Endothelial nitric oxide synthase interactions with G-protein-coupled receptors.
The endothelial nitric oxide synthase (eNOS) is activated in response to stimulation of endothelial cells by a number of vasoactive substances including, bradykinin (BK), angiotensin II (Ang II), endothelin-1 (ET-1) and ATP. In the present study we have used in vitro activity assays of purified eNOS and in vitro binding assays with glutathione S-transferase fusion proteins to show that the capacity to bind and inhibit eNOS is a common feature of membrane-proximal regions of intracellular domain 4 of the BK B2, the Ang II AT1 and the ET-1 ETB receptors, but not of the ATP P2Y2 receptor. Phosphorylation of serine or tyrosine residues in the eNOS-interacting region of the B2 receptor results in a loss of eNOS inhibition due to a decrease in the binding affinity of the receptor domain for the eNOS enzyme. Furthermore, the B2 receptor is transiently phosphorylated on tyrosine residues in cultured endothelial cells in response to BK stimulation. Phosphorylation occurs during the time in which eNOS transiently dissociates from the receptor accompanied by a transient increase in nitric oxide production. Taken together, these data support the hypotheses that eNOS is regulated in endothelial cells by reversible and inhibitory interactions with G-protein-coupled receptors and that these interactions can be modulated by receptor phosphorylation.
Marrero
M B
MB
Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2500, USA.
Venema
V J
VJ
Ju
H
H
He
H
H
Liang
H
H
Caldwell
R B
RB
Venema
R C
RC
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D043782:Receptor, Bradykinin B2; D044023:Receptor, Endothelin B; D011945:Receptors, Angiotensin / Q000737:chemistry / Q000378:metabolism*; D018002:Receptors, Bradykinin / Q000737:chemistry / Q000378:metabolism*; D017466:Receptors, Endothelin / Q000737:chemistry / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D002147:Calmodulin; D008024:Ligands; C546385:P2RY2 protein, human; D010446:Peptide Fragments; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D043782:Receptor, Bradykinin B2; D044023:Receptor, Endothelin B; D011945:Receptors, Angiotensin; D018002:Receptors, Bradykinin; D017466:Receptors, Endothelin; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D009569:Nitric Oxide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019204:GTP-Binding Proteins; D001920:Bradykinin