099242:Atrial Natriuretic Factor Receptor B
chemical_list: D000255:Adenosine Triphosphate; D000409:Alanine; D009320:Atrial Natriuretic Factor; D018698:Glutamic Acid; D006162:Guanylate Cyclase; D017830:Octoxynol; D010748:Phosphopeptides; D017461:Receptors, Atrial Natriuretic Factor; D012694:Serine; D013912:Threonine; D000099242:Atrial Natriuretic Factor Receptor B; C091715:atrial natriuretic factor receptor A