Regulation of natriuretic peptide system activation:effects of spontaneous natriuretic peptide mutations and identification of novel natriuretic peptide receptor phosphorylation sites.

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Regulation of natriuretic peptide system activation:effects of spontaneous natriuretic peptide mutations and identification of novel natriuretic peptide receptor phosphorylation sites.

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2011-08

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Guanylyl cyclase A and B (GC-A and GC-B) are transmembrane guanylyl cyclase receptors that mediate the physiologic effects of natriuretic peptides, including reduction of blood pressure and blood volume, stimulation of long bone growth, and regulation of cardiac size. Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) signal through GC-A while C-type natriuretic peptide (CNP) signals through GCB. This thesis explores several aspects of the natriuretic peptide system, including ligand-receptor interactions, ligand proteolysis, identification of receptor protein-protein interactions, as well as receptor regulation by phosphorylation. Our studies show that even small alterations in the primary sequence of natriuretic peptides can drastically alter receptor binding and activation leading to severe physiologic defects. A naturally occurring point mutation in mouse CNP (CNPlbab) leads to a dwarfism phenotype caused by reduced binding to and activation of GC-B. A naturally occurring familial mutation correlated with early onset atrial fibrillation which alters the length of ANP (fsANP) does not alter ligand-receptor activation but inhibits degradation of the peptide. GC-A and GC-B are most active when fully phosphorylated, and loss of phosphate content is associated with a loss of enzymatic activity. Here, we used mass spectrometry to identify phosphorylation sites in GC-A and GC-B from both rat and human species. Novel, uncharacterized phosphorylation sites were observed at S487 in GC-A and T529 in GC-B. In addition to our mass spectrometry approach, we employed a functional screen to identify a putative phosphorylation site within the N-terminal portion of the kinase homology domains of GC-A and GC-B at S473 and S489. Consistent with the function of known guanylyl cyclase phosphorylation sites, glutamate or alanine substitutions for this site increased or decreased hormone-dependent guanylyl cyclase activity, respectively. Attempts to detect phosphorylation at S473/S489 from cells stably overexpressing GC-A or GC-B by mass spectrometry were unsuccessful due to chemical properties of the tryptic peptide. Additionally, studies on the effect of phosphorylation status on homologous desensitization showed that, dephosphorylation of known phosphorylation sites is not required for homologous desensitization in either GC-A or GC-B as previously thought.

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University of Minnesota Ph.D. dissertation. August 2011. Major: Pharmacology. Advisor:Lincoln R. Potter. 1 computer file (PDF); xii, 221 pages.

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Yoder, Andrea Rae. (2011). Regulation of natriuretic peptide system activation:effects of spontaneous natriuretic peptide mutations and identification of novel natriuretic peptide receptor phosphorylation sites.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/116169.

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