A sensitive method for determining the phosphorylation status of natriuretic peptide receptors: cGK-Ialpha does not regulate NPR-A.
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.
Bryan
Paula M
PM
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, Minnesota 55455, USA.
Smirnov
Dmitri
D
Smolenski
Albert
A
Feil
Susanne
S
Feil
Robert
R
Hofmann
Franz
F
Lohmann
Suzanne
S
Potter
Lincoln R
LR
D000818:Animals; D002478:Cells, Cultured; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D051379:Mice; D010766:Phosphorylation; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors; D014162:Transfection; D000099242:Atrial Natriuretic Factor Receptor B
D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D047908:Intracellular Signaling Peptides and Proteins; D017461:Receptors, Atrial Natriuretic Factor; D000099242:Atrial Natriuretic Factor Receptor B; C015482:protein kinase modulator; C569720:Prkg1 protein, mouse; C091715:atrial natriuretic factor receptor A