Guanylyl cyclase (GC)-A and B are homologous, peptide-activated, cGMP synthesizing enzymes that regulate blood pressure, heart size, long bone growth and oocyte maturation. Hence, they are desirable drug targets. However, lack of regulatory information prevents maximal therapeutic utilization of these enzymes for the treatment of cardiovascular, skeletal and reproductive diseases. Adenine nucleotides regulate GC-A and GC-B by binding unidentified intracellular high affinity activation and low affinity inhibitory sites through undefined mechanisms. Both receptors are highly phosphorylated in resting cells and dephosphorylated receptors are unresponsive to natriuretic peptides (NPs). Hormones that oppose the actions of NPs elevate intracellular calcium, which causes the dephosphorylation of all receptor phosphorylation sites. In contrast, activated protein kinase C (PKC) is hypothesized to phosphorylate a conserved receptor consensus site that reduces phosphorylation of a separate critical regulatory site. The long-term objective of this application is to determine how hormones, adenine nucleotides and phosphorylation regulate GC-A and GC-B. We intend to accomplish this objective by pursuing the following four specific aims: 1) Determine how adenine nucleotides regulate GC- A and GC-B, 2) Identify how PKC inhibits GC-B, 3) Determine how disease-causing missense mutations affect GC-B function, and 4) Identify how hormones inhibit GC-B.
The first aim will measure the effects of structurally unique and reactive purines on the kinetic properties and binding sites of GC-A and GC-B. Receptors containing mutations in purine binding sites will determine whether the catalytic domains are symmetric or asymmetric homodimers.
The second aim will determine how PKC inhibits GC-B by identifying the requisite PKC isoform and phosphorylation sites using siRNA knockdown, in vitro kinase assays and phosphomimetic mutants.
The third aim will determine how each of the twelve dwarfism causing missense mutations inactivate GC-B as well as how a newly discovered mutation that leads to skeletal overgrowth constitutively activates GC-B. Effects of these mutations on 125I-CNP binding, guanylyl cyclase activity, post-translational processing and cellular localization will be determined. Finally, the fourth aim will investigate how hormones inactivate GC- B in mouse follicles, granulosa cells, chondrocytes and smooth muscle cells by assessing the requirements for calcium elevations, PKC activation, and various receptor phosphorylation sites. The proposed research is significant because the successful completion of these specific aims will advance understanding of hormone-, adenine nucleotide- and phosphorylation-dependent regulation of GC-A and GC-B and may reveal new therapeutic targets.

Public Health Relevance

The proposed research is relevant to public health because it investigates activation and inactivation mechanisms for receptors that compensate for heart failure, stimulate skeletal growth and regulate ovulation in humans. Hence, it is expected to lead to new therapeutics for the treatment of cardiovascular, skeletal and reproductive diseases. The proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will help reduce the burden of human disability.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Dunsmore, Sarah
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University of Minnesota Twin Cities
Schools of Medicine
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