Novel Mechanisms of G Protein-Dependent Regulation of Endothelial Nitric-Oxide Synthase

Abstract

Endothelial nitric-oxide synthase (eNOS) plays a crucial role in the regulation of a variety of cardiovascular and pulmonary functions in both normal and pathological conditions. Multiple signaling inputs, including calcium, caveolin-1, phosphorylation by several kinases, and binding to the 90-kDa heat shock protein (Hsp90), regulate eNOS activity. Here, we report a novel mechanism of G protein-dependent regulation of eNOS. We demonstrate that in mammalian cells, the ␣ subunit of heterotrimeric G12 protein (G␣ 12 ) can form a complex with eNOS in an activation-and Hsp90-independent manner. Our data show that G␣ 12 does not affect eNOS-specific activity, but it strongly enhances total eNOS activity by increasing cellular levels of eNOS. Experiments using inhibition of protein or mRNA synthesis show that G␣ 12 increases the expression of eNOS by increasing half-life of both eNOS protein and eNOS mRNA. Small interfering RNA-mediated depletion of endogenous G␣ 12 decreases eNOS levels. A quantitative correlation can be detected between the extent of down-regulation of G␣ 12 and eNOS in endothelial cells after prolonged treatment with thrombin. G protein-dependent increase of eNOS expression represents a novel mechanism by which heterotrimeric G proteins can regulate the activity of downstream signaling molecules.

A Signaling Pathway Linking Nitric Oxide Production to Heterotrimeric G Protein and Hydrogen Peroxide Regulates Extracellular Calmodulin Induction of Stomatal Closure in Arabidopsis

“…However, Ga regulation of NO generation in guard cells has not been reported. In mammalian cells, heterotrimeric G protein regulates endothelial NOS activity by increasing cellular levels of endothelial NOS (Andreeva et al, 2006). If such G protein-dependent regulation of NO generation exists in Arabidopsis, NO Figure 8.…”

Endothelial Nitric-Oxide Synthase Reveals a New Face in G Protein Signaling: Fig. 1.

“…First, what are the specificities of eNOS for G␣ 12 versus other G␣ and, conversely, of G␣ 12 for eNOS versus other NOS isoforms? Andreeva et al (2006) reveal that in addition to G␣ 12 , G␣ 13 and possibly G␣ s can affect eNOS expression and that G␣ q , G␣ z , and G␤␥ lack effects on eNOS expression (A. V. Andreeva R. Vaiskunaite, M. A. Kutuzov, J. Profirovic, R. A. Skidgel, T. VoynoYasenetskaya, unpublished observations). Others have shown that G␣ 13 increases inducible NOS expression in a renal epithelial cell line by transcriptional and possibly posttranscriptional mechanisms (G␣ 13 effects on inducible NOS mRNA and protein stability were not measured; Kitamura et al, 1996).…”

“…Based on this study and other published data, Andreeva et al (2006) hypothesized a macromolecular interaction between G␣ 12 and eNOS and sought to characterize how this might impact eNOS activity and how formation of such a complex might be regulated. Using coimmunoprecipitation of overexpressed G␣ 12 and eNOS, the authors provide evidence for the existence of a complex containing the two proteins.…”

“…In this issue of Molecular Pharmacology, Andreeva et al (2006) explore a proposed functional link between G␣ 12 and endothelial nitric-oxide synthase (eNOS). eNOS is one of three NOS isoforms in mammalian cells that generate nitric oxide (NO) from L-arginine.…”

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

“…endothelial nitric oxide synthase (Andreeva et al 2006), a part of cellular signal transduction machineries. Thus, we are far away from understanding the complexity of heterotrimeric G proteinmediated signal transduction pathways on the cellular level.…”