Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i).
Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17beta (E(2), 10(-8) m) and acetylcholine (10(-5) m) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E(2)-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E(2) in COS-7 cells expressing ERalpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERalpha and specific Galpha proteins demonstrated E(2)-stimulated interaction between ERalpha and Galpha(i) but not between ERalpha and either Galpha(q) or Galpha(s); the observed ERalpha-Galpha(i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E(2)-stimulated ERalpha-Galpha(i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Galpha(i) into COS-7 cells expressing ERalpha and eNOS yielded a 3-fold increase in E(2)-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E(2) response. These findings indicate that eNOS stimulation by E(2) requires plasma membrane ERalpha coupling to Galpha(i) and that activated Galpha(i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERalpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.
Wyckoff
M H
MH
Departments of Pediatrics and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Chambliss
K L
KL
Mineo
C
C
Yuhanna
I S
IS
Mendelsohn
M E
ME
Mumby
S M
SM
Shaul
P W
PW
D000818:Animals; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011233:Precipitin Tests; D011960:Receptors, Estrogen / Q000378:metabolism*; D015398:Signal Transduction
D008565:Membrane Proteins; D011960:Receptors, Estrogen; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019206:GTP-Binding Protein alpha Subunits, Gi-Go