S-nitrosylation of PDE5 contributes to the desensitization of the NO/cGMP signaling pathway

The "classical" mode of signaling by NO is via activation of soluble guanylyl cyclase (sGC) and accumulation of intracellular cGMP. Increasingly, signaling by NO is being shown to result from covalent addition of NO to Cys-thiols in proteins, a posttranslational protein modification termed S-Nitrosylation that can modify protein structure and function. Using a proteomic method for unbiased identification of S-nitrosoproteins, we discovered C220 in PDE5 (human) as a preferred site for NO addition. This cysteine resides within a recognized regulatory loop of PDE5 (in the GAFA domain) and is poised to modulate PDE5 activity. Importantly, PDE5 is the cGMP-binding cGMP-specific PDE that catalyses the rapid conversion of cGMP to 5'GMP. Binding of cGMP to the GAFA domain of PDE5 acts via an allosteric mechanism to enhance phosphodiesterase activity, providing for rapid termination of the NO/cGMP signaling pathway in many cells. We found that exposure of PDE5 to an NO-donor decreases catalytic activity and mutagenesis experiments confirmed that this effect requires S-nitrosylation of C220. PDE5 activity was previously shown to be upregulated by phosphorylation of S102 (human)---although this effect on catalysis is modest, phosphorylation was inferred to be responsible for physiological desensitization of the NO/cGMP pathway. Results of activity studies of phosphorylated PDE5 and C220/S102 mutants revealed that C220 may play a key role in the activity of phosphorylated PDE5 and that phosphorylation and nitrosylation act in concert to synergistically accelerate PDE5 desensitization and thereby shape cGMP signaling. Indeed, using an engineered cell system that reconstitutes the NO/cGMP/PDE5 signaling pathway, we demonstrate that S-nitrosylation and phosphorylation are required for desensitization of the NO/cGMP signaling pathway. Thus, in addition to NO activating the cGMP/PDE5 signaling pathway by nitrosylation of heme-iron in sGC, NO also promotes desensitization of the pathway through S-nitrosylation of C220 in PDE5. Activation of the cGMP/PDE5 pathway by NO and regulation by S-nitrosylation reveals NO to have an even tighter control on this pathway than previously appreciated and expands our understanding of the molecular mechanisms for desensitization of cGMP signaling in blood vessels and the brain.