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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 intracellular calcium mobilization by GPR30. (a) The effect of G15 on the subsequent mobilization of calcium by G1, E2 or ATP was evaluated using indo1-AM-loaded SKBr3 cells. G15 (1 μM, red line) or vehicle (ethanol, black line) was added at 20 sec. (first arrow). G-1 (200 nM), 17β-estradiol (E2, 100 nM) or ATP (1μM, a purinergic receptor control) was added at 80 sec. (second arrow). (b) Dose response profile of G-1-stimulated SKBr3 cells to increasing concentrations of G15. (c) Dose response profile of 17β-estradiol-stimulated SKBr3 cells to increasing concentrations of G15. In panels b and c, G-1 and 17β-estradiol were used at 100 nM and 30 nM, respectively, concentrations that yield approximately the half-maximal calcium response for each ligand (approximately 25% that of the full ATP response). Data in panel a are representative of at least three independent experiments. Data in panels b and c represent the mean ± s.e.m. from at least three separate experiments.
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Figure 3: G15 antagonism of intracellular calcium mobilization by GPR30. (a) The effect of G15 on the subsequent mobilization of calcium by G1, E2 or ATP was evaluated using indo1-AM-loaded SKBr3 cells. G15 (1 μM, red line) or vehicle (ethanol, black line) was added at 20 sec. (first arrow). G-1 (200 nM), 17β-estradiol (E2, 100 nM) or ATP (1μM, a purinergic receptor control) was added at 80 sec. (second arrow). (b) Dose response profile of G-1-stimulated SKBr3 cells to increasing concentrations of G15. (c) Dose response profile of 17β-estradiol-stimulated SKBr3 cells to increasing concentrations of G15. In panels b and c, G-1 and 17β-estradiol were used at 100 nM and 30 nM, respectively, concentrations that yield approximately the half-maximal calcium response for each ligand (approximately 25% that of the full ATP response). Data in panel a are representative of at least three independent experiments. Data in panels b and c represent the mean ± s.e.m. from at least three separate experiments.

Mentions: Evaluation of the functional capabilities of G15 with respect to the rapid mobilization of intracellular calcium demonstrated that G15 alone was incapable of inducing a response in SKBr3 breast cancer cells, which are ERα and ERβ negative but express GPR30, whereas stimulation by either estrogen or G-1 induced a response (Fig. 3a). In contrast, stimulation of the cells with G-1 or estrogen subsequent to G15 exposure substantially reduced the response to G-1 or estrogen (Fig. 3a). There was however no inhibition of the calcium response mediated by ATP through endogenous purinergic receptors, indicating the antagonistic effect is specific to GPR30. Inhibition of G-1-mediated calcium mobilization in SKBr3 cells by G15 was dose-dependent, yielding an IC50 of approximately 185 nM (Fig. 3b), whereas inhibition of E2-mediated calcium mobilization yielded a similar IC50 of approximately 190 nM (Fig. 3c).


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 intracellular calcium mobilization by GPR30. (a) The effect of G15 on the subsequent mobilization of calcium by G1, E2 or ATP was evaluated using indo1-AM-loaded SKBr3 cells. G15 (1 μM, red line) or vehicle (ethanol, black line) was added at 20 sec. (first arrow). G-1 (200 nM), 17β-estradiol (E2, 100 nM) or ATP (1μM, a purinergic receptor control) was added at 80 sec. (second arrow). (b) Dose response profile of G-1-stimulated SKBr3 cells to increasing concentrations of G15. (c) Dose response profile of 17β-estradiol-stimulated SKBr3 cells to increasing concentrations of G15. In panels b and c, G-1 and 17β-estradiol were used at 100 nM and 30 nM, respectively, concentrations that yield approximately the half-maximal calcium response for each ligand (approximately 25% that of the full ATP response). Data in panel a are representative of at least three independent experiments. Data in panels b and c represent the mean ± s.e.m. from at least three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864230&req=5

Figure 3: G15 antagonism of intracellular calcium mobilization by GPR30. (a) The effect of G15 on the subsequent mobilization of calcium by G1, E2 or ATP was evaluated using indo1-AM-loaded SKBr3 cells. G15 (1 μM, red line) or vehicle (ethanol, black line) was added at 20 sec. (first arrow). G-1 (200 nM), 17β-estradiol (E2, 100 nM) or ATP (1μM, a purinergic receptor control) was added at 80 sec. (second arrow). (b) Dose response profile of G-1-stimulated SKBr3 cells to increasing concentrations of G15. (c) Dose response profile of 17β-estradiol-stimulated SKBr3 cells to increasing concentrations of G15. In panels b and c, G-1 and 17β-estradiol were used at 100 nM and 30 nM, respectively, concentrations that yield approximately the half-maximal calcium response for each ligand (approximately 25% that of the full ATP response). Data in panel a are representative of at least three independent experiments. Data in panels b and c represent the mean ± s.e.m. from at least three separate experiments.
Mentions: Evaluation of the functional capabilities of G15 with respect to the rapid mobilization of intracellular calcium demonstrated that G15 alone was incapable of inducing a response in SKBr3 breast cancer cells, which are ERα and ERβ negative but express GPR30, whereas stimulation by either estrogen or G-1 induced a response (Fig. 3a). In contrast, stimulation of the cells with G-1 or estrogen subsequent to G15 exposure substantially reduced the response to G-1 or estrogen (Fig. 3a). There was however no inhibition of the calcium response mediated by ATP through endogenous purinergic receptors, indicating the antagonistic effect is specific to GPR30. Inhibition of G-1-mediated calcium mobilization in SKBr3 cells by G15 was dose-dependent, yielding an IC50 of approximately 185 nM (Fig. 3b), whereas inhibition of E2-mediated calcium mobilization yielded a similar IC50 of approximately 190 nM (Fig. 3c).

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