PHOSPHORYLATION OF THE KINASE HOMOLOGY DOMAIN IS ESSENTIAL FOR ACTIVATION OF THE A-TYPE NATRIURETIC PEPTIDE RECEPTOR

Natriuretic peptide receptor A (NPB-A) is the biological receptor for atrial natriuretic peptide (ANP). Activation of the NPR-A guanylyl cyc lase requires ANP binding to the extracellular domain and ATP binding to a putative site within its cytoplasmic region. The allosteric inter action of ATP with the intracellular kinase homology domain (KHD) is h ypothesized to derepress the carboxyl-terminal guanylyl cyclase cataly tic domain, resulting in the synthesis of the second messenger, cyclic GMP. Here, we show that phosphorylation of the KHD is essential for r eceptor activation, Using a combination of phosphopeptide mapping tech niques, we have identified six residues within the ATP-binding domain (S497, T500, S502, S506, S510, and T513) which are phosphorylated when MPR-A is expressed in HEK 293 cells, Mutation of any one of these Ser or Thr residues to Ala caused reductions in the receptor phosphorylat ion state, the number and pattern of phosphopeptides observed in trypt ic maps, and ANP-dependent guanylyl cyclase activity. The reductions w ere not explained by decreases in NPR-A protein levels, as indicated b y immunoblot analysis and determinations of cyclase activity in the pr esence of detergent, Conversion of Ser-497 to Ala resulted in the most dramatic decrease in cyclase activity (similar to 20% of wild-type ac tivity), but conversion to an acidic residue (Glu), which mimics the c harge of the phosphoserine moiety, had no effect. Simultaneous mutatio n of five of the phosphorylation sites to Ala resulted in a dephosphor ylated receptor which was unresponsive to hormone and had potent domin ant negative inhibitory activity. We conclude that phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR -A.