Novel mechanisms of G protein-dependent regulation of endothelial nitric-oxide synthase.
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 alpha subunit of heterotrimeric G12 protein (G alpha12) can form a complex with eNOS in an activation- and Hsp90-independent manner. Our data show that G alpha12 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 alpha12 increases the expression of eNOS by increasing half-life of both eNOS protein and eNOS mRNA. Small interfering RNA-mediated depletion of endogenous G alpha12 decreases eNOS levels. A quantitative correlation can be detected between the extent of down-regulation of G alpha12 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.
Andreeva
Alexandra V
AV
Department of Pharmacology, University of Illinois, College of Medicine, Chicago, IL 60612, USA.
Vaiskunaite
Rita
R
Kutuzov
Mikhail A
MA
Profirovic
Jasmina
J
Skidgel
Randal A
RA
Voyno-Yasenetskaya
Tatyana
T
D000818:Animals; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation; D004795:Enzyme Stability; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D020871:RNA Stability; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D018841:HSP90 Heat-Shock Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D052250:Nitric Oxide Synthase Type III; D013917:Thrombin; D044365:GTP-Binding Protein alpha Subunits, G12-G13