A sensitive method for determining the phosphorylation status of natriuretic peptide receptors: cGK-Iα does not regulate NPR-A

Paula M. Bryan, Dmitri Smirnov, Albert Smolenski, Susanne Feil, Robert Feil, Franz Hofmann, Suzanne Lohmann, Lincoln R. Potter

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

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 32PO4 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 32PO4 labeling method. The two approaches were highly correlated (R2 = 0.98). We exploited these techniques to measure the effect of cGMP-dependent protein kinase Iα on the phosphate content and guanylyl cyclase activity of NPR-A. Neither value was significantly affected in cells overexpressing cGK-Iα 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.

Original languageEnglish (US)
Pages (from-to)1295-1303
Number of pages9
JournalBiochemistry
Volume45
Issue number4
DOIs
StatePublished - Jan 31 2006

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