Differential regulation of NPR-B/GC-B by protein kinase c and calcium.

C-type natriuretic peptide (CNP) activation of the guanylyl cyclase-linked natriuretic peptide receptor-B (NPR-B) stimulates vasorelaxation and bone growth. Hormones and phorbol esters (PMA) inhibit NPR-B in calcium and protein kinase c-dependent manners, respectively. Here, we characterize the kinetic properties of NPR-B in membranes from cells exposed to PMA, the calcium ionophore, ionomycin, or sphingosine-1-phosphate (S1P). PMA and ionomycin primarily increased the K(m) and decreased the V(max) of NPR-B for GTP, respectively, whereas S1P caused modest changes in both parameters. PMA and S1P treatment increased the EC50 for CNP activation by eight- and three-fold, whereas ionomycin was ineffective. All three agents caused NPR-B dephosphorylation, but the basis for the loss of phosphate differed between treatments. In vitro phosphorylation of NPR-B in membranes was markedly diminished by prior whole cell PMA or S1P exposure, whereas ionomycin pretreatment had no effect. The involvement of the known phosphorylated residues in each process was tested with a mutant receptor containing glutamates substituted for these sites. While the effect of PMA was lost on this receptor, the effects of S1P and ionomycin were only partially blocked. Our data suggest that the molecular bases for PMA- and calcium-dependent inhibition of NPR-B are unique. The former results from reduced phosphorylation of a known site and primarily affects the affinity of NPR-B for CNP and GTP. The latter is associated with reductions in maximal velocities by a mechanism that does not involve inhibition of NPR-B phosphorylation and requires a process in addition to the dephosphorylation of the known sites.

ATP-independent activation of natriuretic peptide receptors.

Natriuretic peptide receptor A (NPR-A) is an essential cardiovascular regulator that is stimulated by atrial natriuretic peptide and B-type natriuretic peptide, whereas natriuretic peptide receptor B (NPR-B) stimulates long bone growth in a C-type natriuretic peptide-dependent manner. Many reports indicate that ATP is essential for NPR-A and NPR-B activation. Current models suggest that natriuretic peptide binding to receptor extracellular domains causes ATP binding to intracellular kinase homology domains, which derepresses adjacent catalytic domains. Here, we report 100-fold activations of natriuretic peptide receptors in the absence of ATP. The addition of a nonhydrolyzable ATP analog had no effect at early time periods (measured in seconds) but increased cGMP production about 2-fold after longer incubations (measured in minutes), consistent with a stabilization, not activation, mechanism. These data indicate that ATP does not activate natriuretic peptide receptors as has been repeatedly reported. Instead, ATP increases activity primarily by maintaining proper receptor phosphorylation status but also serves a previously unappreciated enzyme stabilizing function.

Calcium-dependent dephosphorylation mediates the hyperosmotic and lysophosphatidic acid-dependent inhibition of natriuretic peptide receptor-B/guanylyl cyclase-B.

C-type natriuretic peptide binding to natriuretic peptide receptor-B (NPR-B) stimulates cGMP synthesis, which regulates vasorelaxation, cell proliferation, and bone growth. Here, we investigated the mechanistic basis for hyperosmotic and lysophosphatidic acid-dependent inhibition of NPR-B. Whole cell cGMP measurements and guanylyl cyclase assays indicated that acute hyperosmolarity decreased NPR-B activity in a reversible, concentration- and time-dependent manner, whereas chronic exposure had no effect. Acute hyperosmolarity elevated intracellular calcium in a concentration-dependent fashion that paralleled NPR-B desensitization. A calcium chelator, but not a protein kinase C inhibitor, blocked both calcium elevations and desensitization. Hyperosmotic medium stimulated NPR-B dephosphorylation, and the receptor was rapidly rephosphorylated and resensitized when the hypertonic media was removed. Lysophosphatidic acid also inhibited NPR-B in a calcium- and phosphorylation-dependent process, consistent with calcium being a universal regulator of NPR-B. The absolute requirement of dephosphorylation in this process was demonstrated by showing that a receptor with glutamates substituted at all known NPR-B phosphorylation sites is unresponsive to hyperosmotic stimuli. This is the first study to measure the phosphorylation state of an endogenous guanylyl cyclase and to link intracellular calcium elevations with its dephosphorylation.

Sphingosine-1-phosphate inhibits C-type natriuretic peptide activation of guanylyl cyclase B (GC-B/NPR-B).

C-type natriuretic peptide (CNP) binds and activates the transmembrane guanylyl cyclase B receptor (NPR-B), which decreases vascular tone and inhibits cell proliferation and migration. In contrast, the bioactive lipid sphingosine-1-phosphate (S1P) elicits the opposite physiological effects. Here, we demonstrate a potent acute inhibitory effect of S1P on NPR-B activity in NIH3T3 fibroblasts and A10 vascular smooth muscle cells. In fibroblasts, S1P reduced CNP-dependent cGMP elevations to the same levels as 10% fetal bovine serum, the most potent NPR-B desensitizing agent known. The reduction was dose-dependent (IC50=0.08 micromol/L) and due to decreased NPR-B activity because CNP-dependent guanylyl cyclase activities were markedly diminished in membranes prepared from S1P-treated cells. Similarly, in A10 cells, S1P inhibition was rapid (t1/2=2 to 5 minutes), dose-dependent (IC50=0.3 micromol/L S1P), and mediated by a cell surface receptor. The mechanism of the S1P-dependent desensitization in A10 cells did not require NPR-B degradation or protein kinase C activation, but did require elevated calcium concentrations because a nonspecific calcium ionophore also inhibited NPR-B and an intracellular calcium chelator blocked a significant portion of the S1P response. These are the first data demonstrating cross-talk between the natriuretic peptide and S1P signaling systems. They suggest that the effects of S1P on vascular disease and wound healing may be mediated in part through inhibition of NPR-B.