A Sensitive Method for Determining the Phosphorylation Status of Natriuretic Peptide Receptors:cGK-Ialpha Does Not Regulate NPR-A

摘要

Natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B) are transmembrane guanylyl cyclases that catalyze the synthesis of cGMP in response to natriuretic peptides.Phosphorylation and dephosphorylation regulate these receptors and have been traditionally studied by ~(32)PO_4 labeling of transfected cells.However,this approach cannot be used to determine the phosphorylation state of receptors isolated from unlabeled sources.Here,we use Pro-Q Diamond and SYPRO Ruby dyes to quantify the phosphorylation status and protein levels,respectively,of natriuretic peptide receptors from tissues and cells.Strong Pro-Q Diamond signals for NPR-A and NPR-B were obtained when receptors were isolated from lung tissue,liver tissue and overexpressing cells.The level of NPR-A Pro-Q staining was also high in kidney but was much lower in heart tissue.In contrast,the SYPRO Ruby protein signal was weaker and more variable.In a direct comparison,Pro-Q Diamond staining was as sensitive as but more specific than the ~(32)PO_4 labeling method.The two approaches were highly correlated (R~2 = 0.98).We exploited these techniques to measure the effect of cGMP-dependent protein kinase Ialpha on the phosphate content and guanylyl cyclase activity of NPR-A.Neither value was significantly affected in cells overexpressing cGK-Ialpha or in tissues from mice lacking cGK-I.We conclude that cGK-I does not regulate the cyclase activity or phosphorylation state of NPR-A.Furthermore,we find that Pro-Q Diamond staining is a sensitive method for measuring the phosphate levels of natriuretic peptide receptors,but protein levels are best detected by Western blot analysis,not SYPRO Ruby staining.