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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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Ligand binding properties of G15. Ligand binding affinities of 17β-estradiol, G-1 and G15 for GPR30, ERα and ERβ. For GPR30 (a), Hec50 cells, which endogenously express GPR30 but neither ERα nor ERβ, were incubated with trace quantities of an iodinated G-1 derivative and the indicated concentration of either G-1 (μ) or G15 (τ) as competitor. For ERα and ERβ, COS7 cells were transfected with either ERα-GFP (b) or ERβ-GFP (c). For the latter, competitive ligand binding assays were performed using 10 nM E2-Alexa633 and the indicated concentration of either 17β-estradiol (μ) or G15 (τ). Data indicate the mean ± s.e.m. of at least three separate experiments.
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Figure 2: Ligand binding properties of G15. Ligand binding affinities of 17β-estradiol, G-1 and G15 for GPR30, ERα and ERβ. For GPR30 (a), Hec50 cells, which endogenously express GPR30 but neither ERα nor ERβ, were incubated with trace quantities of an iodinated G-1 derivative and the indicated concentration of either G-1 (μ) or G15 (τ) as competitor. For ERα and ERβ, COS7 cells were transfected with either ERα-GFP (b) or ERβ-GFP (c). For the latter, competitive ligand binding assays were performed using 10 nM E2-Alexa633 and the indicated concentration of either 17β-estradiol (μ) or G15 (τ). Data indicate the mean ± s.e.m. of at least three separate experiments.

Mentions: Chemically synthesized G15 was subjected to multiple cellular and physiological assays in order to characterize its biological effects. Competitive binding assays using endogenous GPR30 and a novel iodinated GPR30-selective G-1 analog (manuscript in preparation), demonstrated that G15 binds to GPR30 with an affinity of approximately 20 nM (Fig. 2a). This compares to an affinity for G-1, utilizing the same assay, of approximately 7 nM, similar to our previously reported affinity of G-1 for recombinant GPR30 of 11 nM 10 and reported affinities for 17β-estradiol between 3-6 nM 6,7. Thus removal of the ethanone moiety resulted in a decrease in relative binding affinity of approximately 3 fold. Additional competitive binding studies to assess interactions with ERα and ERβ revealed that similar to G-1, G15 displays little binding to ERα or ERβ at concentrations up to 10 μM, where estrogen competes with a Ki of approximately 0.3-0.5 nM (Figs. 2b and c). These results reveal that G15, like G-1, displays high affinity for GPR30 with minimal binding to ERα and ERβ (Ki > 10 μM).


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)

Ligand binding properties of G15. Ligand binding affinities of 17β-estradiol, G-1 and G15 for GPR30, ERα and ERβ. For GPR30 (a), Hec50 cells, which endogenously express GPR30 but neither ERα nor ERβ, were incubated with trace quantities of an iodinated G-1 derivative and the indicated concentration of either G-1 (μ) or G15 (τ) as competitor. For ERα and ERβ, COS7 cells were transfected with either ERα-GFP (b) or ERβ-GFP (c). For the latter, competitive ligand binding assays were performed using 10 nM E2-Alexa633 and the indicated concentration of either 17β-estradiol (μ) or G15 (τ). Data indicate the mean ± s.e.m. of at least three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Ligand binding properties of G15. Ligand binding affinities of 17β-estradiol, G-1 and G15 for GPR30, ERα and ERβ. For GPR30 (a), Hec50 cells, which endogenously express GPR30 but neither ERα nor ERβ, were incubated with trace quantities of an iodinated G-1 derivative and the indicated concentration of either G-1 (μ) or G15 (τ) as competitor. For ERα and ERβ, COS7 cells were transfected with either ERα-GFP (b) or ERβ-GFP (c). For the latter, competitive ligand binding assays were performed using 10 nM E2-Alexa633 and the indicated concentration of either 17β-estradiol (μ) or G15 (τ). Data indicate the mean ± s.e.m. of at least three separate experiments.
Mentions: Chemically synthesized G15 was subjected to multiple cellular and physiological assays in order to characterize its biological effects. Competitive binding assays using endogenous GPR30 and a novel iodinated GPR30-selective G-1 analog (manuscript in preparation), demonstrated that G15 binds to GPR30 with an affinity of approximately 20 nM (Fig. 2a). This compares to an affinity for G-1, utilizing the same assay, of approximately 7 nM, similar to our previously reported affinity of G-1 for recombinant GPR30 of 11 nM 10 and reported affinities for 17β-estradiol between 3-6 nM 6,7. Thus removal of the ethanone moiety resulted in a decrease in relative binding affinity of approximately 3 fold. Additional competitive binding studies to assess interactions with ERα and ERβ revealed that similar to G-1, G15 displays little binding to ERα or ERβ at concentrations up to 10 μM, where estrogen competes with a Ki of approximately 0.3-0.5 nM (Figs. 2b and c). These results reveal that G15, like G-1, displays high affinity for GPR30 with minimal binding to ERα and ERβ (Ki > 10 μM).

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