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Browsing by Subject "Guanylyl Cyclase"

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    Activation and Inhibition Mechanisms of Membrane Guanylyl Cyclases
    (2013-04) Robinson, Jerid
    Guanylyl cyclase (GC)-A and GC-B are homologous enzymes that catalyze the formation of cyclic guanosine monophosphate and pyrophosphate from GTP. GC-A is activated by atrial natriuretic peptide and B-type natriuretic peptide and regulates the cardiovascular system. GC-B is activated by C-type natriuretic peptide and regulates the skeletal and female reproductive systems. Activation of GC-A and GC-B was hypothesized to occur through two steps, binding of natriuretic peptide and subsequent binding of ATP to the kinase homology domain. However, our group reported that ATP binding does not increase maximal velocity but reduces the Michaelis constant. My work revealed an allosteric ATP binding site in the catalytic domain. Mutation and structure/function studies indicated that the allosteric and catalytic sites are different and that GC-A and GC-B are asymmetric homodimers, not symmetric homodimers as had been previously suggested. Interestingly, a constitutively active mutant of GC-B mimicked an ATP bound state. ATP inhibits soluble guanylyl cyclases (sGC), and I demonstrated that physiological concentrations of ATP inhibit GC-A and GC-B. I went on to determine that the mechanism of inhibition was through binding the pyrophosphate-product site. Together, these data revealed how low and high concentrations of ATP activate and inhibit GC-A and GC-B, respectively.
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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.
    (2011-08) Yoder, Andrea Rae
    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.