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In vivo effects of a GPR30 antagonist.

Dennis MK, Burai R, Ramesh C, Petrie WK, Alcon SN, Nayak TK, Bologa CG, Leitao A, Brailoiu E, Deliu E, Dun NJ, Sklar LA, Hathaway HJ, Arterburn JB, Oprea TI, Prossnitz ER - Nat. Chem. Biol. (2009)

Bottom Line: Here we describe the identification and characterization of G15 (2), a G-1 analog that binds to GPR30 with high affinity and acts as an antagonist of estrogen signaling through GPR30.In vivo administration of G15 revealed that GPR30 contributes to both uterine and neurological responses initiated by estrogen.The identification of this antagonist will accelerate the evaluation of the roles of GPR30 in human physiology.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology & Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA.

ABSTRACT
Estrogen is central to many physiological processes throughout the human body. We have previously shown that the G protein-coupled receptor GPR30 (also known as GPER), in addition to classical nuclear estrogen receptors (ER and ER), activates cellular signaling pathways in response to estrogen. In order to distinguish between the actions of classical estrogen receptors and GPR30, we have previously characterized G-1 (1), a selective agonist of GPR30. To complement the pharmacological properties of G-1, we sought to identify an antagonist of GPR30 that displays similar selectivity against the classical estrogen receptors. Here we describe the identification and characterization of G15 (2), a G-1 analog that binds to GPR30 with high affinity and acts as an antagonist of estrogen signaling through GPR30. In vivo administration of G15 revealed that GPR30 contributes to both uterine and neurological responses initiated by estrogen. The identification of this antagonist will accelerate the evaluation of the roles of GPR30 in human physiology.

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G15 antagonism of PI3K activation by GPR30. The activity of G15 was evaluated using COS7 cells transfected with Akt-PH-mRFP1 and either GPR30-GFP (a), ERα-GFP or ERβ-GFP (b) or SKBr3 cells transfected with Akt-PH-mRFP1 (c). 17β-estradiol, G-1 and G15 were used at the indicated concentrations. The white bar in upper panel of (a-c) denotes 10 μm for all images. Data are representative of three independent experiments.
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Figure 4: G15 antagonism of PI3K activation by GPR30. The activity of G15 was evaluated using COS7 cells transfected with Akt-PH-mRFP1 and either GPR30-GFP (a), ERα-GFP or ERβ-GFP (b) or SKBr3 cells transfected with Akt-PH-mRFP1 (c). 17β-estradiol, G-1 and G15 were used at the indicated concentrations. The white bar in upper panel of (a-c) denotes 10 μm for all images. Data are representative of three independent experiments.

Mentions: In addition to intracellular calcium mobilization, we have demonstrated that estrogen stimulation of ERα, ERβ or GPR30 results in the nuclear accumulation of PIP3 as a result of PI3K activation6, revealed by the translocation of an Akt PH domain-fluorescent protein fusion protein reporter24. To determine whether G15 similarly inhibits GPR30-mediated PI3K activation, we examined the activation of PI3K in receptor-transfected COS7 cells, where estrogen stimulates the nuclear accumulation of PIP3 through all three receptors and G-1 selectively activates GPR30 but not ERα or ERβ. Not only was G15 capable of inhibiting the G-1-mediated activation of PI3K in GPR30-transfected cells, it also effectively blocked the estrogen-mediated response in GPR30-transfected cells (Fig. 4a) but had no effect on the estrogen-mediated response in ERα or ERβ-transfected cells, even at concentrations 100-fold greater than that required to inhibit GPR30 (Fig. 4b). To determine whether G15 inhibits PI3K activation in cells endogenously expressing GPR30, we examined PI3K activation in SKBr3 breast cancer cells. As in GPR30-transfected cells, G15 was able to inhibit both estrogen and G-1 stimulation of PI3K (Fig. 4c). In total, these results demonstrate that G15 can selectively inhibit GPR30.


In vivo effects of a GPR30 antagonist.

Dennis MK, Burai R, Ramesh C, Petrie WK, Alcon SN, Nayak TK, Bologa CG, Leitao A, Brailoiu E, Deliu E, Dun NJ, Sklar LA, Hathaway HJ, Arterburn JB, Oprea TI, Prossnitz ER - Nat. Chem. Biol. (2009)

G15 antagonism of PI3K activation by GPR30. The activity of G15 was evaluated using COS7 cells transfected with Akt-PH-mRFP1 and either GPR30-GFP (a), ERα-GFP or ERβ-GFP (b) or SKBr3 cells transfected with Akt-PH-mRFP1 (c). 17β-estradiol, G-1 and G15 were used at the indicated concentrations. The white bar in upper panel of (a-c) denotes 10 μm for all images. Data are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 4: G15 antagonism of PI3K activation by GPR30. The activity of G15 was evaluated using COS7 cells transfected with Akt-PH-mRFP1 and either GPR30-GFP (a), ERα-GFP or ERβ-GFP (b) or SKBr3 cells transfected with Akt-PH-mRFP1 (c). 17β-estradiol, G-1 and G15 were used at the indicated concentrations. The white bar in upper panel of (a-c) denotes 10 μm for all images. Data are representative of three independent experiments.
Mentions: In addition to intracellular calcium mobilization, we have demonstrated that estrogen stimulation of ERα, ERβ or GPR30 results in the nuclear accumulation of PIP3 as a result of PI3K activation6, revealed by the translocation of an Akt PH domain-fluorescent protein fusion protein reporter24. To determine whether G15 similarly inhibits GPR30-mediated PI3K activation, we examined the activation of PI3K in receptor-transfected COS7 cells, where estrogen stimulates the nuclear accumulation of PIP3 through all three receptors and G-1 selectively activates GPR30 but not ERα or ERβ. Not only was G15 capable of inhibiting the G-1-mediated activation of PI3K in GPR30-transfected cells, it also effectively blocked the estrogen-mediated response in GPR30-transfected cells (Fig. 4a) but had no effect on the estrogen-mediated response in ERα or ERβ-transfected cells, even at concentrations 100-fold greater than that required to inhibit GPR30 (Fig. 4b). To determine whether G15 inhibits PI3K activation in cells endogenously expressing GPR30, we examined PI3K activation in SKBr3 breast cancer cells. As in GPR30-transfected cells, G15 was able to inhibit both estrogen and G-1 stimulation of PI3K (Fig. 4c). In total, these results demonstrate that G15 can selectively inhibit GPR30.

Bottom Line: Here we describe the identification and characterization of G15 (2), a G-1 analog that binds to GPR30 with high affinity and acts as an antagonist of estrogen signaling through GPR30.In vivo administration of G15 revealed that GPR30 contributes to both uterine and neurological responses initiated by estrogen.The identification of this antagonist will accelerate the evaluation of the roles of GPR30 in human physiology.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology & Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA.

ABSTRACT
Estrogen is central to many physiological processes throughout the human body. We have previously shown that the G protein-coupled receptor GPR30 (also known as GPER), in addition to classical nuclear estrogen receptors (ER and ER), activates cellular signaling pathways in response to estrogen. In order to distinguish between the actions of classical estrogen receptors and GPR30, we have previously characterized G-1 (1), a selective agonist of GPR30. To complement the pharmacological properties of G-1, we sought to identify an antagonist of GPR30 that displays similar selectivity against the classical estrogen receptors. Here we describe the identification and characterization of G15 (2), a G-1 analog that binds to GPR30 with high affinity and acts as an antagonist of estrogen signaling through GPR30. In vivo administration of G15 revealed that GPR30 contributes to both uterine and neurological responses initiated by estrogen. The identification of this antagonist will accelerate the evaluation of the roles of GPR30 in human physiology.

Show MeSH