Direct interaction between endothelial nitric-oxide synthase and dynamin-2. Implications for nitric-oxide synthase function.
Endothelial nitric-oxide synthase (eNOS) is regulated in part through specific protein interactions. Dynamin-2 is a large GTPase residing within similar membrane compartments as eNOS. Here we show that dynamin-2 binds directly with eNOS thereby augmenting eNOS activity. Double label confocal immunofluorescence demonstrates colocalization of eNOS and dynamin in both Clone 9 cells cotransfected with green fluorescent protein-dynamin and eNOS, as well as in bovine aortic endothelial cells (BAEC) expressing both proteins endogenously, predominantly in a Golgi membrane distribution. Immunoprecipitation of eNOS from BAEC lysate coprecipitates dynamin and, conversely, immunoprecipitation of dynamin coprecipitates eNOS. Additionally, the calcium ionophore, a reagent that promotes nitric oxide release, enhances coprecipitation of dynamin with eNOS in BAEC, suggesting the interaction between the proteins can be regulated by intracellular signals. In vitro studies demonstrate that glutathione S-transferase (GST)-dynamin-2 quantitatively precipitates both purified recombinant eNOS protein as well as in vitro transcribed (35)S-labeled eNOS from solution indicating a direct interaction between the proteins in vitro. Scatchard analysis of binding studies demonstrates an equilibrium dissociation constant (K(d)) of 27.6 nm. Incubation of purified recombinant eNOS protein with GST-dynamin-2 significantly increases eNOS activity as does overexpression of dynamin-2 in ECV 304 cells stably transfected with eNOS-green fluorescent protein. These studies demonstrate a direct protein-protein interaction between eNOS and dynamin-2, thereby identifying a new NOS-associated protein and providing a novel function for dynamin. These events may have relevance for eNOS regulation and trafficking within vascular endothelium.
Cao
S
S
GI Research Unit, Anesthesia Research and Department of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
Yao
J
J
McCabe
T J
TJ
Yao
Q
Q
Katusic
Z S
ZS
Sessa
W C
WC
Shah
V
V
D000818:Animals; D001011:Aorta / Q000166:cytology; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D034284:Dynamin I; D034281:Dynamins; D004730:Endothelium, Vascular / Q000166:cytology; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D007476:Ionophores; D011993:Recombinant Fusion Proteins; D000001:Calcimycin; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D005982:Glutathione Transferase; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins