The binding of atrial natriuretic peptide and C-type natriuretic peptide to the guanylyl cyclase-linked natriuretic peptide receptors A and B (NPR-A and NPR-B), respectively, results in decreases in extracellular volume, vascular tension and cell proliferation. Both NPR-A and NPR-B are extensively phosphorylated in resting cells and receptor dephosphorylation is correlated with ligand-induced homologous desensitization. To understand the role of phosphorylation in the regulation of these receptors, we identified the in vivo phosphorylation sites of NPR-A and NPR-B and found that the phosphorylation of multiple sites within their kinase homology domains is absolutely required for their activation. In this review, we give a detailed description of the phosphopeptide mapping techniques that were used to identify and characterize these sites and discuss the potential pitfalls that are associated with them.
Bibliographical noteFunding Information:
This work was initiated in David L. Garbers’ laboratory in the Department of Pharmacology at the University of Texas South-western Medical Center. We thank Dr. Garbers for helpful comments during the early stages of this work and for the generous donation of numerous reagents that were necessary for these studies. We also thank Jill Meisenhelder for peptide synthesis and purification. L.R.P. was supported by Fellowship CA-67452 from the National Cancer Institute. T.H. is an American Cancer Society Research Professor. This work was supported by USPHS Grants CA14195 and CA39780 to T.H.