Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation

Abstract

The molecular mechanism of desensitization of soluble guanylyl cyclase (sGC), the NO receptor, has long remained unresolved. Posttranslational modification and redox state have been postulated to affect sGC sensitivity to NO but evidence has been lacking. We now show that sGC can be S-nitrosylated in primary aortic smooth muscle cells by S-nitrosocysteine (CSNO), an S-nitrosylating agent, in human umbilical vein endothelial cells after vascular endothelial growth factor treatment and in isolated aorta after sustained exposure to acetylcholine. Importantly, we show that S-nitrosylation of sGC results in decreased responsiveness to NO characterized by loss of NO-stimulated sGC activity. Desensitization of sGC is concentration-and time-dependent on exposure to CSNO, and sensitivity of sGC to NO can be restored and its S-nitrosylation prevented with cellular increase of thiols. We confirm in vitro with semipurified sGC that S-nitrosylation directly causes desensitization, suggesting that other cellular factors are not required. Two potential S-nitrosylated cysteines in the ␣-and ␤-subunits of sGC were identified by MS. Replacement of these cysteines, C243 in ␣ and C122 in ␤, created mutants that were mostly resistant to desensitization. Structural analysis of the region near ␤-C122 in the homologous Nostoc H-NOX crystal structure indicates that this residue is in the vicinity of the heme and its S-nitrosylation could dampen NO activation by affecting the positions of key residues interacting with the heme. This study suggests that S-nitrosylation of sGC is a means by which memory of NO exposure is kept in smooth muscle cells and could be a mechanism of NO tolerance.cGMP ͉ tolerance ͉ redox ͉ S-nitrosothiols I n the cardiovascular system, nitric oxide (NO) is critical for regulation of vascular tone and homeostasis (1, 2). In mammals, the main sensor of NO is the soluble guanylyl cyclase (sGC), a heme-containing heterodimer formed by an ␣-and a ␤-subunit. When NO binds to the heme, catalytic activity of sGC increases several hundredfold to produce the second messenger cGMP (3). Despite the importance of the NO-cGMP pathway in the biology and pathology of the cardiovascular and neuronal systems, modulation of sGC is poorly understood (4, 5). In particular, the mechanism of desensitization of sGC has remained unresolved despite 30 years of effort. Desensitization is the transition to a state in which sGC's response to a new NO stimulation is reduced or abolished. This direct effect on sGC differs from the desensitization of the NO-cGMP pathway caused by decrease in cGMP levels [e.g., because of increased phosphodiesterase activity (6)] or decrease in NO availability (7). Desensitization of sGC itself has been reported in various cell types and tissues after exposure to NO (8-11) but its mechanism is unknown. Ser/Thr phosphorylation was proposed to be involved but evidence is still lacking (12), and Tyr phosphorylation was ruled out recently (13). Elucidating the mechanism of sGC desensitization is crucial considering i...

Aldosterone Increases Oxidant Stress to Impair Guanylyl Cyclase Activity by Cysteinyl Thiol Oxidation in Vascular Smooth Muscle Cells

“…Analysis of non-mammalian proteins that are structurally similar to GC in which Cys-122 is highly conserved has revealed that subtle conformational changes at Cys-122 may limit NO ⅐ binding at the sixth coordinating position of reduced heme (at His-105). This, in turn, would prevent the nitrosyl-heme formation that is necessary for GC activation (18,28).…”

“…Site-directed Mutagenesis and Transfection-Expression constructs in pCMV5 were used to overexpress ␣ 1 -, ␤ 1 -, and ␤ 1 -C122A subunits of rat GC as described previously (18). COS-7 cells, which do not express endogenous GC, were plated in 6-well tissue culture dishes and transfected with 5 g of DNA for 4 h with Lipofectamine 2000 TM (Invitrogen) (24).…”

“…These cysteines were selected for analysis based on prior studies that implicated them as integral to normal NO ⅐ sensing by GC (10,18). Immunoprecipitation of GC using a ␤ 1 -subunit antibody was performed on VSMC exposed to aldosterone for 24 h, and after separation of peptides by capillary column liquid chromatography, MS1 scanning suggested potential oxidative modification of Cys-122 (peptide sequence: CTDADKGK).…”

“…In the presence of excess ROS, it has been suggested that GC enzymatic function may be decreased by: (i) a peroxynitrite (ONOO Ϫ )-mediated decrease in GC specific activity (15); (ii) oxidation of the ␤ 1 -subunit-associated prosthetic heme group that converts GC to an NO ⅐ -insensitive state (14); (iii) oxidation-induced disulfide formation of ␤ 1 -subunit-thiol groups (16,17); or (iv) NO ⅐ -dependent posttranslational modification (e.g. S-nitrosation) of a ␤ 1 -subunit-thiol (18). Based on these observations, we hypothesized that aldosterone-mediated ROS formation promotes oxidative posttranslational modification of GC and conversion of the enzyme to an NO ⅐ -insensitive state, thereby disrupting a key pathway essential for normal VSMC relaxation.…”

Dynamic Ligand Exchange in Soluble Guanylyl Cyclase (sGC)

“…These include oxidation of sGC heme and subsequent protein degradation (51,52) and reduced sGC activity in response to S-nitrosylation (30,31). Although it is clear that these mechanisms play an important role in desensitization of sGC signaling under chronic oxidative conditions, their contribution to rapid desensitization of sGC in response to NO (46) is unclear.…”