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