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

Outline of the chloramphenicol (Cat) gene mutagenesis and assembly experiment

<p><b>Copyright information:</b></p><p>Taken from "USER™ friendly DNA engineering and cloning method by uracil excision"</p><p></p><p>Nucleic Acids Research 2007;35(6):1992-2002.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1874603.</p><p>© 2007 The Author(s)</p> () PCR primer design strategy. Lines with the arrowheads symbolize priming sequences in the PCR primers. Left and Right cloning primers contain 5′ extensions compatible with the single-stranded extensions on the linearized pNEB206A vector. The overlapping primers P1/P2 and P3/P4 are selected in the vicinity of the targeted mutations. Asterisks designate the point mismatches in the primers compared to the template sequence. Overlapping primers are complementary to each other within the boxed sequences. The 3′ dT of the boxed sequence in the primer sequences is replaced by dU. () Schematic representation of six PCR products amplified using the indicated primers. Cycling conditions are described in Materials and Methods. The label, Cat926 etc. shows the PCR fragment size in bp. The ends of the PCR products are flanked by the compatible overlapping sequences with a single dU residue at the junction. () Four assembly reactions were carried out using the indicated PCR fragments to assemble a full-length Cat gene carrying the indicated phenotype into pNEB206A