The Research Advances in G-Protein-Coupled Estrogen Receptor

Abstract

Estrogen binds to the typical estrogen receptor (ER) ERα or ERβ and is translocated to the nucleus, where it binds directly to the estrogen response element of the target gene to induce transcription and regulate gene expression, and the whole process is completed in several hours to several days. The G protein-coupled estrogen receptor (GPER), a type that is structurally distinct from typical ERα and ERβ, rapidly induces most non-genomic effects within seconds to minutes. GPER regulates cell growth, migration, and programmed cell death in a variety of tissues and has been associated with the progression of estrogen-associated cancers. Here, the characteristics, cell signal transduction, and the latest research progress of GPER in estrogen-associated tumors and retinal diseases are reviewed.

1. Introduction

Estrogen, the main sex hormone in cisgender women, plays an important role in regulating various physiological and pathological processes in the body and affects mood, bone strength, and even heart health beyond fertility and sex-related functions. Typical estrogen receptor (ER) includes ERα and ERβ. After combined with ERα or ERβ, estrogen transfers in nucleus and directly binds to the estrogen response element in the promoter region of target gene and then induces transcription and regulates gene expression, and this process is called estrogen genomic effect, which usually takes several hours to several days to complete this process [1].

Besides, by combined with a novel type of ER, G protein-coupled ER (GPER), an alternate ER with a structure distinct from the two canonical ERα and ERβ, can also induce rapid non-genomic effects in seconds to minutes and mediate most of the non-genomic effects of estrogen.

GPER triggers several downstream pathways that exert vital biological roles in the regulation of cell growth, migration, and programmed cell death in a variety of tissues. In particular, there is a significant correlation between GPER and the progression of estrogen-related cancers. Therefore, better understanding the role of GPER in physiological function, especially in estrogen-related cancers, may help identify new biological regulatory targets, as well as cancer therapeutic targets. In this review, we summarize the characteristic and cell signaling and detail the functions of GPER, as well as the latest research progress of GPER in estrogen-related cancers.

2. Brief overview of GPER

2.1 G-protein and GPCR

G proteins, also called as guanosine nucleotide-binding proteins, are proteins that are involved in delivering a variety of stimuli from extracellular environment to intracellular organelle. G protein-coupled receptors (GPCRs) are a general name of membrane protein receptors, which locate on cell membranes and are the largest family of proteins encoded by the human genome [2]. The unique feature of this kind of GPCRs is that there are seven transmembrane α helices in the stereo-structure of the receptor, and there are G protein-binding sites on the C-terminal of the peptide chain and the intracellular ring (the third intracellular ring) connecting the fifth and sixth transmembrane helices (starting from the N-terminal of the peptide chain). Endogenous ligands of GPCR include many compounds, such as amines, carbohydrates, lipids, peptides, proteins, hormones, neurotransmitters, chemokines, and even photons and odors. GPCR has been linked to type 2 diabetes, obesity, depression, cancer, Alzheimer’s disease, and other diseases [3].

2.2 ER

Estrogen receptor (ER) is a steroid receptor, belonging to a superfamily of proteins, whose function is to regulate transcription of gene pools in other cells. The estrogen receptors such as ERα, ERβ, and GPER are located in the tissues of the female reproductive tract and breast, as well as in a variety of tissues such as bone, brain, liver, colon, skin, and salivary glands, exerting the effects of estrogen compounds on their target tissues [4].

2.3 GPER

In 1996, the third estrogen receptor, G-protein coupled receptor 30 (GPR30), was discovered in breast cancer tissues for the first time [5, 6]. GPR30 is a member of GPCR family, which can be exclusively bound to by estrogen and estrogen-like molecules, and it is involved in rapid non-genomic estrogen effects [7, 8, 9]. GPR30 also has another name GPER or GPER1 [10]. The function of this receptor provides an important basis for expounding the rapid response of estrogen. The human GPER gene, including a 1128 bp open reading frame that encodes 375-amino acid receptors, is located on chromosome 7 [6, 11].

GPER is expressed in various tissues, such as nerve, reproductive, digestive, and muscle organs [12], as well as in brain pericellular, cortex, dentate gyrus, anterior pituitary, and adrenal medulla cells [13]. The expression of GPER is not limited to the cell surface, and the binding domain of this receptor is also expressed in the endoplasmic reticulum [14].

In normal tissue, GPER was expressed markedly in the cortex and the anterior pituitary, islets and pancreatic ducts, fundic glands of the stomach, the epithelium of the duodenum and gallbladder, hepatocytes, proximal tubules of the kidney, the adrenal medulla, and syncytiotrophoblasts and decidua cells of the placenta [15].

2.4 GPER agonists and antagonists

There are synthetic and natural estrogen compounds that can interact with GPER. GPER agonists can be categorized as follows: (1) natural estrogens including 17β-estradiol (E2, a general ER agonist) and 2-methoxy-estradiol; (2) phytoestrogens including genistein, quercetin, equol, and resveratrol; (3) selective estrogen receptor modulators (SERMs), which includes tamoxifen (TMX), 4-hydroxytamoxifen, and raloxifene; (4) selective estrogen receptor downregulation agents (SERDs), which include fulvestrant and pesticides; (5) plastic compounds as endocrine disruptors, which include atrazine, bisphenol A, zearalenone, nonylphenol, and ketone. GPER antagonists can be categorized as follows: (1) natural estrogens, which include 2-hydroxyoestradiol and estriol; and (2) synthesis of compounds such as MIBE [15, 16, 17].

In addition, there are highly selective GPER ligands, which include GPER agonist G-1 and GPER antagonists G-15 and G-36 [18, 19]. G-1, also known as Tespria, is a nonsteroidal compound and a selective GPER1 agonist. In vitro cell experiments have shown that G-1 has no binding affinity for ERα and ERβ [20, 21]. Moreover, G-1 regulation is involved in intracellular calcium [Ca2+] and phosphoadenosine 3-kinases (PI3Ks) signaling pathways. G-15 is a selective GPER1 antagonist with high binding affinity to GPER1 and less binding to ERα and ERβ [19, 22, 23]. As another selective GPER1 antagonist, G36 also owns poor binding ability to ERα and ERβ as well as G15, whereas G36 is more powerful than G15 in binding to GPER1 and then modulates Ca2+ mobilization and PI3K signaling [18].

3. GPER-related signaling pathway

3.1 Nuclear factor-Kappa B (NF-κB) signaling pathway

When epithelial cells undergo epithelial-mesenchymal transition (EMT), mastitis may be aggravated. GPER1 activation inhibits EMT of goat mammary epithelial cells through NF-κB signaling pathway, thus impeding the occurrence of mastitis [24]. Okamoto et al. [25] delineated that lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) overexpression in mouse macrophages is negatively regulated by GPER agonist G-1 via NF-κB signaling pathway, which is associated with anti-inflammatory effects of estrogen.

3.2 Hippo signaling pathway

Zhou et al. [26] determined that, compared with adjacent normal tissue, the expression of GPER is highly upregulated in the cancer cells of breast invasive ductal carcinoma, and key downstream signals of GPER are associated with the Hippo/YAP (Yes-associated protein 1)/TAZ (transcriptional coactivator with a PDZ-binding domain) pathway, which plays an important role in breast tumorigenesis. YAP is a pivotal effector of Hippo pathway, and Deng et al. [27] exhibited that bisphenol S activates YAP, upgrades YAP nuclear accumulation, and regulates its downstream genes in triple-negative breast cancer cells. By inhibiting GPER, the effect of bisphenol S-induced Hippo/YAP signal pathway can be weakened.

3.3 Mitogen-activated protein kinase (MAPK) signaling pathway

Triclosan can act on GPER to induce estrogen effect. It has been found that triclosan can upregulate the expression of mir-144 by activating GPER, which leads to abnormal regulation of neuro-related genes and neurodevelopmental toxicity, and this process is closely related to the activation of downstream protein kinase C (PKC)/MAPK signaling pathway [28]. In ER-positive breast cancer cells, the activation of GPER and its downstream MAPK/ERK signal pathway increase the expression level of TRIM2 protein, which leads to the reduction of Bim protein in tamoxifen-resistant breast cancer cells, providing a new idea for solving tamoxifen resistance in ER-positive breast cancer cells [29].

3.4 Phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway

The nuclear ER is negative in breast cancer SKBR-3 cells, while GPER is positive. Using the SKBR-3 cells, Shi et al. [30] demonstrated that the expression of PI3K and p-AKT can be downregulated by cryptotanshinone (an active compound of traditional Chinese medicine Danshen), and the inhibitory effect of cryptotanshinone on SKBR-3 cells is carried out by inhibiting GPER-mediated PI3K/AKT pathway. Additionally, cryptotanshinone can mediate the downregulation of PI3K/AKT pathway and induce apoptosis in breast cancer MCF-7 cells by activating GPER [31].

3.5 Extracellular signal-regulated kinase (ERK) signaling pathway

Hepatocellular carcinoma (HCC) is a malignant tumor, whose cell lines HCCLM3 and SMMC-7721 express high levels of GPER. The research by Qiu et al. [32] presented that compared with GPER-negative patients, GPER-positive HCC patients were closely related to the following factors, such as female, negative hepatitis B surface antigen, smaller tumor size, lower serum alpha-fetoprotein level and longer overall survival, and treatment of HCC cell lines HCCLM3 and SMMC-7721 with GPER-specific agonist G1 resulted in the activation of epidermal growth factor receptor (EGFR)/ERK and EGFR/AKT signaling pathways. In a murine Crohn’s disease model induced by trinitrobenzene sulfonic acid, activation of GPER exerts the anti-inflammatory effects and lowers the mortality of mouse and C-reactive protein level, and this activation is accompanied by regulating ERK signaling pathway [33].

4. Physiological effects of GPER

4.1 GPER roles in inflammation

In a mouse model, the GPER selective agonist G-1 was found to reduce circulating levels of inflammatory cytokines tumor necrosis factor α (TNFα), monocyte chemoattractant protein-1 (MCP-1), and IL-6 and to lower the expression of inflammatory genes in a variety of metabolic tissues, when administered over a long period of time [34]. In microglia, GPER is involved in the anti-inflammatory process when genistein was administered to inhibit lipopolysaccharide (LPS)-induced microglia activation [35].

4.2 GPER roles in cardiovascular system

The blood pressure of male Sprague-Dawley (SD) rats can be significantly lowered by GPER1 activation with G1 [36]. Blood pressure is lowered in ovariectomy (OVX) SD rats and OVX mRen2 Lewis rats after chronic systemic administration with G-1 [37, 38]. The downstream of GPER1 activation involves signaling pathways such as nitric oxide (NO), which can mediate vasodilation occurrence and is a well-known vasodilator [39, 40, 41]. Downstream of GPER1 activation is also involved in renin angiotensin aldosterone system (RAAS), which controls blood pressure and glomerular filtration rate through renin and angiotensin I, II and aldosterone [42, 43, 44].

It is GPER1, while not ERα and ERβ, plays an important role in estrogen-responded heart rate regulation [45, 46]. GPER1 activation has a cardioprotective effect on heart failure in male mice and myocardial inflammation in male spontaneously hypertensive rats (SHRs), adriamycin-induced cardiotoxicity in male rats can be alleviated by GPER1 activation, and myocardial cell death can also be protected by GPER1 activation [47, 48, 49, 50]. Moreover, in stress-induced cardiomyopathy, also known as Takotsubo syndrome, GPER activation exerts a protective role through balancing β2 adrenergic receptor (β2AR) with the Gαs and Gαi signaling pathways [51].

In short, it is worth noting that the current studies on the physiological effects of GPER are mainly from GPER knockout (KO) mice, which may have certain limitations. Therefore, when using GPER KO mice to study the mechanism of GPER-mediated action, the possibility of systemic compensation in mice should be taken into account [52].

4.3 GPER roles in stress

Endoplasmic reticulum stress (ERS) has been linked to several diseases such as cancer and diabetes and may be one of the possible inducers of pathological cell death and dysfunction [53, 54]. Icariin, an active constituent of epimedium, can inhibit ERS pathway through promoting the expression of GPER in diabetic kidney disease, and GPER is negatively related to the expression of endoplasmic reticulum response stress protein [55]. ERS pathways can be activated by GPER agonist, which leads to cancer cell death [56, 57]. GPER agonist G-1 can inhibit the increase of colonic crypt cell apoptosis in the colitis model [58] and improve epithelial cells after intestinal injury [59], and the protective effect of GPER is at least partially correlated with the inhibition of ERS.

Oxidative stress refers to the imbalance between oxidation and antioxidant effects in the body. Oxidative stress can occur when overexposed to reactive oxygen and nitrogen species, leading to damage of proteins, lipids, and DNA [60, 61]. Icariin also inhibits oxidative stress and promotes experimental diabetic nephropathy repair through GPER-mediated degradation of Keap1 protein and activation of Nrf2 protein [62]. Studies using a male rat model with traumatic brain injury-induced liver injury found that oxidative stress was significantly inhibited by 17β-estradiol [63, 64, 65]. In human skin fibroblasts, keratinocytes, and hepatic cells’ 17 β-estradiol is also involved in inhibiting oxidative stress through the participation of GPER [66, 67, 68].

The range of cytoskeletal variation can be modulated by GPER, which is thought to be involved in mechanical transduction [69, 70]. When mechanical stress-mediated chondrocyte apoptosis occurs, chondrocyte apoptosis in osteoarthritis can be attenuated by GPER via causing the inhibition of Piezo 1 protein [71].

5. GPER in cancer

5.1 GPER in gynecological tumors

Bubb et al. [15] found that GPER is expressed in hepatocellular, pancreatic, renal and endometrial cancers, pancreatic neuroendocrine tumors, and pheochromocytomas by using GPER antibody 20H15L21. GPER is found to be expressed in 50–60% of breast cancer tissues [72, 73, 74], and is thought to be involved in the development of tamoxifen resistance in ERα-positive breast cancer [75, 76]. GPER can inhibit YAP1 phosphorylation by shutting down the Hippo pathway in breast cancer [27]. An immunohistochemical study of 1245 patients with primary invasive breast cancer showed that low expression of GPER was significantly related not only to clinicopathological and molecular characteristics of invasive behavior but also to poor survival in patients with breast cancer [77]. Chan et al. [78] showed that GPER induces phosphorylation of PKA and BAD-Ser118 to maintain breast cancer stem cells through activation of its ligands, which include tamoxifen (TMX) and GPER agonists increase cell proliferation and the number of breast cancer stem cells through the p-PKA/p-BAD signaling pathway.

GPER is expressed in cell lines such as human ovarian cancer cell line SKOV3, OVCAR-3, and OVCAR5 [79, 80], in both malignant and benign ovarian tumors, and overexpressed in some malignant ovarian tumors [81]. GPER is found in serous and mucinous ovarian adenocarcinoma biopsies. G-1 controls ovarian cancer cell proliferation, inhibits G2/M cell cycle progression, and promotes ovarian cancer cell apoptosis [79]. Zhu et al. showed that nuclear GPER is an independent negative marker for the prognosis of ovarian cancer patients, particularly for patients with highly malignant ovarian cancer [82].

Fujiwara et al. [83, 84] found that high GPER expression is related to poor prognosis in ovarian cancer patients, and lower GPER expression was positively correlated with overall survival time. However, in contrast to the results mentioned earlier, the study by Ignatov et al. found that high GPER expression positively affected 2-year disease-free survival in ovarian cancer patients [79]. The relation between GPER and ovarian cancer needs more studies to clarify.

5.2 GPER in tumor of male reproductive system

Concentrations of 17B-estradiol (E2) in the testis are 10 to 100 times higher than in the blood [85]. Although the molecular mechanisms by which estrogen and its receptors play a role in spermatogenesis are not fully understood, it is no doubt that estrogen is crucial in spermatogenesis [86]. GPER regulates the balance between testosterone and estrogen and is associated with the male reproductive system. GPER is predominantly located in the basal epithelium and mediates estrogen action, and the level of GPER expression is low in prostate tumor cells and in benign prostate tissue [87]. The expression of GPER was negatively correlated with the differentiation of prostate tumor cells [88]. In addition, it has been reported that GPER is a tumor suppressor that activates ERK1/2 and C-Jun/C-FOS signaling and thus inhibits PC3 cells, a cell line characteristic of prostatic small cell carcinoma and from arresting in G2 phase [89]. Lau et al. reported that GPER-specific agonist G1 binds to GPER to maintain ErK1/2 activation, thereby inhibiting prostate cancer cell growth and regulating metastasis characteristics [90].

Testicular germ cell carcinoma (TGCC) is a malignant solid tumor and is account for the main reason of death in young men. 17β-estradiol inhibits the proliferation of human seminoma cell lines by an ERβ-dependent mechanism, which is stimulated in vitro by GPER activation of ERK1/2 and protein kinase A. GPER is not expressed in non-seminoma, whereas GPER is exclusively overexpressed in seminoma, which is linked to the downregulation of ERβ, and may also be associated with genetic variations such as single-nucleotide polymorphisms [91].

It is worth mentioning that, regarding the potential sex-specific effects of GPER-1, there was no significant prognostic value of GPER-1 in the group of male breast cancers, which is contrary to what was expected in the studies of female breast cancers [92].

5.3 GPER in other cancer

Liu et al. [93, 94, 95] demonstrated that both cytoplasm-GPER (80.49%) and nucleus-GPER (53.05%) were detected in non-small-cell lung cancer (NSCLC) samples through immunohistochemical analysis, GPER activation promotes the growth of NSCLC cells via YAP1/QKI/circNOTCH1/m6A-methylated NOTCH1 pathway, and GPER antagonist G15 administration can reverse estrogen-induced progress of NSCLC cells, and these results suggest that blocking GPER signaling by G15 may be a new therapeutic target in NSCLC. In addition, the role of GPER in adrenal cortical carcinoma is related to mitochondria-related signal transduction, and GPER positively regulates mitochondrial apoptotic pathways through the EGR-1 pathway and ROS/EGR-1/BAX pathway, thereby inhibiting adrenocortical cancer cell growth [96].

6. GPER in retinal disease

Retinopathy of prematurity (ROP), characterized by abnormal growth of immature retinal blood vessels, is one of the leading causes of blindness and visual impairment in children worldwide. High incidence of ROP is intimately related to improved perinatal life care and poor ophthalmological special management in many developing countries, which lifts the survival rate of infants with ROP, and simultaneously, total infants with ROP [97].

Our research group has studied the role of GPER in ROP. The expression of GPER was found in endoplasmic reticulum of mouse retinal microglia, retinal ganglion cells, and retinal astrocytes. By GPER activation, hyperoxia-induced autophagy and apoptosis in these cells were reduced markedly, hyperoxia-induced decrease of the viability in these cells was reversed significantly, the high activity of inositol-1,4, 5-triphosphate receptor in these cells was reduced significantly also, and simultaneously, high calcium levels induced by hyperoxia in the endoplasmic reticulum was lowered again in these cells. These changes were reversed after the use of GPER antagonist G15. Thus, ERS irritated by hyperoxia was mitigated by G1 administration. These results suggest that GPER agonists may have therapeutic potential in the early stage of ROP [98, 99, 100].

7. Future directions

The key scientific issues that GPER needs to be further addressed as a key node are as follows: (1) the relationship between drugs (such as phytoestrogens, active ingredients of phytochemicals) and GPER and its signaling pathways needs to be systematically studied; (2) the interaction parameters and kinetics between GPER and ligands need to be further studied; (3) more work needs to be done to fully understand the potential of GPER1 in health regulatory mechanisms; and (4) the relationship between GPER and estrogen-related cancers, and the research on GPER as a therapeutic target for estrogen-related cancers needs to be carried out, which may unveil new therapies aimed at improving clinical outcomes of the diseases related to estrogen.

Acknowledgments

This work was supported by grants from the Key Project of Natural Science Basic Research Plan of Shaanxi Province (China, No. 2021JZ-60).

Conflict of interest

The authors declare no conflict of interest.

Abbreviations

EGFR	epidermal growth factor receptor
E2	17-β-estradiol
ER	estrogen receptor
GPCR	G protein-coupled receptor
GPER	G protein-coupled estrogen receptor
GPR30	G protein-coupled receptor 30
HCC	hepatocellular carcinoma
KO	knockout
SHR	spontaneously hypertensive rats
SD rat	Sprague-Dawley rats
SERM	selective estrogen receptor modulator
TGCC	testicular germ cell cancers
NSCLC	non-small-cell lung cancer