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A sensitive method for determining the phosphorylation status of natriuretic peptide receptors: cGK‐I alpha does not regulate NPR‐A
Author(s) -
Bryan Paula Marie,
Smirnov Dmitri,
Lohmann Suzanne M,
Feil Suzanne,
Feil Robert,
Hofmann Franz,
Potter Lincoln Ross
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a967-b
Subject(s) - receptor , phosphorylation , npr2 , natriuretic peptide , npr1 , dephosphorylation , chemistry , peptide , atrial natriuretic peptide , microbiology and biotechnology , cyclase , biochemistry , signal transduction , protein kinase a , adenylate kinase , biology , medicine , endocrinology , phosphatase , heart failure
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/dephosphorylation regulates these receptors and has 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 ProQ Diamond and SYPRO Ruby dyes to quantify the phosphorylation status and protein levels, respectively, of natriuretic peptide receptors from tissues and cells. Strong ProQ Diamond signals for NPR‐A and NPR‐B were obtained when receptors were isolated from kidney, lung, liver tissue and overexpressing cells. In contrast, the SYRO Ruby protein signal was weaker and more variable. In a direct comparison, ProQ Diamond staining was as sensitive but more specific than the 32 PO 4 labeling method. 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 ProQ Diamond staining is a sensitive method for measuring the phosphate levels of natriuretic peptide receptors.