Novel Galpha13 signaling partners: Serotonin 5HT4a receptor and vasodilator-stimulated phosphoprotein.

Novel Galpha13 signaling partners: Serotonin 5HT4a receptor and vasodilator-stimulated phosphoprotein

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

Zyxin is involved in thrombin signaling via interaction with PAR-1 receptor

Protease-activated receptor 1 (PAR-1) mediates thrombin signaling in human endothelial cells. As a G-protein-coupled receptor, PAR-1 transmits thrombin signal through activation of the heterotrimeric G proteins, Gi, Gq, and G12/13. In this study, we demonstrated that zyxin, a LIM-domain-containing protein, is involved in thrombin-mediated actin cytoskeleton remodeling and serum response element (SRE)-dependent gene transcription. We determined that zyxin binds to the C-terminal domain of PAR-1, providing a possible mechanism of involvement of zyxin as a signal transducer in PAR-1 signaling. Data showing that disruption of PAR-1-zyxin interaction inhibited thrombin-induced stress fiber formation and SRE activation supports this hypothesis. Similarly, depletion of zyxin using siRNA inhibited thrombin-induced actin stress fiber formation and SRE-dependent gene transcription. In addition, depletion of zyxin resulted in delay of endothelial barrier restoration after thrombin treatment. Notably, down-regulation of zyxin did not affect thrombin-induced activation of RhoA or Gi, Gq, and G12/13 heterotrimeric G proteins, implicating a novel signaling pathway regulated by PAR-1 that is not mediated by G-proteins. The observation that zyxin targets VASP, a partner of zyxin in regulation of actin assembly and dynamics, to focal adhesions and along stress fibers on thrombin stimulation suggests that zyxin may participate in thrombin-induced cytoskeletal remodeling through recruitment of VASP. In summary, this study establishes a crucial role of zyxin in thrombin signaling in endothelial cells and provides evidence for a novel PAR-1 signaling pathway mediated by zyxin.—Han, J., Liu, G., Profirovic, J., Niu, J., Voyno-Yasenetskaya, T. Zyxin is involved in thrombin signaling via interaction with PAR-1 receptor