Abstract
Receptor desensitization is a ubiquitous regulatory mechanism that defines the activatable pool of receptors, and thus, the ability of cells to respond to environmental stimuli. In recent years, the molecular mechanisms controlling the desensitization of a variety of receptors have been established. However, little is known about the molecular mechanisms that underlie desensitization of natriuretic peptide receptors, including natriuretic peptide receptor-A (NPR-A). Here we report that calcineurin (protein phosphatase 2B, PP2B, PPP3C) regulates homologous desensitization of NPR-A in murine Leydig tumor (MA-10) cells. We demonstrate that both pharmacological inhibition of calcineurin activity and siRNA-mediated suppression of calcineurin expression potentiate atrial natriuretic peptide (ANP)-induced cGMP synthesis. Treatment of MA-10 cells with inhibitors of other phosphoprotein phosphatases had little or no effect on ANP-induced cGMP accumulation. In addition, overexpression of calcineurin blunts ANP-induced cGMP synthesis. We also present data indicating that the inhibition of calcineurin potentiates ANP-induced testosterone production. To better understand the contribution of calcineurin in the regulation of NPR-A activity, we examined the kinetics of ANP-induced cGMP signals. We observed transient ANP-induced cGMP signals, even in the presence of phosphodiesterase inhibitors. Inhibition of both calcineurin and phosphodiesterase dramatically slowed the decay in the response. These observations are consistent with a model in which calcineurin mediated dephosphorylation and desensitization of NPR-A is associated with significant inhibition of cGMP synthesis. PDE activity hydrolyzes cGMP, thus lowering intracellular cGMP toward the basal level. Taken together, these data suggest that calcineurin plays a previously unrecognized role in the desensitization of NPR-A and, thereby, inhibits ANP-mediated increases in testosterone production.
Highlights
Atrial natriuretic peptide (ANP) is classically described as a cardiac hormone primarily stored within atrial granules
Hormonal regulation of steroidogenesis in MA-10 cells is similar to that observed in primary Leydig cells, making them well suited as a model system for this study [9,16,17]
The natriuretic peptide clearance receptor, NPR-C was not detected by western blot analysis. These results indicate that the guanylyl cyclase activity of natriuretic peptide receptor-A (NPR-A) is primarily responsible for cGMP production in MA-10 cells, and that activity of NPR-C is unlikely to alter the extracellular ANP level during in vitro assays
Summary
Atrial natriuretic peptide (ANP) is classically described as a cardiac hormone primarily stored within atrial granules. ANP is nearly as effective as luteinizing hormone in triggering testosterone production [1,9]. At the molecular/cellular level, the effects of ANP are primarily mediated through the particulate guanylyl cyclase activity of NPR-A [10,11,12]. It is known that in the basal state NPR-A is phosphorylated on six key residues, four Ser and two Thr [13]. A previous study has shown that binding of ANP triggers marked increases in guanylyl cyclase activity and facilitates subsequent dephosphorylation of the six key residues, resulting in homologous NPR-A desensitization [5]. The kinases(s) responsible for phosphorylating NPR-A and the phosphoprotein phosphatase(s) responsible for dephosphorylating NPR-A and the inhibition of NPR-A activity have yet to be identified [5]
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