| C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family, which also includes atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). ANP and BNP are cardiac hormones that lower blood pressure by stimulating natriuresis, diuresis and vasorelaxation. CNP signals in a paracrine manner to inhibit cell contraction and proliferation in the cardiovascular system and to stimulate chondrocyte proliferation and long bone growth. CNP binds and activates guanylyl cyclase B receptor (NPR-B), which converts GTP into the second messenger cGMP. In contrast, pressor hormones such as arginine-vasopressin (AVP), and the bioactive lipids, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), elicit the opposite physiologic effects. The goal of this thesis was to evaluate the regulation of NPR-B by vasoactive agents that antagonizing the actions of natriuretic peptides. Specifically, I determined whether AVP, LPA and SIP inhibited NPR-B activity in physiologically relevant cell systems, and then investigated the mechanism behind these effects. I demonstrate a potent acute inhibitory effect of AVP, SIP and LPA on NPR-B activity. In all cases, the inhibition is concentration-dependent, reversible, rapid, and is a direct result of decreased enzymatic activity. The mechanism of the AVP, LPA and S1P-dependent desensitization did not require NPR-B degradation or protein kinase C activation, but did require elevated calcium concentrations. The most well studied mechanism of heterologous desensitization of NPR-B is phorbol ester-induced activation of protein kinase C (PKC), which results in the specific dephosphorylation of Serine-523. I compared the effects of calcium elevations and PKC activation on the kinetic properties and phosphorylation state of NPR-B. I found that while PMA-induced inhibition leads to the dephosphorylation of Serine-523, calcium elevations result in global dephosphorylation. Furthermore, the major kinetic effect of PKC activation is an increase in Km of substrate binding, whereas calcium-dependent inhibition decreases the maximal enzymatic velocity (Vmax). The final goal of my thesis project was to identify proteins that interact with NPR-B in order to determine the identity of natriuretic peptide receptor regulatory proteins. I found that male enhanced antigen-2 and a predicted protein product, FLJ46072, interact specifically with NPR-B in immunoprecipitations from 293T cells overexpressing NPR-B. |
