A Sensitive Method for Determining the Phosphorylation Status of Natriuretic Peptide Receptors: cGK-I Does Not Regulate NPR-A
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- 作者：Paula M. Bryan ; Dmitri Smirnov ; Albert Smolenski ; Susanne Feil ; Robert Feil ; Franz Hofmann ; Suzanne Lohmann ; and Lincoln R. Potter
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：January 31, 2006
- 年：2006
- 卷：45
- 期：4
- 页码：1295 - 1303
- 全文大小：482K
- 年卷期：v.45,no.4(January 31, 2006)
- ISSN：1520-4995
文摘
Natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B) aretransmembrane guanylyl cyclases that catalyze the synthesis of cGMP in response to natriuretic peptides.Phosphorylation and dephosphorylation regulate these receptors and have been traditionally studied by32PO4 labeling of transfected cells. However, this approach cannot be used to determine the phosphorylationstate of receptors isolated from unlabeled sources. Here, we use Pro-Q Diamond and SYPRO Ruby dyesto quantify the phosphorylation status and protein levels, respectively, of natriuretic peptide receptorsfrom tissues and cells. Strong Pro-Q Diamond signals for NPR-A and NPR-B were obtained when receptorswere isolated from lung tissue, liver tissue and overexpressing cells. The level of NPR-A Pro-Q stainingwas also high in kidney but was much lower in heart tissue. In contrast, the SYPRO Ruby protein signalwas weaker and more variable. In a direct comparison, Pro-Q Diamond staining was as sensitive as butmore 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 I
on the phosphatecontent and guanylyl cyclase activity of NPR-A. Neither value was significantly affected in cellsoverexpressing cGK-I or in tissues from mice lacking cGK-I. We conclude that cGK-I does not regulatethe cyclase activity or phosphorylation state of NPR-A. Furthermore, we find that Pro-Q Diamond stainingis a sensitive method for measuring the phosphate levels of natriuretic peptide receptors, but protein levelsare best detected by Western blot analysis, not SYPRO Ruby staining.
on the phosphatecontent and guanylyl cyclase activity of NPR-A. Neither value was significantly affected in cellsoverexpressing cGK-I or in tissues from mice lacking cGK-I. We conclude that cGK-I does not regulatethe cyclase activity or phosphorylation state of NPR-A. Furthermore, we find that Pro-Q Diamond stainingis a sensitive method for measuring the phosphate levels of natriuretic peptide receptors, but protein levelsare best detected by Western blot analysis, not SYPRO Ruby staining.