Membrane-Initiated Estrogen, Androgen, and Progesterone Receptor Signaling in Health and Disease.

Rapid effects of steroid hormones were discovered in the early 1950s, but the subject was dominated in the 1970s by discoveries of estradiol and progesterone stimulating protein synthesis. This led to the paradigm that steroid hormones regulate growth, differentiation, and metabolism via binding a receptor in the nucleus. It took 30 years to appreciate not only that some cellular functions arise solely from membrane-localized steroid receptor (SR) actions, but that rapid sex steroid signaling from membrane-localized SRs is a prerequisite for the phosphorylation, nuclear import, and potentiation of the transcriptional activity of nuclear SR counterparts. Here, we provide a review and update on the current state of knowledge of membrane-initiated estrogen (ER), androgen (AR) and progesterone (PR) receptor signaling, the mechanisms of membrane-associated SR potentiation of their nuclear SR homologues, and the importance of this membrane-nuclear SR collaboration in physiology and disease. We also highlight potential clinical implications of pathway-selective modulation of membrane-associated SR.

Membrane-localized estrogen (ER), androgen (AR) and progesterone (PR) receptors activate signaling pathways that potentiate the actions of their nuclear steroid receptor (SR) counterparts, usually by phosphorylation, allowing steroid hormone early membrane effects to enhance the late SR-dependent nuclear actions.

SRs have both membrane-initiated rapid signaling and classical transcriptional nuclear properties that may function either separately as distinct pathways, or together as a fully integrated network.

Ligand-activated membrane SRs initiate signaling by activating the nonreceptor tyrosine kinase Src or via G protein coupling, leading to the activation of kinase cascades including MAPK, Akt, PKA, and AMPK.

Transmembrane SRs unrelated to the nuclear ERs (GPER), ARs (TRPM8, ZIP9, OXER1, and GPRC6A) and PRs (PAQRs and PGMRCs), can cross talk and collaborate with classical membrane-associated and nuclear SR.

The rapid effects of steroid hormones in the adaptive response to stress were first described for glucocorticoids in the early 1950s by the physiologist Hans Selye in Hungary (1). It took nearly 20 years for the laboratory of Clara Szego to show that rapid effects of 17ß-estradiol (E2) administration in rats increased cyclic adenosine monophosphate (cAMP) in the uterus within seconds (2). The same laboratory showed rapid stimulation of endometrial cell calcium resulting from E2 binding with high affinity to a surface protein of undetermined nature (3, 4). These studies promoted strong interest in the effects of potential membrane-localized steroid receptors (SRs) to impact cell and organ biology and function. However, the field was dominated by powerful discoveries by Bert O’Malley and coworkers showing that estradiol and progesterone stimulated protein synthesis in the chick oviduct. This led to the concept that steroid hormones regulate growth, differentiation, and metabolism in target tissues following steroid permeation of the cell membrane, binding to a specific cytoplasmic receptor, transfer of this steroid-receptor complex to the nucleus, binding of the steroid-receptor complex to specific “acceptor sites” on the nuclear chromatin, and synthesis of new RNA specific to a protein (5-7). At the same time, studies by Jean Wilson and coworkers showed binding of testosterone to a nuclear protein in prostate (8), followed by the cloning of the androgen receptor (AR) and analysis of its structure (9-11), leading to the establishment of a paradigm in which AR is a nuclear ligand-activated transcription factor. In the early 1980s, studies revealed that testosterone induces a rapid (< 1 minute) stimulation of endocytosis, amino acid, and sugar transport in mouse kidney cortex slices in an AR-dependent manner (12). The essential role of rapid and dynamic movement of progesterone receptors (PRs) between the cytoplasmic and nuclear compartments has been appreciated since the early 1990s, with the discovery by Giuochon-Mantel and Milgrom that nuclear PRs appeared to diffuse into the cytoplasm but were actively transported back into the nucleus, an action mediated by nuclear localization signals within the hinge region and DNA-binding domains (13). These studies, and others that further mapped sequences within the nuclear localization signals that mediate bidirectional movement (14), marked the beginning of a much broader understanding of the mechanisms by which SRs are implicated in the regulation of transcription, sensing and responding to activated membrane and cytoplasmic signaling pathways, and the realization that dynamic crosstalk with and exposure to cytoplasmic signaling molecules and chaperones is essential for the nuclear function of ligand-activated transcription factors. It is now widely accepted that SRs have both transcriptional (classical nuclear) and membrane-initiated rapid signaling properties that may function either separately as distinct pathways or together as fully integrated networks (15, 16). Here, we provide an update on the current state of knowledge of membrane-initiated estrogen, androgen, and progesterone receptor signaling, their collaboration with nuclear receptors, their role in physiology and disease, and clinical implications of pathway-selective activation of membrane SRs. When details from previous studies on mechanisms of extranuclear SR signaling are needed, we refer to recent reviews.

Membrane Estrogen Signaling

Membrane Estrogen Receptor-α and -β

Following their discovery of E2 rapid effects on cAMP production, Szego and coworkers reported the existence of an estrogen receptor at the cell membrane (4, 17). Studies using antibodies to the estrogen receptor suggested that the protein to which E2 binds at the plasma membrane has homology to classical estrogen receptor-α (ERα) (18). Expression of ERα and ERβ in ER-null cells showed localization of estrogen-binding proteins at the plasma membrane and in the nucleus and provided evidence that both receptor pools derive from the same gene and transcript (19). Later studies in breast cancer and other cells with knockdown or knockout of ERα or ERβ, thus eliminating E2 binding, confirmed these results (20, 21). Ultimately, affinity column isolation and mass spectrometry of membrane and nuclear E2-binding proteins in breast cancer cells definitively showed that the membrane and nuclear proteins are the same classical ERα (22). The same holds true for ERβ, as rapid signaling by the membrane-localized receptor is not present in ERβ knockout mice (19, 23). Although most rapid actions of E2 are mediated via full-length ERα (ER66) at the membrane (24), Bender and coworkers showed that an N-terminus truncated ERα isoform, ER46, plays a key role in rapid membrane-initiated endothelial responses to E2 (25). In addition, a 50kDa ERα isoform was identified in hypothalamic neurons and astrocytes, and an ER36 variant was found in breast cancer cells (26). ERα isoforms originate from different ERα mRNA transcripts regulated by alternative promoter usage and alternative splicing, as well as from alternative translation initiation (26).

Trafficking to the Cell Membrane and Early Signaling Events

Many proteins that translocate to the plasma membrane require myristylation and/or palmitoylation for membrane targeting. Highly conserved cysteine palmitoylation sites were identified for ERα and ERβ in mice and humans (23, 27), as were cysteine palmitoylation sites for the classical androgen and progesterone receptor proteins as discussed in the corresponding sections (23). Palmitoylation of ERα at cysteine-451 in the mouse and cysteine-447 in humans is required for 5% of the total cellular ERα pool to localize to the plasma membrane (23). The human palmitoyl acyltransferase enzymes ZDHHC7 and 21 promote palmitoylation of all 3 sex steroid receptors, aided by heat shock protein 27 (Hsp27), both of which are necessary for membrane localization (28, 29). Mutation of a single cysteine palmitoylation site in ERα or small interfering RNA (siRNA) knockdown of Hsp27 eliminates ERα trafficking to the membrane and rapid signal transduction by E2 (30). Interestingly, only the receptor monomer is palmitoylated; thus, E2-induced dimerization, which is critical for membrane ER signaling (20, 21), also limits the number of receptors that can traffic to the membrane.

ERα and all sex steroid receptors interact with the protein caveolin-1, an essential process for transport of palmitoylated ERα to the membrane caveolae (23). Caveolin-1 serves as a scaffold for assembling various signaling molecules like G protein subunits, kinases, and cyclic nucleotides as part of a functional signalosome (31-33). G protein coupling enables a subpopulation of membrane ERα to initiate rapid signal transduction from the cell surface (34). For example, in endothelial cells, membrane ERα coupling to Gα 13 activates a cascade of RhoA, Rho kinase, and moesin to induce rapid remodeling of the actin cytoskeleton and migration (35). The same Gα 13 coupling and RhoA-moesin cascade activation promotes migration and invasion of breast cancer cells (36). However, coupling to Gα i-2/3, transactivates particulate guanylate cyclase-A (pGC-A) to produce cGMP, thereby activating protein kinase G type I (PKG-I) and resulting in the phosphorylation of cystathionine γ-lyase, the key enzyme in vascular endothelial hydrogen sulfide (H2S) release (37). ERα-Gα i coupling activates c-Src, inducing nitric oxide synthase (NOS) activation and the resulting attenuation of monocyte adhesion (38). Apart from G proteins, ERs can also directly bind membrane-associated signaling molecules, including mitogenic protein kinases such as c-SRC and phosphoinositide-3-kinase (PI3K), and ion channels to mediate context-dependent rapid activation of downstream signaling pathways. For example, Simoncini et al initially showed that in endothelial cells, following E2 stimulation, ERα rapidly binds the p85α subunit of PI3K, leading to the activation of endothelial NOS (eNOS) (39) (Fig. 1). In fact, a subpopulation of ERα is localized to membrane caveolae/lipid rafts where they are organized into functional signaling modules (40).

Figure 1.

Membrane-initiated ERα signaling. Ligand-dependent interactions of membrane-associated ERα with Src lead to rapid activation of Src and downstream kinases in the Ras/Raf/MEK/ERK and PI3K/AKT kinase cascades, producing signals ranging from vasodilation and insulin synthesis to cell migration. G protein coupling enables membrane-associated ERα to initiate signals leading to migration or lipogenesis inhibition. In adipose stem cells, membrane-initiated ERα signaling initiates a cascade leading to cytosolic ERα phosphorylation and nuclear import and binding to the PPARγ promoter for inhibition of adipogenesis. The figure represents a compilation of some of the multiple binding proteins and signaling pathways of membrane-associated ERα actions that have been described in a variety of different cell types.

Functions of Membrane-Localized ERα

The clinical relevance of studying the contribution of cellular pools of ERα is the fine-tuning of estrogen therapy for contraception and the relief of menopausal symptoms. As estrogen deficiency is also associated with increased risk of cardiovascular disease as well as alteration in lipid and glucose metabolism, we discuss the most relevant studies related to reproduction and cardiometabolism. In the case of ERα, the development of multiple genetic mouse models and pharmacological probes has provided important insights into the relative importance of membrane-initiated and nuclear ERα signaling in vivo. Levin and Arnal’s groups independently developed mice carrying a mutation in the palmitoylation site of ERα (C451A-ERα) resulting in a loss of membrane-localized ERα. Female C451A-ERα mice exhibited abnormal breast and ovarian development, leading to infertility (41, 42). Uterine development, however, was impaired in only 1 study (41), likely due to persistence of membrane ERα expression in the endometrium of the C451A-ERα mice in the other study (42). Indeed, since mice with heterozygous loss of membrane ERα (41) were phenotypically similar to wild-type mice, the partial expression of membrane ERα (42) may have reversed the effects of homozygous deletion of membrane ERα (41). Nevertheless, together these results indicated the importance of the cellular pool of ERα in reproduction. Male mice also exhibited abnormal sperm development and decreased fertility (43). These studies demonstrated for the first time the importance of collaborations between membrane and nuclear pools of ERα to in vivo physiology.

Subsequent functional studies in mice lacking membrane-localized or nuclear ERα revealed several important functions of membrane and nuclear ERα pools working together, especially for gene expression. Some cellular functions appear to arise solely from membrane-initiated ERα action. As an example, E2 inhibition of insulin-stimulated triglyceride synthesis and deposition in cultured mature adipocytes resulted entirely from membrane ERα-stimulated activation of several kinases (44). The signaling involves cytoplasmic sequestration of the lipogenic transcription factor carbohydrate response element–binding protein (ChREBP), thereby limiting insulin-stimulated expression of key lipogenic genes. Selective activation of membrane-associated ERα in mice lacking nuclear ERα (MOER mice) decreased insulin-stimulated lipogenesis in hepatocytes, decreasing fatty acid synthesis and triglyceride deposition in the liver (45). This occurred exclusively from membrane ERα activation of AMP-activated protein kinase (AMPK), which phosphorylates and thereby promotes cytoplasmic sequestration of another lipogenic transcription factor, the sterol regulatory element–binding protein 1c (SREBP1c), inhibiting lipid synthesis. Accordingly, in vivo, E2 and an estrogen dendrimer conjugate (EDC, comprising an estrogen molecule linked to a poly(amido)amine dendrimer that selectively activates extranuclear ER) had a comparable favorable effect in preventing diet-induced hepatic steatosis via downregulation of fatty acid and triglyceride synthesis genes in the liver (46). Similarly, in INS-1 insulin-secreting cells, E2 and EDC inhibited lipogenesis via membrane ERα and activated AMPK to suppress SREBP1c protein expression (47). Activation of membrane-localized ERα also inhibited lipogenesis in pancreatic islets in vivo by stimulating the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in a Src-dependent manner. This promoted STAT3 nuclear translocation and transcriptional repression of master regulators of lipogenesis, liver X receptors (LXRs), which suppresses the transcription of SREBP1c and ChREBP lipogenic genes (47, 48) (Fig. 1). Despite these antilipogenic effects of membrane-localized ERα in adipocytes and hepatocytes, mice lacking membrane ERα (C451A-ERα) exhibited a mild obesity phenotype (44, 49). In addition, in vivo treatment with EDC that selectively activates nonnuclear ER did not prevent Western diet-induced obesity in wild-type ovariectomized female mice (46). In contrast, male and female mice lacking nuclear ERα (MOER) (49) or the transcriptional domain AF-2 (ERα AF-20) (50) developed obesity as reported in ERα-deficient mice. Thus, cellular pools of nuclear ERα outside adipocytes and hepatocytes, or promoting actions other that inhibition lipogenesis, are predominant in whole-body control of energy homeostasis and adipose tissue mass. These may include activation of nuclear ERα in hypothalamic neurons preventing obesity (49), in hepatocytes promoting FGF-21 gene transcription/FGF21 production and increasing energy expenditure (51), or in muscle and brown adipocytes promoting mitochondrial function (52, 53).

In pancreatic insulin-producing β cells, ERα exhibits a predominantly extranuclear location and is critical to islet survival from diabetic stresses (54). In cultured mouse and human islets, E2, EDC, or E2-BSA similarly prevented apoptosis, demonstrating the importance of membrane-localized or extranuclear ERα to this process (55). Membrane-localized ERα also potentiated the effect of glucose on insulin biosynthesis. This involved ERα binding to Src, followed by ERK activation and leading to nuclear localization, as well as binding of the transcription factor NeuroD1 to the insulin promoter, thereby amplifying insulin gene transcription (56). The nuclear pool of ERα plays a predominant role in glucose homeostasis. Male and female MOER mice lacking nuclear ERα exhibited both fasting and fed hyperglycemia and glucose intolerance (49). In this case, female MOER mice exhibited impaired hypothalamic insulin action leading to increased hepatic glucose production. In contrast, male MOER mice exhibited reduced brain glucose sensing leading to impaired glucose-stimulated insulin secretion. Still, female mice lacking membrane ERα (NOER), exhibited mild glucose intolerance, indicating that a membrane pool of ERα is necessary for glucose homeostasis (49). Together, these studies demonstrated the physiological importance of interactions and collaborations between membrane-initiated and nuclear ERα signaling to preserve energy and glucose homeostasis and to prevent obesity and type 2 diabetes.

Membrane-initiated ERα signaling is necessary and sufficient to promote the rapid and short-term actions of E2 on the endothelium. Thus, E2 vascular actions such as eNOS phosphorylation and rapid vasodilatation, as well as the acceleration of endothelial repair, were abrogated in C451A-ERα mice lacking membrane ERα (42, 57). Accordingly, in vivo treatment of mice with EDC promoted eNOS activation and artery reendothelialization (58). In contrast, treatment with estetrol (E4), a natural estrogen synthesized by human fetal liver that selectively activates nuclear ER actions, failed to promote eNOS activation and to accelerate endothelial repair (59). A second mouse model harboring a point mutation of the murine arginine 264 into alanine (R264A-ERα), which is important to membrane ERα action, showed a loss of rapid dilation of mesenteric arteries and the acceleration of endothelial repair of the carotid artery (60).

Studies from the Arnal and Shaul groups have revealed the dispensable role of membrane-initiated over nuclear ERα signaling for estrogen’s chronic vascular protection. Membrane-initiated ERα was neither necessary nor sufficient for eliciting 4 long-term protective effects of E2: against neointimal hyperplasia, atheroma deposition, hypertension, and induction of flow-mediated remodeling in response to increased blood flow and shear stress (46, 57, 59, 61). Indeed, in contrast to E2, EDC did not blunt atherosclerosis in hypercholesterolemic apoE-/- mice (46), while selective activation of nuclear ERα with E4 prevented atheroma (59) and hypertension, and restored flow-mediated arteriolar remodeling (57). Additionally, ERαC451A mice were fully responsive to estrogens to prevent atheroma and AngII-induced hypertension and allow flow-mediated arteriolar remodeling, while ERαAF20 mice were unresponsive to these estrogens’ beneficial vascular effects. Note that residual membrane-localized ERα expression in ERαC451A mice (42) could have contributed to the effects of E2. EDC treatment improved cardiac ischemia-reperfusion injury (a model of myocardial infarction) in ovariectomized wild-type female mice to an extent similar to that of E2, and this protection was lost in mice deficient in endothelial cell ERα (62). Additionally, treatment of ovariectomized wild-type female mice with a nonnuclear ER agonist, pathway-preferential estrogen (PaPE)-1, decreased stroke severity and improved functional recovery after transient middle cerebral artery occlusion (63).

Female mice express higher levels of membrane-localized ERα in hippocampal field CA1, a region critical for spatial learning, where membrane-localized ERα is required for the activation of kinases that support long-term potentiation, a form of synaptic plasticity thought to underlie learning (64). Finally, the membrane-localized ERα is involved in the perinatal programming and sexual differentiation of the male brain (65), and is required for the effect of E2 in hypothalamic neurons to respond to hypoglycemia and promote efficient refeeding in response to starvation (66).

Functions of Membrane ERβ

As discussed above in the case of membrane-localized ERα, a subpopulation of ERβ is localized to membrane caveolae (67), and membrane-initiated ERβ signaling regulates the cellular localization and function of transcription factors relevant to estrogen action. However, these 2 receptors seem to promote differential engagement of downstream effectors. For example, in human prostate stem and progenitor cells, Prins and coworkers showed that while membrane-initiated ERα signaling preferentially activated AKT, membrane-initiated ERβ signaling selectively activated mitogen-activated protein kinase (MAPK) cascades (68).

In humans, angiotensin II (AngII) stimulates cardiac hypertrophy and progression toward cardiac failure. In female mice, E2 action via membrane-localized ERβ inhibited the nuclear localization of the transcription factor NFATc2, a key player in cardiomyocyte hypertrophy stimulated by AngII, thus preventing AngII-induced cardiac hypertrophy (69). The signaling cascade involved membrane ERβ stimulating AKT-induced calcineurin-interacting protein (MCIP) 1 gene and protein expression, inhibiting AngII activation of the phosphatase calcineurin and resulting in the dephosphorylation and cytoplasmic sequestration of NFATC2. Since NFATC2 plays a prominent role in hypertrophic gene expression, sequestration prevents this AngII action. Another example is the cardiomyocyte hypertrophy inhibitory transcription factor KLF15 (70). AngII acting through TGFβ stimulated a TAK1-p38α kinase axis, inhibiting expression and nuclear localization of KLF15, which increases cardiomyocyte hypertrophic gene expression. ERβ acting through protein kinase A (PKA) opposed TAK1-p38α activation, which restored KLF15 expression to the control state and nuclear localization, contributing in part to inhibition of AngII-induced gene expression and cardiomyocyte hypertrophy.

Another important function of membrane-localized ERβ in preventing cardiac hypertrophy involves the bidirectional regulation of histone-deacetylases (HDAC). AngII stimulated the expression, phosphorylation, and nuclear localization of pro-hypertrophic HDAC2. E2 action via membrane-localized ERβ blocked AngII activation of casein kinase 2 and its ability to promote HDAC2 nuclear translocation and pro-hypertrophic gene expression, thus limiting cardiac hypertrophy (71). In contrast, E2 and membrane-localized ERβ blocked the multiple kinases involved in the phosphorylation and nuclear exclusion of the antihypertrophic HDAC4 and 5. This results in HDAC4 and 5 nuclear translocation and additional inhibition of hypertrophic gene expression (71).

E2 signaling from membrane-localized ERβ inhibited AngII-induced cardiac fibrosis in vitro and in vivo (72). This prevented the ventricular stiffness that diminishes cardiac outflow of blood. The major targets were AngII-stimulated TGFβ expression that required Rho kinase activation, opposed by E2 and ERβ (73). TGFβ activated c-Jun kinase phosphorylation of SMAD 2 and 3 transcription factors, causing nuclear localization and resulting in enhanced collagen and other fibrotic proteins. This was blocked by membrane-localized ERβ signaling through PKA (72, 73). The transition of fibroblasts to myo-fibroblasts was promoted by AngII but inhibited by E2/ERβ signaling. These studies indicate that in the 2 major cell types of the heart, myocytes and fibroblasts, AngII-regulated transcription factor cell localization was opposed by E2/membrane ERβ-initiated signaling (74).

G Protein–Coupled Estrogen Receptor

In 2005, Peter Thomas (75) and Eric Prossnitz (76) independently reported that E2 directly binds to GPR30, which was then considered to act as a membrane-bound ER. In 2007, Chaudry and coworkers reported that administration of E2 induced GPR30 expression and attenuated trauma-hemorrhage-induced hepatic injury via PKA and Bcl-2 expression in vivo in rats (77). As knockdown of GPR30 (but not ERα) attenuated the E2-dependent activation of PKA and Bcl-2 expression in isolated hepatocytes, the study concluded that GPR30 (but not ERα) mediated the effect of E2 in reducing hepatic injury. The same year, the International Union of Basic and Clinical Pharmacology officially named GPR30 as G protein–coupled estrogen receptor (GPER).

Ligand binding studies revealed that GPER binds E2 with much lower binding affinities (Kd = 3-6nM) (75, 76) than classical ERs (Kd = 0.1-1.0nM) (78), raising the question as to whether GPER can mediate E2 physiological actions in vivo. In addition to E2, ER ligands, and SERMs like tamoxifen, 4-hydroxytamoxifen, ICI182,780, and raloxifene elicited cellular responses via GPER in ERα-deficient cells (76, 79-81). Numerous synthetic estrogenic compounds and phytoestrogens were shown to bind and/or activate GPER (82, 83). It is important to consider that these studies were mostly performed in in vitro–based assays, using cancer cells or clonal cells with artificially overexpressed GPER and pharmacological concentrations of E2. The concept of GPER as a membrane ER has been challenged by other groups. Otto et al reported that E2 only shows specific saturated binding to ERα, but not GPER, in GPER-overexpressing COS-7 and CHO cells (84). Most importantly, in primary endothelial cells from ERα/β double knockout mice, E2 failed to activate classical estrogen targets like cAMP, ERK, or PI3K as observed in MCF7 cells expressing ERα (22). In addition, in MCF7 cells, these E2 responses were maintained following siRNA knockdown of GPER. The controversy has been further fueled by a report that the selective GPER agonist G-1 recognizes ERα-36 and may not be specific for GPER (85), although there is little evidence supporting a role of ERα-36 in membrane ERα signaling (24).

In vivo, in clear contrast to the phenotype of ERα deficiency, deletion of GPER in mice produced no effect on reproductive organs (86), demonstrating that GPER is not involved in mediating E2 reproductive actions and raising the possibility that it may not be activated by endogenous E2. Over the years, evidence has accumulated for the importance of GPER in mediating selective E2 actions in vivo in multiple organs (83, 87). It should be noted, however, that these studies do not provide evidence that E2 binds GPER to promote these actions in vivo. GPER-deficient female mice were susceptible to streptozotocin (STZ)-induced islet β-cell death and diabetes, a phenotype like that of ERα-deficient mice but less severe (55). Notably, ERα/ERβ-deficient mice were still protected from STZ-induced diabetes by endogenous and exogenous E2, suggesting for the first time that E2 still signaled in vivo in the absence of ERα/ERβ, possibly via GPER (55). In fact, G-1 was as powerful as E2 in protecting cultured human islet survival. Importantly, the effect of G-1 was lost in GPER-deficient islets, demonstrating its selectivity for GPER (55). In diabetic mouse models of islet transplantation, treatment with E2, the ERα agonist PPT, the ERβ agonist DPN, or G-1 all improved blood glucose and eventually promoted islet engraftment, thus reversing diabetes (88). Similarly, E2, PPT, DPN, and G-1 suppressed islet de novo fatty acid synthesis and esterification into triglycerides in cultured rodent and human islets via ERα, ERβ, and GPER (47, 48). These surprising observations that G-1 produces the same effects as PPT or DPN suggest that both membrane ERα and ERβ could engage GPER as a signaling partner. GPER has been implicated in the expansion of β-cell mass observed during pregnancy. In female rodents, GPER expression is markedly upregulated during pregnancy, which is associated with decreased expression of islet microRNA miR-338-3p (89) and accordingly downregulation of miR-338-3p promoted rodent β-cell proliferation. In isolated rat islets, exposure to E2 or G1 also decreased miR-338-3p to levels observed in gestation, which increased β-cell proliferation via cAMP and PKA pathways (89). Unfortunately, the upstream mechanism of activation of GPR30 was not assessed. Several groups have examined the role(s) of GPER in adiposity. In some studies, GPER deficiency was associated with an increase in overall adiposity in male and female mice (90-92). In others, GPER deficiency produced no phenotype of a slight decrease in adiposity in female mice (93, 94). Differences in environmental factors (chow, bedding, microbiome, temperature of the vivarium) are likely to have contributed to the differences reported (95). Still, in ovariectomized and diet-induced obese female mice, G-1 treatment increased energy expenditure, which normalized the increased adiposity, indicating the importance of GPER activation in reversing metabolic dysfunction induced by E2 deficiency in mice (96). Ovary-intact GPER-deficient female mice exhibited increased atherosclerosis, total and LDL cholesterol levels, and vascular inflammation, with reduced vascular NO bioactivity, effects that were aggravated by ovariectomy. Chronic G-1 treatment also reduced postmenopausal atherosclerosis and vascular inflammation in ovariectomized wild-type mice (97). In hypertensive ovariectomized female rats, chronic G-1 treatment mimicked E2’s effect in decreasing blood pressure, suggesting that activation of GPER protects E2 deficiency-induced hypertension in females (98). The blood pressure lowering effect of GPER could involve NO production, inhibition of endothelin-mediated vasoconstriction, or prevention of AngII-induced hypertension. GPER also promoted glycolysis in endothelial cells (99). In contrast, basal activity of GPER promoted age-dependent impairment of endothelial, NO-mediated relaxation in the renal artery via excessive generation of reactive oxygen species by NADPH oxidases (100). In the brain, G-1 reversed ovariectomy-induced memory impairment in female rats, suggesting that GPER activation can also mimic E2’s effects on memory (101). In the dorsal hippocampus, GPER agonists also enhanced memory in ovariectomized female mice (102); however, GPER modulated memory independently from ERα and ERβ by activating JNK signaling (rather than ERK signaling, like E2) (102). In any case, GPER is considered to mediate rapid E2 effects on neuroprotection (103). In the kidney, GPER is believed to mediate the actions of E2 on natriuresis (104) and seems to mediate E2 lithogenic actions independently of ERα to increase gallstone formation susceptibility in female mice (105). GPER is expressed in peripheral B and T lymphocytes as well as in monocytes, eosinophils, and neutrophils and is believed to mediate E2’s effects in immune cells (106). Finally, GPER is reported to promote endocrine resistance to the antiestrogenic effects of tamoxifen (107-109).

In summary, multiple studies provide evidence for the importance of GPER in E2 actions in vivo in multiple organs. However, these studies do not provide evidence (or exclude) that E2 binds GPER, and it remains to be determined whether GPER functions as a specific endogenous E2 receptor in vivo. Indeed, there may be signaling collaboration between E2-activated membrane-localized ERα (and ERβ) and GPER as part of a signalosome at the membrane (110). It is possible that one or more other endogenous ligands may be the natural ligand for GPER, such as a lipid, an E2 metabolite, or another estrogenic compound, but this remains undetermined.

Mechanisms of Membrane and Nuclear ER Collaborations

During stem cell commitment to organ development and function, both membrane-localized and nuclear ER are required, but the nature of the collaboration of the ER pools is poorly understood. Both bone marrow-derived (44) and adipose tissue-derived (111) pluripotent stem cells are limited in commitment to mature adipocyte formation by estrogen/ER action, thus preventing adipocyte hyperplasia and hypertrophy, and wild-type female mice show little abdominal fat. However, female mice lacking nuclear ERα (MOER), and to a lesser extent female mice lacking membrane-localized ERα (NOER mice), develop visceral adiposity (44, 49). Additionally, the ability of E2 to stimulate the nuclear translocation of ERα in adipose stem cells requires membrane ERα-initiated activation of PI3K-AKT signaling. AKT then phosphorylates cytoplasmic ERα at several sites that are required for nuclear localization (111). This allows nuclear ERα to bind an estrogen response element on the promoter of a key adipogenic gene, PPARγ and repress the maturation of progenitor cells to mature adipocytes. Similarly, the recruitment of functional co-repressors, such as GATA3 and TCF4, to the PPARγ promoter requires E2-stimulated AKT phosphorylation of these proteins (111). Thus, membrane-initiated ERα signaling initiates a phosphorylation cascade required for cytosolic ERα nuclear import, binding to the PPARγ promoter, and recruitment of corepressors, with the end result of inhibiting adipogenesis. This exemplifies the importance of membrane and nuclear ERα collaboration in biology (in this case, for the prevention of obesity). Another interesting example of membrane and nuclear ERα collaboration concerns misfolded protein degradation. In mouse and human pancreatic islets, during endoplasmic reticulum stress, E2 binding to ERα stabilized the endoplasmic reticulum–associated protein degradation (ERAD) system and promoted the proteasomal degradation of misfolded proinsulin (112). This involved E2 activation of membrane-localized ERα, promoting the rapid proteasomal degradation of the ubiquitin-conjugating enzyme and ERAD degrader UBC6e. Additionally, E2 activation of nuclear ERα also promoted the transcriptional repression of the UBC6e gene (112). This provides another example of the importance of membrane-localized and nuclear ERα collaborations in biology, in this case by activating different and synergistic pathways that target the same endpoint: the repression of UBC6e and resulting prevention of endoplasmic reticulum stress (Fig. 1).

Membrane Androgen Signaling

Membrane Androgen Receptor Isoforms

In addition to the canonical full-length 110 kDa androgen receptor (AR), at least 2 AR isoforms originating from alternative splicing at different promoters have been found on the plasma membrane. AR8 is a novel AR splice variant that contains the N-terminal domain and a unique 33–amino acid C-terminus. It lacks a DNA-binding domain and is therefore unlikely to function as a transcription factor, but it is upregulated in castration-resistant prostate cancer cells (113). Another AR splice variant found on the plasma membrane is AR45, which lacks the N-terminal domain (114). A recent study identified AR45 in the plasma membrane lipid rafts of neuronal cells (115).

In LNCaP cells, within minutes of androgen stimulation, AR localizes to lipid rafts membrane microdomains and activates AKT (116). As in the case of ERs and PR (discussed above), palmitoylation on cysteine residue at position 807 is critical for AR membrane translocation. It is inhibited by treatment with 2-bromopalmitate (2-BP), a palmitoylation inhibitor, and by substituting the cysteine residue at position with an alanine (AR–C807A) (23, 117). In prostate cells, the pool of membrane-associated AR is estimated to represent 8% to 10% of total AR after 20 minutes of androgen stimulation, with the remaining AR being translocated to the nucleus. In islet β cells, however, following dihydrotestosterone (DHT) stimulation, AR remained predominantly at the membrane and extranuclear compartments with minimal nuclear localization (118, 119). As in the case of ERs, caveolin-1 is important in prostate cells to promote the transport of palmitoylated AR to the caveolae at the membrane (23, 120), and downregulation of caveolin-1 decreases AR membrane localization (120, 121). AR transport to the membrane is also dependent on microtubules, as it is inhibited by microtubule inhibitors (117, 122). The motor protein kinesin 5B (KIF5B) transports protein cargos to the plasma membrane and disruption of its function via expression of a dominant negative KIF5B (or gene silencing) in LNCaP cells, interferes with AR membrane association (117). Ligand binding to AR promotes AR interaction with KIF5B. In the absence of androgen, AR was associated with the microtubules, which retain AR in the cytoplasm (122). Following androgen stimulation, AR undergoes conformational changes that reduce AR binding to the microtubules, thus allowing it to interact with KIF5B via the N-terminal domain. KIF5B then promotes anterograde transport of AR to the plasma membrane (117) (Fig. 2).

Figure 2.

Membrane-initiated AR signaling. Ligand-dependent interactions of membrane-associated AR with Src lead to rapid activation of Src and downstream kinases in the Ras/Raf/MEK/ERK and PI3K/AKT kinase cascades that enhance cell proliferation and survival in prostate cancer cells or migration and invasiveness in breast cancer cells. Activation of Src can also lead to tyrosine phosphorylation and activation of the EGFR followed by activation of the MAPK and Akt pathways. Collaboration between membrane-associated and nuclear ARs involves membrane-localized AR activating PI3K through Gα s and MAPK through Gα i, resulting in phosphorylation and nuclear import of cytosolic AR and activation of AR transcriptional activity. The figure represents a compilation of some of the multiple binding proteins and signaling pathways of membrane-associated ERα actions that have been described in a variety of different cell types.

Growth factor signaling from the membrane (IR, IGF-1R, GHR) elicits tyrosine kinase activity to signal downstream. The AR does not exhibit intrinsic tyrosine kinase activity, but as in the case of ERs and PRs (discussed above and below), AR recruits the nonreceptor tyrosine kinase Src. Migliaccio et al were the first to show that treatment of human prostate cancer cells with an AR agonist and E2 induces cell proliferation through induction of a membrane ER–AR–Src ternary complex where AR binds the SH3 while ERα binds the SH2 domain of Src (123). Since then, a large body of scientific evidence has demonstrated that ligand-activated AR binds and activates Src in various cell types near the plasma membrane, in lipid raft signalosomes. For example, in prostate cancer cells, binding of AR to Src leads to Src autophosphorylation, activating Src tyrosine kinase (123-125). This is followed by Src phosphorylation of membrane-localized AR on phosphotyrosine residues, which mediates AR recruitment of the p85α regulatory subunit of PI3K and triggers downstream MAPK and Akt pathways to enhance cell proliferation and survival (125-128). In breast cancer cells, androgen treatment similarly drives the assembly of the AR/Src complex that recruits p85α and drives migration and invasiveness (129). In Sertoli cells, upon testosterone stimulation, a population of AR localized to the plasma membrane rapidly associated with and activated Src kinase phosphorylation, leading to tyrosine phosphorylation and activation of the epidermal growth factor receptor (EGFR), followed by activation of the MAPK pathway (130) and Akt (121). These effects are important for spermatogenesis. Notably, Marcelli and coworkers showed that in prostate cancer cells, the stimulatory effects of AR on Src and MAPK activation is observed at low physiological DHT concentrations (0.1-10nM) and were inhibited by higher concentrations (100nM) (131). Accordingly, in NIH3T3 fibroblasts, low concentrations of androgens induced formation of the complex AR/Src/p85. In these cells, AR expression was sufficient to activate cytoplasmic functions but inadequate to stimulate gene transcription (132). Together, these studies are consistent with a model where low concentrations of androgens activate membrane-initiated AR signaling, while nuclear translocation of AR and ARE-dependent transcription is detected in cells with high AR expression and in the presence of high androgen concentrations.

Membrane-localized ARs can signal from the membrane in a Src-independent manner via G protein coupling. For example, AR45 interacted with membrane-associated G proteins Gα q and Gα o in male rat dopaminergic neurons (115). In human prostate cells, androgen induced PI3K activation through Gα s and Gα 12 (133). Additionally, in insulin-producing ß cells, DHT acting on membrane AR amplified the insulinotropic effect of glucagon-like peptide-1 (GLP-1) (118, 119). This involved ligand-activated membrane-localized AR amplification of the ability of the GLP-1 receptor, a G protein–coupled receptor coupled to Gα s, to produce cAMP at the plasma membrane and endosomes and to enhance insulin secretion (119). In this model, DHT-activated AR signaled via cAMP-dependent protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC). In addition, in female mouse liver, DHT increased membrane-localized AR binding to the p85 regulatory subunit of PI3K, resulting in p85 dissociation from the catalytic subunit p110, leading to reduced PI3K and AKT activity, thus producing hepatic insulin resistance (134). In bladder cancer cells lacking nuclear AR, DHT acted on a membrane-localized AR via Gα i protein-mediated MAPK/MMP9 intracellular signaling to increase cancer cell migration and invasion (135). This pathway was insensitive to classical nuclear AR antagonists including enzalutamide, bicalutamide, and hydroxyflutamide. Figure 2 summarizes membrane-initiated AR actions.

Candidates for Transmembrane AR

Four proposed candidates for potential transmembrane androgen receptors, transient receptor potential cation channel subfamily M member 8 (TRPM8), Zrt- Irt-like protein-9 (ZIP9), oxoeicosanoid receptor 1 (OXER1), and G protein–coupled receptor family C group 6 member A (GPRC6A), have been reviewed recently (136).

TRPM8

TRPM8 is the molecular transducer of cold somatosensation in humans and an androgen-regulated cation channel with selectivity for Ca2+. Testosterone increased intracellular Ca2+ influx and channel current activity at low picomolar concentrations in a variety of TRPM8-transfected cells, whereas DHT exhibited a much lower potency and was effective in the low nanomolar range (137, 138). The effect of testosterone in regulating Ca2+ oscillations in HEK-293 cells expressing recombinant TRPM8 was only partially reversed by AR small interfering RNA (siRNA), indicating that in the absence of TRPM8, the classical AR is important in regulating these oscillations. Still, most of the criteria for establishing TRMP8 as a receptor for androgens, such as steroid specificity, limited capacity, high affinity binding, and dissociation, have not yet been met (136).

OXER1

OXER1 is a GPCR activated by the 5-lipoxygenase metabolites of arachidonic acid, 5-oxoeicosatretraenoic acid (5-oxo-ETE) and 5-eicosatretraenoic acid derivatives. 5-oxo-ETE inhibits cAMP production in cells and induces rapid calcium mobilization through activation of an inhibitory Gi protein. Kalyvianaki et al reported that testosterone binds OXER1 in the same grove as 5-OxoETE in prostate cancer cells and antagonizes the inhibitory effects of 5-oxo-ETE on cAMP production, cell migration, and actin cytoskeleton reorganization (139). However, testosterone treatment alone did not alter cAMP levels in OXER1-transfected cells, indicating that an important criterion (activation of AR signaling pathway) was not met for considering OXER1 as a membrane AR (136).

ZIP9

ZIP9 belongs to the ZIP zinc-transporter family that regulates zinc transport into the cytoplasm from outside the cell and from intracellular stores. Using plasma membranes isolated from croaker ovarian cells, human breast and prostate cancer cell lines with high human ZIP9 expression, as well as cancer cells expressing recombinant ZIP9, Thomas and collaborators revealed that ZIP9 displays specific, high affinity, limited capacity, displaceable testosterone binding, characteristic of a membrane AR (136, 140). Testosterone activation of ZIP9 in gonadal and cancer cells signaled through different G proteins (Gs, Gi, and Gq) (136). For example, ZIP9 was a specific Gi coupled-mAR mediating testosterone-induced MAP kinase and zinc signaling in PC3-ZIP9 cells (140). However, testosterone induced apoptosis via activation of Gα s, adenylyl cyclase, protein kinase A, and MAP kinase (Erk1/2) in croaker ovarian cells (141). Testosterone-induced survival or apoptotic processes in teleost ovarian cells occur via Gα s or Gα i -dependent mechanisms in a ZIP9-dependent and AR-independent manner (142).

GPRC6A

GPRC6A is a Gq/11-coupled receptor widely expressed in human and rodent tissues. GPRC6A is activated by multiple ligands, including cations, basic amino acids, and the bone-derived peptide osteocalcin (143). GPRC6A was proposed by Quarles and coworkers to mediate some rapid effects of testosterone in peripheral tissues, including Leydig cells, pancreatic β-cells, and skeletal muscle (144). The effect of testosterone to enhance insulin secretion in mouse cultured pancreatic islets was abolished in GPRC6A-deficient islets, but the glucose-stimulated insulin secretion assay was performed in nonstandard conditions. In fact, the effect of testosterone to increase insulin secretion is abolished in vivo in mice lacking AR in β-cells and in cultured human islets using AR antagonists (118, 119). Testosterone has low affinity for GPRC6A (KD: 9nM) (144) compared with AR (0.2-0.5nM) (145). Surprisingly, testosterone, but not the potent androgen dihydrotestosterone, activated downstream signaling by the GPRC6A such as ERK, Akt, and mTORC1 signaling (146). It is also extraordinary that a GPCR binds such a broad range of endogenous compounds with such different structures, so there is considerable controversy over the proposed role of GPRC6A as an androgen sensor (136). GPRC6A’s broad ligand specificity complicates the interpretation of results, particularly in vivo where it may be modulated simultaneously by multiple ligands. Therefore, there is a need for a more comprehensive characterization of testosterone binding to GPRC6A to determine whether it meets the criteria for its designation as membrane AR.

Mechanisms of Membrane and Nuclear AR Collaborations

The membrane-associated AR pool plays a critical role in potentiating the nuclear import of AR, as the use of a palmytoilation inhibitor or a palmytoilation-deficient mutant AR (AR-C807A) impaired AR transcriptional activity in prostate cells (117). Further, ligand-activated AR induced the phosphorylation of heat shock protein 27 (HSP27), which in turn facilitated AR nuclear translocation (147). However, androgen failed to induce HSP27 phosphorylation in cells expressing a dominant negative of the motor protein KIF5B (discussed above) or in COS-7 cells expressing AR-C807A (117). Thus, membrane-associated AR initiates a signaling cascade, possibly through interaction with AKT, leading to the phosphorylation and activation of ERK and HSP27, the latter of which further facilitates AR nuclear transport (117) (Fig. 2).

Another proposed mechanism of membrane-nuclear AR collaboration involved G proteins (Fig. 2). Low concentrations of androgen acting on a membrane-localized AR signals though Gα s to produce cAMP and activate protein kinase A, which is required for the nuclear translocation and transactivation of AR in prostate cancer cells in the environment of reduced androgen levels (148). In human prostate cancer cells, DHT activation of membrane-bound AR also activates a Gα s and Gα 12 PI3K pathway, which is important for androgen-induced AR interaction with the AR target gene’s promoter region (133). Thus, the collaboration between membrane-associated and nuclear ARs is important in prostate cancer where membrane AR activates different signaling cascades altogether, resulting in nuclear import of cytosolic AR, activation of AR transcriptional activity, and prostate cancer growth.

Membrane Progesterone Signaling

Membrane-Localized Progesterone Receptors

Below, we discuss the signaling actions of 3 major classes of progesterone-binding receptors (PR, PAQRs, PGRMCs; Fig. 3) and provide illustrative examples from physiology. The PGR gene encodes 2 DNA-binding PR isoforms: full-length PR-B, and N-terminally truncated but otherwise identical PR-A. A third, further truncated PR-C isoform, expressed in the fundal myometrium during the onset of labor, lacks part of the DNA-binding domain but may modulate the activity of PR-A or PR-B by competitive binding to cofactors (149). Historic studies in PR knockout mouse models (PRKO) demonstrated that PR-A is required for uterine development, while PR-B is required for mammary gland development (150); PGRs expressed in the male and female brain are also important for a host of integrated neuroendocrine processes involved in normal sexual behavior and reproduction (151). Unlike in the case of ERα, for which the importance of membrane-associated and nuclear pools in vivo has been facilitated by the development of genetic mouse models and pharmacological probes (discussed above), the molecular and biochemical mechanisms of membrane-initiated PR actions have been largely defined using in vitro models. Among these, one of the first examples of direct participation of PR isoforms with membrane-localized or cytoplasmic signaling complexes was included a report by Migliaccio et al of membrane-associated PR-B/ERα/c-Src kinase ternary complexes that could, in the presence of hormone (either estradiol or progesterone), rapidly (3-5 mins) activate both c-Src and downstream MAPKs, independently of PR or ER nuclear transcription (152). Since that time, the biochemical details of these interactions have been further delineated, and their roles in both transcription and cell biology have been defined primarily in breast cancer cell models (16, 153). As discussed above for membrane-localized ERs and AR, membrane-localized PRs can interact with Src via the classical SH2 (p-Tyr-binding) and SH3 (poly-Pro-binding) domains. Notably, a poly-Pro-rich region in the N-terminus of PR-B (but not PR-A) interacts directly with the SH3-domain of Src (154), while ER or other SRs such as AR interact with the Src SH2-domain via phosphorylated Tyr residues (155, 156). PR-B also directly interacts with ERs via 2 ER interacting domains (ERID1 and ERID2) that flank the N-terminal poly-Pro region (157). Ligand-dependent interactions of membrane-associated PR or ER with Src lead to rapid (seconds to minutes) activation of Src and downstream kinases in the Ras/Raf/MEK/MAPK and PI3K/AKT/mTOR/S6 kinase cascades. Importantly, stimulation of MAPK signaling by progesterone in ER/PR-null models (COS cells) required co-expression of membrane-associated PR and ERα, suggesting that a ternary complex (PR/ERα/c-Src) is required to transduce the signal (154). The endpoint of membrane-initiated PR signaling is frequently the regulation of kinase cascades that culminate in changes in nuclear PR activity or stability, as reflected in altered gene regulation and associated biological responses (i.e., proliferation, prosurvival, endocrine resistance, cancer stem cell expansion), most of which have been defined in breast, uterine, or ovarian cancer cell models (16). Thus, liganded membrane-associated PRs also rapidly activate EGFR and JAKs in breast cancer models (158-161). The resulting phosphorylated and activated nuclear PRs regulate STAT expression and activity (158, 162). For example, once PR-B is phosphorylated on Ser81 (pSer81), STAT5A becomes an important target gene of the complex pSer81PR-B/DUSP6/ck2 (162). Nuclear PR and STATs (STAT5, STAT3) co-assemble at PR target genes (exp. wnt1, NOTCH3, KLF4) that are important for breast cancer cell proliferation, survival, and stemness properties (163). As such, targeting phosphorylated PRs may provide a means to block endocrine therapy-resistant cancer cells, as will be discussed in the final section.

Figure 3.

Membrane-initiated PR signaling. Ligand-dependent interactions of membrane-associated PR with Src lead to rapid activation of Src and downstream kinases in the Ras/Raf/MEK/MAPK and PI3K/AKT kinase cascades. Src- and MAPK-dependent phosphorylation of PRs modulate PR location and transcriptional activity, and regulate posttranslational modifications needed for efficient protein trafficking and receptor turnover. MAPK-induced phosphorylation of PRs also promotes the interaction of pPRs with ERαto coregulate target genes as competing or cooperative transcriptional complexes that depend on the hormonal context. Phosphorylation of PRs dramatically alters PR promoter selection and gene expression. In addition, mPRα also likely cross talks with classical PRs (PGR) via changes in cAMP levels and MAPK or other kinase activities. The endpoint of membrane-initiated PR signaling is frequently the regulation of kinase cascades that culminate in changes in nuclear PR activity or stability. The figure represents a compilation of some of the multiple binding proteins and signaling pathways of membrane-associated PR actions that have been described in a variety of different cell types.

Transmembrane Progesterone Receptors—mPRs of the PAQR Gene Family

In addition to nuclear PRs encoded by the PGR gene that function in part at the membrane (discussed above), at least 2 additional types of membrane proteins that bind progesterone with high affinity were described in the early 2000s (164). One class of receptors, collectively termed the mPRs (MW ~40kDa) was initially discovered and characterized in spotted seatrout ovaries ( (165); mPR subtypes α, β,and γ) and is encoded by distinct genes belonging to a large family of seven-transmembrane progesterone and adiponectin Q receptors (PAQR1-11 wherein PAQR5 (γ), PAQR7 (α), and PAQR8 (β) bind progesterone). The second class (further discussed below) includes single transmembrane proteins termed progesterone receptor membrane components 1 and 2 (PGRMC1 and PGRMC2, members of the membrane-associated progesterone receptor (MAPR) family). Both types of membrane PRs (PARQ-5, -7, and -8 and PGMRCs) have been described in several vertebrate (including human) reproductive tissues (endometrium, myometrium, ovaries, sperm) as well as nonreproductive tissues including blood (lymphocytes), gut, and brain (164, 166).

Membrane PRs of the PAQR gene family were first discovered in fish by Peter Thomas and coworkers (165). Since then, mPRs have been extensively characterized in diverse organisms and tissues (167, 168) and are believed to be implicated in important physiological processes, including recently in Schwann cells as targets for the promotion of neuroregeneration (169, 170). The general binding characteristics and signaling functions of mPRs appear to be similar across species and were validated in nuclear PR-null (i.e., PGR gene) cell models including human breast and ovarian cancer cells (166, 171). These receptors bind progesterone with high affinity (Kd ~4-8 nM) as well as androgens with moderate affinity, but not corticosteroids or estrogens. Interestingly, while these receptors primarily bind endogenous progestogens, synthetic PR agonists (R5020, promegestone) or antagonists (RU486, mifepristone) show negligible/low affinity binding (172, 173). The reasons for this specificity are unknown but likely involve the structural alterations of the synthetic PR agonists preventing their binding in the mPR pocket. The progesterone-binding mPRs signal by directly coupling to G proteins and have primarily been shown to activate pertussis-sensitive inhibitory G proteins (Gαi) that downregulate adenylyl cyclase activity and lower intracellular cAMP concentrations, thereby limiting the activation of cAMP-dependent PKA. This family of receptors (namely mPRα) also mobilize calcium and rapidly activate ERK1/2 and p38 MAPKs, as well as PI3K/Akt pathways (likely via trimeric G protein–dependent mechanisms wherein βγ subunits activate downstream MAPKs). Notably, mPRα can also induce (rather than inhibit) cAMP production via activation of stimulatory olfactory G proteins expressed in fish (Atlantic croaker, southern flounder) teleost sperm and is required for progestin-induced hypermotility (174).

The most well-studied mPR family member is mPRα, whose levels are regulated by hormonal treatments and change during the reproductive cycle. Mechanistic studies have supported a requirement for mPRα in oocyte maturation; injection of phosphothioate and morpholino antisense oligonucleotides specifically targeting mPRα into either zebrafish (167) or goldfish (175) blocked oocyte maturation. Studies of multiple mPR knockouts in zebrafish have confirmed a role in both oocyte maturation and ovulation (176). In Xenopus oocytes, progesterone binding to mPRβ induced clathrin-dependent endocytosis of mPRβ into the signaling endosome, where mPR interacted transiently with APPL1 and Akt2 to induce meiosis (177). Additional physiological functions of mPRα include the promotion of sperm motility and the regulation of prolactin secretion. Both mPRα and mPRβ have been implicated in rapid progesterone signaling via activation of inhibitory G proteins, thus lowering cAMP production in human myometrial cells, as a means to alter progesterone action through nuclear PRs during labor (reviewed in (164)). Progesterone-binding mPRs (α, β, and γ) have now been characterized in a variety of mammalian animal models (178, 179) and have different tissue distributions and expression patterns, suggestive of different functions. For example, Drucker and coworkers reported a novel role for progesterone-binding PAQRs in glucose homeostasis (180). Using both in vitro and in vivo models of the intestinal gut enteroendocrine system, they demonstrated that progesterone induces rapid activation of MAPK and increases the expression and secretion of GLP-1 in a PAQR5- and PAQR7-dependent manner. Accordingly, enteral but not systemic administration of progesterone increased plasma levels of GLP-1, glucose-dependent insulinotropic polypeptide (GIP), and insulin, and improved oral glucose tolerance in an RU486-insensitive manner in mice. The authors concluded that intestine-restricted activation of mPRs may allow for stimulation of incretin hormone secretion to control glucose homeostasis. Selective knockdown of mPRα and mPRβ expressed in the brain has revealed their complex roles in sexual behavior (181). Recently, Pang and Thomas showed that the rapid effects of progesterone to enhance the relaxation of vascular smooth muscle cells through mPRα (PAQR7) and increased cytosolic Ca2+ levels involved an increase in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression and activity through inhibitory G proteins Gα i, MAP kinase, and Akt signaling pathways, as well as the downregulation of RhoA activity (182). Finally, changes in the expression levels and localization of all 3 mPRs (α, β, and γ) were associated with early endometrial cancer development and progression, suggesting that these receptors (namely loss of mPR expression) may be an important prognostic biomarker of poor outcome (183).

Progesterone Receptor Membrane Components

In the mid-1990s, PGRMC1 monomers (28 kDa) and dimers (56 kDa) were initially detected in microsomal membranes prepared from rat and porcine livers (184, 185) and the gene encoding a 194–amino acid transmembrane protein was later cloned from porcine smooth muscle (reviewed in (164)). These single-pass transmembrane receptors bind progesterone with high affinity followed by corticosterone/cortisol and testosterone, but do not bind estrogens. A closely related PGRMC2 was later found to be expressed on human sperm; an important signaling function of PGRMC1 and PGRMC2 includes the induction of calcium mobilization/rapid calcium signaling that is associated with the acrosome reaction (186). PGMRCs belong to the MAPR family studied in diverse eukaryotes and have been given several gene names (Hpr6, VemaA, IZA). In addition to progesterone and glucocorticoids, PGMRCs bind an additional 21 carbon steroids, cholesterol, and heme (187). A well-studied physiological role for the progesterone-binding actions of PGRMC1/2 includes antiapoptotic functions in ovarian granulosa cells that occur via regulation of NFKB/p65 signaling (188). The intracellular domain of PGRMC1 contains a cytochrome b5 binding site that mediates receptor interactions with cytochrome P450 enzymes (CYPs). PGRMC1 is thus a positive regulator of several cytochrome P450 (CYP)-catalyzed reactions critical for intracellular sterol metabolism, including the cholesterol synthetic pathway and steroid hormone biosynthesis (189). Genetic translocations that result in greatly reduced expression of PGRMC1 or missense mutations (H165R) in the CYP-binding domain of PGRMC1 (which abrogates binding to CYP7A1 and attenuates receptor antiapoptotic activity) are associated with premature ovarian failure (POF), a condition marked by hypergonadotropic hypogonadism (lack of sex steroid production and elevated gonadotropin levels as a compensatory endocrine mechanism) and amenorrhea before age 40 (190). Altered expression and function of PGRMC1 have also been linked to numerous cancers, including deadly ovarian (190) and cervical (191) cancers. Interestingly, upon heme binding, PGRMC1 monomers convert into stable dimers by forming a heme-heme stacking structure that activates EGFR signaling and confers chemoresistance in colorectal cancer cells; this reaction can be reversed by carbon monoxide (CO) binding, which dissociates the heme-stacking dimer of PGRMC1 (187). Recent studies in knockout mice revealed a new role for PGRMC1 in lipid metabolism: PGRMC1 inhibited hepatocyte de novo lipogenesis via interaction with SREBP1c, thus protecting mice from nonalcoholic fatty liver disease (NAFLD) and hepatic steatosis/steatohepatitis (192). PGRMC1 also enhanced mitochondrial respiration and fatty acid oxidation in cardiomyocytes to prevent cardiac lipotoxicity (193). Zhang et al reported that PGRMC1 is a component of the GLP-1R complex, which potentiates GLP-1-induced cAMP accumulation and insulin secretion (194). The mechanism underlying PGRMC1 enhancement of GLP-1-induced insulin secretion is not clearly understood. It is proposed that PGRMC1 acts as a membrane adaptor protein in β cells to enhance GLP-1R transactivation of the epidermal growth factor receptor, thus increasing GSIS via calcium influx (195-197). Finally, PGRMC1 expression occurs in and is required for mammary gland development during puberty and pregnancy, independently of modulation of nuclear PRs/PGR levels (198).

Mechanisms of Membrane and Nuclear PR Collaborations

Much of the field initially focused on the cytoplasmic or rapid signaling actions of classical nuclear SRs (i.e., the ability to activate diverse signaling pathways) as entirely separate from their functions as ligand-activated transcription factors. While examples of independent actions exist (discussed above), nuclear PRs are also important substrates of the same signaling pathways they rapidly activate (Fig. 3). Thus, PR phosphorylation is an important consequence of PR membrane-initiated rapid signaling events. Indeed, all SRs are heavily posttranslationally modified in response to activation of mitogenic signaling pathways (MAPK, AKT, CK2, CDKs). In the case of PRs, c-Src- and MAPK-dependent phosphorylation events, by promoting PRs phosphorylation, modulate PR location, transcriptional activity, and regulate the degree of other complex posttranslational modifications (ubiquitination, sumoylation, acetylation) needed for efficient protein trafficking and receptor turnover (199-206). Importantly, PR phosphorylation events dramatically alter PR promoter selection (i.e., both DNA binding [cistrome] and gene expression [transcriptome]) (207, 208). The impact of SR phosphorylation and other posttranslational modifications on global gene expression has been modeled for glucocorticoid receptor (GR) (209), AR (210, 211), ER (212, 213), and PR (16, 207, 208).

Studies of the integrated actions of SRs with signaling pathways are relevant to both health and disease states. For example, in the context of ER+ breast cancer, MAPK-induced phosphorylation of PRs on Ser294 (pSer294-PRs) promotes the interaction of pSer294-PRs with ERα and IRS-1 and pSer294-PRs entry into the nucleus to alter the patterns of gene expression, drive increased insulin sensitivity and metabolic plasticity linked to endocrine therapy resistance, and promote cancer stem cell expansion (214, 215). Indeed, ER and PR interact extensively and coregulate target genes as part of either competing or cooperative transcriptional complexes that depend on the hormonal context (216-218). PR-B/ERα interaction requires PR phosphorylation on Ser294, a Pro-directed kinase (MAPK or CDK2) phosphorylation site common to both PR isoforms but well-studied in PR-B (16). Interestingly, the presence of unliganded PR-B (218), PR antagonists (216), or PR agonists (217) can each dramatically alter the E2-regulated transcriptome. PR expression (but not progesterone) is required for the E2-induced regulation of numerous ER-target genes; progestins as well as antiprogestins block E2-induced anchorage-independent breast cancer cell growth (214, 216-218). Functionally redundant (i.e., to PR) glucocorticoid receptors (GRs) are similarly key substrates for p38 MAPK in the absence of GR ligands (209). In triple-negative breast cancer (TNBC) models cultured in steroid-striped serum (i.e., lacking corticosteroids), p38-induced pSer134-GR species bind 14-3-3ζ and are essential mediators of TGFβ1 (and other cytokine) signaling to cell migration that regulate distinct gene sets from those induced/repressed by liganded GRs in the same cells (219). Notably, pSer134 GR target genes also include essential components (MAP3K5) of the p38 MAPK pathway. Similarly, MAPK-dependent phosphorylation of PR on Ser294 induces the expression of unique PR target genes that include essential MAPK pathway components needed to maintain sensitive and robust pathway activation (208).

The regulation of signaling pathway components by phosphorylated SRs is a recurring theme that illustrates the remarkable depth of pathway integration: membrane-localized SRs activate signaling pathways that in turn potentiate the actions of their nuclear SR counterparts. Such a feed-forward system design allows for decisive and sustained biological responses to small (i.e., physiological) changes in hormonal flux. This theme can be expanded to include the concept that distinct membrane PRs (namely mPRα) also likely cross talk extensively with classical nuclear PRs (PGR) via regulation of changes in cAMP levels and MAPK or other kinase activities that are invoked downstream of these surface progesterone-binding receptors. For example, potent mPR-dependent transactivation of nuclear PR-B in human myometrium models has been reported (220), albeit using a reporter gene readout. Nuclear PRs are regulated by changes in cAMP levels: Treatment of PR+ breast cancer cells with cAMP analogues promotes the partial agonist activities of PR antagonists (221, 222). Notably, mPRs and PGMRCs are co-expressed with nuclear PRs (PGR gene product) in breast and reproductive tissues as well as brain.

Conclusions, Clinical Implications, and Future Directions

Plasma membrane SRs are the first point of contact of sex steroids with the cellular signaling machinery. Thus, rapid sex steroid signaling is a logical prerequisite for downstream nuclear receptor nuclear translocation and transcriptional activity. In this paradigm, membrane-localized SRs activate cytosolic signaling pathways that in turn potentiate the actions of their nuclear SR counterparts, usually via phosphorylation events. This integrated model of SR signaling allows steroid hormone-dependent early membrane effects to enhance late SR-dependent nuclear actions. This includes the effect of membrane SRs (unrelated to the nuclear SR) that cross talk and collaborate with classical membrane and nuclear SR. Plasma membrane rapid SR action can also be independent of SR nuclear actions.

Several therapeutic avenues are emerging from the characterization of membrane and nuclear SR signaling collaborations and pathways discussed in this review. The development of ligands that favor ER- or AR-mediated extranuclear actions at the expense of nuclear pathways, and without minimal reproductive effects or leveraging how relative affinity has differential impact on nuclear vs plasma membrane-associated receptors, may be beneficial for hormone replacement in men and women (223, 224). Preferential activation of nuclear ERα with the estrogen E4 could potentially have beneficial effects in preventing postmenopausal atheroma and hypertension (57, 59). The efficient targeting of membrane ERβ could potentially mitigate cardiac hypertrophy and fibrosis and prevent postmenopausal hypertension-induced heart failure (69-74).

Similarly, activation of GPER with the selective agonist G-1 could provide therapeutic benefits against menopause-associated metabolic abnormalities, such as obesity and diabetes, without reproductive effects (96). Additionally, discoveries regarding the use of G-1 in combination with immune checkpoint inhibitors in melanoma have led to the initiation of the first Phase I clinical trial for G-1 (109). Regarding androgen actions, the discovery that membrane-associated ARs enhance insulin secretion via amplification of GLP-1R actions has implications for the treatment of diabetes in aging hypogonadal men (118, 119). Similarly, the 4 candidates for mARs are potential novel targets for treating prostate cancer because they all mediate either antitumorigenic or protumorigenic effects in prostate cells and tumors (136). As discussed above, in ER+ breast cancer, the phosphorylation of nuclear PRs downstream of rapid signaling events promotes interaction with ERα to drive endocrine therapy resistance and promote breast cancer stem cell expansion (214, 215). Thus, targeting phosphorylated PRs as essential components of active nuclear transcription complexes may provide an effective means to block the emergence of endocrine therapy-resistant breast cancer cell subpopulations capable of early dissemination (16). Due to their high affinity for steroids and drugs, PGMRCs are emerging as exciting targets for treating multiple conditions of altered cholesterol or hormone levels, including infections, liver steatosis and related conditions, cardiovascular diseases, and cancers (189, 225). Therefore, the identification of selective SR pathways offers multiple novel research avenues for therapeutic intervention. Future challenges include the need for identification of specific ligands (agonists and antagonists) and creation of high-quality tools such as specific antibodies to allow a more integrated approach to understanding the actions of the various androgen and progesterone-binding protein receptors (PAQRs, PGMRCs TRPM8, ZIP9, OXER1, and GPRC6A), alone and as potential collaborative partners in both health and disease states. These will be essential to design experiments that will allow distinguishing rapid androgen and/or progesterone actions mediated through the different candidates for mARs and mPRs from each other, since many cells express all 4 proteins.