Eric R. Prossnitz

G-Protein-Coupled Receptor 30 and Estrogen Receptor-[Alpha] Are Involved in the Proliferative Effects Induced by Atrazine in Ovarian Cancer Cells

Background Atrazine, one of the most common pesticide contaminants, has been shown to up-regulate aromatase activity in certain estrogen-sensitive tumors without binding or activating the estrogen receptor (ER). Recent investigations have demonstrated that the orphan G-protein–coupled receptor 30 (GPR30), which is structurally unrelated to the ER, mediates rapid actions of 17β-estradiol and environmental estrogens. Objectives Given the ability of atrazine to exert estrogen-like activity in cancer cells, we evaluated the potential of atrazine to signal through GPR30 in stimulating biological responses in cancer cells. Methods and results Atrazine did not transactivate the endogenous ERα in different cancer cell contexts or chimeric proteins encoding the ERα and ERβ hormone-binding domain in gene reporter assays. Moreover, atrazine neither regulated the expression of ERα nor stimulated aromatase activity. Interestingly, atrazine induced extracellular signal-regulated kinase (ERK) phosphorylation and the expression of estrogen target genes. Using specific signaling inhibitors and gene silencing, we demonstrated that atrazine stimulated the proliferation of ovarian cancer cells through the GPR30–epidermal growth factor receptor transduction pathway and the involvement of ERα. Conclusions Our results indicate a novel mechanism through which atrazine may exert relevant biological effects in cancer cells. On the basis of the present data, atrazine should be included among the environmental contaminants potentially able to signal via GPR30 in eliciting estrogenic action.

Estrogen Signaling through the Transmembrane G Protein–Coupled Receptor GPR30

Steroids play an important role in the regulation of normal physiology and the treatment of disease. Steroid receptors have classically been described as ligand-activated transcription factors mediating long-term genomic effects in hormonally regulated tissues. It is now clear that steroids also mediate rapid signaling events traditionally associated with growth factor receptors and G protein-coupled receptors. Although evidence suggests that the classical steroid receptors are capable of mediating many of these events, more recent discoveries reveal the existence of transmembrane receptors capable of responding to steroids with cellular activation. One such receptor, GPR30, is a member of the G protein-coupled receptor superfamily and mediates estrogen-dependent kinase activation as well as transcriptional responses. In this review, we provide an overview of the evidence for the cellular and physiological actions of GPR30 in estrogen-dependent processes and discuss the relationship of GPR30 with classical estrogen receptors.

The G-protein-coupled estrogen receptor GPER in health and disease

Estrogens mediate profound effects throughout the body and regulate physiological and pathological processes in both women and men. The low prevalence of many diseases in premenopausal women is attributed to the presence of 17β-estradiol, the predominant and most potent endogenous estrogen. In addition to endogenous estrogens, several man-made and plant-derived molecules, such as bisphenol A and genistein, also exhibit estrogenic activity. Traditionally, the actions of 17β-estradiol are ascribed to two nuclear estrogen receptors (ERs), ERα and ERβ, which function as ligand-activated transcription factors. However, 17β-estradiol also mediates rapid signaling events via pathways that involve transmembrane ERs, such as G-protein-coupled ER 1 (GPER; formerly known as GPR30). In the past 10 years, GPER has been implicated in both rapid signaling and transcriptional regulation. With the discovery of GPER-selective ligands that can selectively modulate GPER function in vitro and in preclinical studies and with the use of Gper knockout mice, many more potential roles for GPER are being elucidated. This Review highlights the physiological roles of GPER in the reproductive, nervous, endocrine, immune and cardiovascular systems, as well as its pathological roles in a diverse array of disorders including cancer, for which GPER is emerging as a novel therapeutic target and prognostic indicator.

Distribution and characterization of estrogen receptor G protein-coupled receptor 30 in the rat central nervous system.

The G protein-coupled receptor 30 (GPR 30) has been identified as the non-genomic estrogen receptor, and G-1, the specific ligand for GPR30. With the use of a polyclonal antiserum directed against the human C-terminus of GPR30, immunohistochemical studies revealed GPR30-immunoreactivity (irGPR30) in the brain of adult male and non-pregnant female rats. A high density of irGPR30 was noted in the Islands of Calleja and striatum. In the hypothalamus, irGPR30 was detected in the paraventricular nucleus and supraoptic nucleus. The anterior and posterior pituitary contained numerous irGPR30 cells and terminal-like endings. Cells in the hippocampal formation as well as the substantia nigra were irGPR30. In the brainstem, irGPR30 cells were noted in the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus; a cluster of cells were prominently labeled in the nucleus ambiguus. Tissue sections processed with pre-immune serum showed no irGPR30, affirming the specificity of the antiserum. G-1 (100 nM) caused a large increase of intracellular calcium concentrations [Ca(2+) ](i) in dissociated and cultured rat hypothalamic neurons, as assessed by microfluorometric Fura-2 imaging. The calcium response to a second application of G-1 showed a marked homologous desensitization. Our result shows a high expression of irGPR30 in the hypothalamic-pituitary axis, hippocampal formation, and brainstem autonomic nuclei; and the activation of GPR30 by G-1 is associated with a mobilization of calcium in dissociated and cultured rat hypothalamic neurons.

G Protein–Coupled Receptor 30 (GPR30) Mediates Gene Expression Changes and Growth Response to 17β-Estradiol and Selective GPR30 Ligand G-1 in Ovarian Cancer Cells

Estrogens play a crucial role in the development of ovarian tumors; however, the signal transduction pathways involved in hormone action are still poorly defined. The orphan G protein-coupled receptor 30 (GPR30) mediates the nongenomic signaling of 17beta-estradiol (E2) in a variety of estrogen-sensitive cancer cells through activation of the epidermal growth factor receptor (EGFR) pathway. Whether estrogen receptor alpha (ERalpha) also contributes to GPR30/EGFR signaling is less understood. Here, we show that, in ERalpha-positive BG-1 ovarian cancer cells, both E2 and the GPR30-selective ligand G-1 induced c-fos expression and estrogen-responsive element (ERE)-independent activity of a c-fos reporter gene, whereas only E2 stimulated an ERE-responsive reporter gene, indicating that GPR30 signaling does not activate ERalpha-mediated transcription. Similarly, both ligands up-regulated cyclin D1, cyclin E, and cyclin A, whereas only E2 enhanced progesterone receptor expression. Moreover, both GPR30 and ERalpha expression are required for c-fos stimulation and extracellular signal-regulated kinase (ERK) activation in response to either E2 or G-1. Inhibition of the EGFR transduction pathway inhibited c-fos stimulation and ERK activation by either ligand, suggesting that in ovarian cancer cells GPR30/EGFR signaling relays on ERalpha expression. Interestingly, we show that both GPR30 and ERalpha expression along with active EGFR signaling are required for E2-stimulated and G-1-stimulated proliferation of ovarian cancer cells. Because G-1 was able to induce both c-fos expression and proliferation in the ERalpha-negative/GPR30-positive SKBR3 breast cancer cells, the requirement for ERalpha expression in GPR30/EGFR signaling may depend on the specific cellular context of different tumor types.

In vivo Effects of a GPR30 Antagonist

Estrogen is central to many physiological processes throughout the human body. We have previously shown that the G protein-coupled receptor GPR30/GPER, in addition to classical nuclear estrogen receptors (ERα/β), 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 a selective agonist of GPR30, G-1 (1). 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 a G-1 analog, G15 (2) that binds to GPR30 with high affinity and acts as an antagonist of estrogen signaling through GPR30. In vivo administration of G15 reveals 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.

Regulatory Role of G Protein–Coupled Estrogen Receptor for Vascular Function and Obesity

We found that the selective stimulation of the intracellular, transmembrane G protein-coupled estrogen receptor (GPER), also known as GPR30, acutely lowers blood pressure after infusion in normotensive rats and dilates both rodent and human arterial blood vessels. Stimulation of GPER blocks vasoconstrictor-induced changes in intracellular calcium concentrations and vascular tone, as well as serum-stimulated cell proliferation of human vascular smooth muscle cells. Deletion of the GPER gene in mice abrogates vascular effects of GPER activation and is associated with visceral obesity. These findings suggest novel roles for GPER in protecting from cardiovascular disease and obesity.

Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging

A single-molecule tracking technique coupled with mathematical modeling was developed for fully determining the dynamic monomer–dimer equilibrium of molecules in or on the plasma membrane, which will provide a framework for understanding signal transduction pathways initiated and regulated by dynamic dimers of membrane-localized receptors.

Mechanisms of estrogen signaling and gene expression via GPR30.

The effects of estrogen are widespread throughout the body. Although the classical nuclear estrogen receptors have been known for many years to decades and their primary modes of action as transcriptional regulators is well understood, certain aspects of estrogen biology remain inconsistent with the mechanisms of action of these receptor. More recently, the G protein-coupled receptor, GPR30/GPER, has been suggested to contribute to some of the cellular and physiological effects of estrogen. Not only does GPR30 mediate some of the rapid signal transduction events following cell stimulation, such as calcium mobilization and kinase activation, it also appears to regulate rapid transcriptional activation of genes such as c-fos. Since many cells and tissues co-express classical estrogen receptors and GPR30, there exists great diversity in the possible avenues of synergism and antagonism. In this review, we will provide an overview of GPR30 function, focusing on the rapid signaling events that culminate in the transcriptional activation of certain genes.

GPR30: A G protein-coupled receptor for estrogen.

Estrogen is a critical steroid in human physiology exerting its effect both at the transcriptional level as well as at the level of rapid intracellular signaling through second messengers. Many of estrogens transcriptional effects have long been known to be mediated through classical nuclear steroid receptors but recent studies also demonstrate the existence of a 7-transmembrane G protein-coupled receptor, GPR30 that responds to estrogen with rapid cellular signaling. There is currently controversy over the ability of classical estrogen receptors to recapitulate GPR30-mediated signaling mechanisms and vice versa. This article will summarize recent literature and address the relationship between GPR30 and conventional estrogen receptor signaling.