General and Comparative Endocrinology 195 (2014) 157–163

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General and Comparative Endocrinology

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GPCR-mediated rapid, non-genomic actions of steroids: Comparisonsbetween DmDopEcR and GPER1 (GPR30)

0016-6480/$ - see front matter Crown Copyright � 2013 Published by Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ygcen.2013.10.015

⇑ Corresponding author. Address: The Inositide Laboratory, The BabrahamInstitute, Babraham Research Campus, Babraham, Cambridge CB22 3AT, UK.

E-mail addresses: peter.evans@babraham.ac.uk (P.D. Evans), asha.bayliss@babraham.ac.uk (A. Bayliss), vincenzina.reale@babraham.ac.uk (V. Reale).

Peter D. Evans ⇑, Asha Bayliss, Vincenzina RealeThe Inositide Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK

a r t i c l e i n f o a b s t r a c t

Article history:Available online 1 November 2013

Keywords:G-protein coupled receptorSteroid hormoneCatecholamineRapid, non-genomic actions

Steroid hormones classically mediate their actions by binding to intracellular receptor proteins thatmigrate to the nucleus and act as transcription factors to change gene expression. However, evidenceis now accumulating for rapid, non-genomic effects of steroids. There is considerable controversy overthe mechanisms underlying such effects. In a number of cases evidence has been presented for the directactivation of G-protein coupled receptors (GPCRs) by steroids, either at the plasma membrane, or at intra-cellular locations. Here, we will focus on the non-genomic actions of ecdysteroids on a Drosophila GPCR,DopEcR (CG18314), which can be activated by both ecdysone and the catecholamine, dopamine. We willalso point out parallels between this system and the activation of the vertebrate GPCR, GPER1 (GPR30),which is thought to be activated by 17b-estradiol. We propose that the cellular localization and signallingproperties of both DopEcR and GPER1 may be cell specific and depend upon their interactions with bothaccessory molecules and signalling pathways.

Crown Copyright � 2013 Published by Elsevier Inc. All rights reserved.

1. Introduction

It is now clear that steroids can mediate their actions in cells byboth classical interactions with the mechanisms controlling geneexpression and by much more rapid, and fast, non-genomic inter-actions. In the former case, lipid soluble steroids enter the cell andbind with specific intracellular receptor proteins. These then mi-grate to the nucleus and bind to specific upstream gene promotersites controlling gene expression. Examples of such regulation in-clude the effects of estrogen on its nuclear receptors ERa andERb. Rapid non-genomic actions of steroids, however, can be med-iated by a wide range of mechanisms, including direct actions onion channels, such as GABA or NMDA channels, or effects on mem-brane fluidity. In addition, rapid effects may also be produced incells by steroid-mediated changes in second messenger levels.Such changes have been suggested to be mediated by steroid acti-vation of nuclear receptors, somehow localised at the plasmamembrane by interactions with scaffolding or chaperone proteins.However, increasing evidence is now being presented for the inter-actions of steroids with 7-transmembrane spanning G-protein cou-pled receptors (GPCRs). Since steroids are lipid soluble, it is notclear if such GPCRs need to be expressed on the surface of the plas-ma membrane to facilitate such interactions, or if they could be

activated when expressed on the internal membranes of the endo-plasmic reticulum (ER). There is now increasing evidence for GPCRsignalling from ER and nuclear membranes (Gobeil et al., 2006;Boivin et al., 2008). Examples of steroid activation of GPCRs includethe activation of GPER1 (GPR30) by estrogen (Prossnitz and Barton,2011; Filardo and Thomas, 2012) and the activation of the Dro-sophila DmDopEcR receptor by ecdysone (Srivastava et al., 2005;Evans et al., 2009). In addition, progesterone interacts with mem-brane progesterone receptors (mPRs) which are novel G-protein-coupled receptors belonging to the progestin and adipoQ receptorfamily (PAQR) (Zhu et al., 2003a,b; Thomas and Pang, 2012). Thisshort review will focus on recent studies on the signalling andfunctional properties of DmDopEcR and GPER1 and attempt toidentify parallels between the two different systems.

2. DmDopEcR

2.1. Pharmacology and signalling properties of DmDopEcR

DmDopEcR (CG18314) was first identified as an orphan Dro-sophila GPCR that had a high sequence similarity with vertebrateb-adrenergic receptors (Srivastava et al., 2005). However, extensivepharmacological studies revealed that it had a highly unusualpharmacology. Dopamine was the only biogenic amine capable ofmodestly increasing cyclic AMP levels in clonal cell lines express-ing this receptor. Moreover, both the potency and efficacy of theactions of dopamine showed cellular specificity. Both activationmeasures were increased when the receptor was expressed in

Fig. 1. The Drosophila GPCR, DmDopEcR (CG18314) responds to both ecdysteroidsand to the catecholamine, dopamine (Srivastava et al., 2005). When expressed inDrosophila Sf9 cells, or in CHO cells, DmDopEcR shows ‘‘agonist-specific coupling’’(biased agonism) whereby the catecholamine dopamine can couple the receptor toan increase in cyclic AMP levels together with an activation of the PI3Kinasepathway, as judged by the increased phosphorylation of Akt. Ecdysteroids can alsoproduce rapid non-genomic effects through this receptor by coupling it to theactivation of MAPKinase and by inhibiting the actions of dopamine. The receptorhas a much higher affinity for ecdysteroids compared to dopamine (modified fromEvans et al. (2009)).

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insect Sf9 cells compared to its effects when expressed in Chinesehamster ovary (CHO) cells. This suggests that the signalling prop-erties of the receptor can be modulated by interactions with intra-cellular scaffolding proteins, or with other receptors, expressed inthe Sf9 cells but not in the CHO cells. The pharmacology of thereceptor was also unusual in terms of its interactions with syn-thetic agonists and antagonists. It did not display any consistentadrenergic or dopaminergic characteristics (Srivastava et al., 2005).

Unusual aminergic pharmacologies have been reported forother physiological effects in many tissues. For instance, a so called‘‘c-adrenergic pharmacology’’ has been reported to underlie the ac-tions of catecholamines on neurons in certain regions of the brain(Yawo, 1996), on blood vessels (Hirst and Neild, 1980) and on pan-creatic cells (Nadal et al., 2000; Ropero et al., 2002). In these casesthe effects were mediated by a receptor that binds dopamine, nor-adrenaline and adrenaline and had a pharmacology distinct fromthat of a- adrenergic and b-adrenergic receptors. Interestingly, inthe pancreas it was reported that estrogen might mediate someof its rapid effects by the activation of a receptor with such phar-macological properties (Nadal et al., 2000; Ropero et al., 2002).Thus, we decided to see if any endogenous insect steroids, suchas ecdysone, 20-hydroxy-ecdysone (20E) or makisterone A, hadany effects on DmDopEcR (Srivastava et al., 2005). We found thatmembranes isolated from Sf9 cells transfected with this receptorshowed specific saturable binding for [3H]-ponasterone, a plantsteroid related to ecdysone. This specific binding could be dis-placed by cold steroids with the following rank order of potency;ponasterone > ecdysone > 20-hydroxy-ecdysone, with makisteroneA having no effect. These steroids showed an identical rank order ofpotency in their ability to inhibit dopamine-stimulated increases incyclic AMP levels in Sf9 cells expressing DmDopEcR. Further exper-iments revealed that dopaminergic activation of the receptor couldalso lead to the phosphorylation of Akt via activation of the PI3Ki-nase pathway, but that it could not activate the MAPKinase path-way, as measured by the phosphorylation of ERK1/2. Conversely,activation of the receptor by ecdysone lead to a rapid, and dose-dependent, increase in the phosphorylation of ERK1/2 but had noeffect on the phosphorylation of Akt.

The above results suggest that DmDopEcR shows agonist-spe-cific coupling (biased agonism) (Robb et al., 1994; Evans et al.,1995; Kenakin, 1995; Kenakin and Christopoulos, 2013) in its cou-pling to different second messenger pathways when expressed inboth Sf9 cells and in CHO cells (Srivastava et al., 2005) (seeFig. 1). Thus, different agonists are able to couple the receptor dif-ferentially to different second messenger pathways. In functionalterms, it could mean that the receptor signals via different secondmessenger pathways in different locations in the nervous systemwhere it is preferentially expressed (Robb et al., 1994), dependingon the identity of the ligand released into its immediate chemicalenvironment. In addition, the presence of one ligand, such as ecdy-sone, which is released in a pulsatile fashion to control various as-pects of development, could gate the responses of the receptor toexposure to pulses of dopamine released at the same time.

One outstanding question on the molecular pharmacology ofDmDopEcR relates to the relationship between the binding siteson the receptor for ecdysone and for dopamine. The sites couldbe partially overlapping, with the di-hydroxylated ring structuresof both molecules binding to a common site, but with the morecomplex steroid molecule binding to additional sites on the recep-tor. This would be consistent with the higher affinity of the recep-tor for steroids compared to dopamine. It would also be consistentwith the fact that the steroid appears to be able to displace dopa-mine from the receptor, whereas dopamine appears not to be ableto displace bound steroids from the receptor (Srivastava et al.,2005). However, it is also possible that the two ligands couldbind to completely different, but allosterically interacting, sites

(Christopoulos and Kenakin, 2002). To facilitate studies on thelocalization of ligand binding sites on DmDopEcR, using in vitromutagenesis approaches, we have attempted to find ways to in-crease the size of the signal in the dopamine-mediated increasein cyclic AMP assays. We reasoned that the modest size of thesesignals might be due to a low cell surface expression of the V5 epi-tope C-terminally tagged DmDopEcR in the clonal cell lines used.However, we were unable to substantially increase the cell surfaceexpression pattern of this receptor after extensive studies, includ-ing expression in a number of additional cell lines, including Dro-sophila Schneider S2 cells, the use of alternative tags, e.g., HA-tagsand the use of inducible expression systems, (Hughes and Evans,unpublished). Our current thinking is that DmDopEcR is likely toexhibit only a very low level of cell surface expression in clonal celllines and to be rapidly and constitutively endocytosed to the trans-Golgi network, in parallel with observations on a range of otherGPCRs. These include the leucine-rich GPCR-5 (LGR5) (Snyderet al., 2013), the protease activated receptor 1 (Paing et al.,2002), the human vasopressin receptor (Barak et al., 2001), theparathyroid hormone receptor type 1 (Feinstein et al., 2011), theCC Chemokine receptor CCR5 (Escola et al., 2010) and GPER1(GPR30) (Cheng et al., 2011). However, it seems likely that theexpression of such receptors may be stabilized in various nativecell types by interactions with various scaffolding and chaperoneproteins (see examples for GPER1 below), which might also havecell specific influences on their individual pharmacological re-sponses to agonists. Thus, in vitro mutagenesis studies on agonistbinding sites on DmDopEcR might have to await the identificationof appropriate binding partners that can stabilize the receptor inthe plasma membrane.

2.2. Functional roles of DmDopEcR

In view of the widespread expression of DmDopEcR in the Dro-sophila nervous system (Srivastava et al., 2005; Inagaki et al.,2012), it seems very likely that this receptor will have a wide vari-ety of different functional roles in the animal. Many of these arelikely to be related to the transient peaks in ecdysteroid titre

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observed during insect development (Riddiford, 1993). However, itshould be realised that recent studies suppressing the ecdysteroidpeak preceding a moult in the locust, Schistocerca gregaria, byRNAi of the Halloween genes spook, phantom and shade, whichcode for genes involved in ecdysone biosynthesis, do not impedenormal moulting, challenging the classical concept that such apeak is causally linked to a moult (see De Loof et al., 2013). In addi-tion, it is now becoming increasingly realised that ecdysone mayhave a range of functions in the adult nervous system related tothe control of the sleep/wake cycle, to the response to stressful so-cial interactions, to the formation of long-term courtship memoryand even a contribution to the remodelling of the adult nervoussystem during ageing, at both the functional and structural levels,through epigenetic mechanisms (Ishimoto and Kitamoto, 2010,2011; De Loof et al., 2013). Besides producing effects by the induc-tion of changes in gene expression through the activation of nucle-ar ecdysone receptors, ecdysone can also produce effects in adultinsects by the activation of rapid, non-genomic mechanisms (seeSrivastava et al., 2005; Evans et al.. 2009). These include aspectsof development in the tobacco hornworm, Manduca (Champlinand Truman, 1998a,b; Brennan et al., 1998, 2001), the functioningof the neuromuscular junction (Ruffner et al., 1999; Li et al., 2001),the functioning of the silk gland in the silkworm (Elmogy et al.,2004), and the control of electrical activity in lateral and medianprotocerebral cells in Rhodnius prolixus (Ruegg et al., 1982).

To attempt to identify behavioural phenotypes associated withthe DmDopEcR receptor, we have constructed transgenic Drosoph-ila fly lines either overexpressing the receptor, or where theexpression of the receptor has been substantially knocked down.We generated transgenic fly lines overexpressing the DmDopEcRreceptor (Hughes and Evans, unpublished) using the Gal4/UAS sys-tem (Brand and Perrimon, 1993). We expressed the receptor panneuronally (elav-Gal4), in the eye (Rh1-Gal4), in salivary glandsfrom mid-3rd instar (AB1-Gal4) and ubiquitously (Da-Gal4). How-ever, none of the transgenic fly lines we created exhibited anyobvious behavioural phenotypes. Thus, we next generated a rangeof deficiency and transgenic insertion fly lines for DmDopEcR(Hughes and Evans, unpublished). We initially cleaned up a piggy-back line (BL-10847) and then crossed it with a CG18314 RNAi lineto generate a number of lines with substantially reduced expres-sion of DmDopEcR, as assessed by qPCR analysis. Our initial obser-vations on these lines indicate that they have defects in theinitiation of male courtship and in olfactory conditioning (Hanand Evans, in preparation). Recent evidence has also been pro-vided, using the TANGO-mapping of the activation of neuromodu-latory dopaminergic neurones, for a role for DmDopEcR in thedopaminergic appetite control of sugar sensing (Inagaki et al.,2012). Further possible roles for DmDopEcR have also been sug-gested in the regulation of associative and non-associative learn-ing, in short-lasting courtship memory, in the habituation of thejump and flight escape reflex, in the control of locomotor activityand in behavioural transitions under ‘‘stressful social signals’’(Ishimoto and Kitamoto, 2011). In the latter cases it has yet to beresolved whether the behavioural effects described are mediatedvia activation of the receptor by dopamine or ecdysone, or a com-bination of both agonists.

Recent genetic studies in Drosophila have also shown thatDmDopEcR can function as a steroid hormone receptor in vivoand modulate complex behaviours (Ishimoto et al., 2013). Mutantflies with a reduced expression of DmDopEcR show reduced neuro-nal plasticity with delayed giant fibre habituation responses. Theyalso show reduced experience-dependent courtship suppression.Based on a wide variety of genetic, pharmacological and behav-ioural experiments Ishimoto et al. (2013) propose that the activa-tion of DmDopEcR by 20-hydroxy-ecdysone, which increasesduring courtship conditioning, leads to an increase in cyclic AMP

levels in the mushroom bodies of the fly which is responsible forthe suppression of courtship behaviour. Increasing the effective-ness of the DmDopEcR system by either over expression of thereceptor or feeding 20-hydroxy-ecdysone, can overcome the mem-ory defects in the suppression of courtship behaviour caused by apharmacological reduction of dopamine levels. The authors con-sider the possibility that 20-hydroxy-ecdysone could initially stim-ulate the production or release of dopamine and that this in turncould activate DmDopEcR and elevate cyclic AMP levels to inducecourtship memory. However, they consider this unlikely in viewof their current results. Nevertheless, it is interesting to speculateon whether the effects of overexpression of DmDopEcR in flies mu-tant in rutabaga, the gene for Ca2+/CaM-dependent adenylyl cy-clase, in which courtship suppression is blocked, are due to anecdysone-dependent increase in cyclic AMP levels or due to consti-tutive activity of the DmDopEcR receptor. Cheng et al. (2011) havesuggested that GPER1 may also exhibit constitutive activity since itappears to be spontaneously internalized by a clathrin-dependent,non-b-arrestin-dependent pathway.

Thus, the above genetic studies have demonstrated that DmDo-pEcR can be activated by either dopamine or 20-hydroxy-ecdysoneboth in clonal cell lines (Srivastava et al., 2005) and in vivo (Inagakiet al., 2012; Ishimoto et al., 2013). Interestingly, in clonal cell linesonly dopamine increased cyclic AMP levels whilst ecdysone in-creased ERK 1/2 phosphorylation. In contrast, in the above geneticstudies in vivo 20-hydroxy-ecdysone was also shown to increasecyclic AMP levels. A possible explanation for this difference insignalling capabilities could be due to the presence of differentscaffolding or modulatory proteins in vivo which alter the ago-nist-induced conformations of the receptor and thus its ability tocouple to different second messenger pathways. It will be of muchinterest to determine the role of DmDopEcR in other parts of theDrosophila nervous system and to see how its signalling propertiesmodulate other forms of complex behaviour.

Future studies on DmDopEcR will provide valuable informationabout the interactions of steroids with GPCRs, as well as details ofthe signalling pathways they utilise. In addition, they will generateinformation about the usefulness of this receptor as a potential tar-get for the development of novel insect specific control agents.

3. GPER1 (GPR30)

GPER1 is a putative vertebrate estrogen activated GPCR that iswidely distributed in many tissues, including the pancreas, cancercells, placenta, heart, prostate, blood vessels and lymphoid tissue(Prossnitz and Barton, 2011; Filardo and Thomas, 2012). It is alsoexpressed in several brain regions, suggesting it might play a rolein mediating, rapid, fast actions of 17b-estradiol within the brain,including neuroprotection and the modulation of plasticity of syn-aptic spines (see Srivastava and Evans, 2013). However, there areseveral contentious issues surrounding aspects of the location ofthis receptor, its signalling properties and its pharmacology, whichbring into question whether 17b-estradiol is the true, or sole,ligand for this receptor (Langer et al., 2010; Levin, 2009).

3.1. Cellular location of GPER1

The cell surface expression of GPER1 has been highly controver-sial. Some cell surface binding experiments and immunocytochem-ical studies, have suggested it expresses on the cell surface ofcancer cell lines (Thomas et al., 2005) and of transfected HEK cells(Filardo et al., 2007), as well as on intracellular membranes. It hasalso been reported to be localised in plasma membranes in a vari-ety of transfected cell types and primary tissues, including rat hip-pocampal CA2 pyramidal cells (Funakoshi et al., 2006; Maiti et al.,

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2011; Sanden et al., 2011). In contrast, studies using GFP taggedreceptor expressed in COS cells, immunocytochemistry on somecancer cell lines and the use of fluorescently tagged estradiol insome cancer cell lines, failed to demonstrate a plasma membranelocation for GPER1 (Revankar et al., 2005) and suggested that thereceptor was localised on intracellular ER membranes. It was sug-gested that it could be activated here by its presumed lipid solubleligand, 17b-estradiol, after it had crossed the cell membrane. It isnow becoming accepted that some GPCRs may be functionally ac-tive at intracellular sites on both the endoplasmic reticulum andnuclear membranes (Boivin et al., 2008; Gobeil et al., 2006), espe-cially if they can be activated by lipophilic ligands. A possible res-olution to this controversy has been provided recently by the use of‘‘live-cell cell-surface immunocytochemistry’’ approaches (Chenget al., 2011), which have detected low levels of N-terminally HA-tagged GPER1 in HEK-293 cells exposed to HA-antibodies in thecold prior to fixation and permeabilization. Under these conditionsa plasma membrane localization of GPER1 could be detected ini-tially which was then internalized by a rapid ligand-independentconstitutive internalization process once the cells had beenwarmed up to 37 �C. The receptor could be followed through theendosomal regions of the cell and accumulated in a perinuclearlocation. As mentioned above, GPER1 is one of an increasing num-ber of GPCRs that show low cell surface expression in many clonalcell lines. This raises the question of whether GPER1 can be stabi-lised functionally in ‘‘signalosomes’’ in the plasma membrane ofcells in intact tissues by interactions with scaffolding proteins orby dimerization with other GPCRs. Akama et al. (2013) have pro-vided evidence that GPER1 can interact with post-synaptic den-sity-95 protein (PSD-95) in hippocampal dendritic spines andthat such an interaction can stabilize and increase the amount ofGPER1 at the plasma membrane. They also provide evidence thatGPER1 can interact with a range of other GPCRs, including themembrane progestin receptor, the corticotropin releasing hormonereceptor and the 5HT1a serotonin receptor. In addition, GPER1 hasalso been shown to interact with the Receptor Activity Modifying

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Fig. 2. The time course of action of 17b-estradiol (E2) (A), aldosterone (B) and G1 (C) ontagged GPER1 were grown as previously described (Filardo et al., 2007). Cells were seruagonist concentrations giving maximal effects were chosen from preliminary dose–respo100%. Equal quantities of cell lysates were separated by SDS–PAGE and analysed for phos(Bayliss and Evans, 2013). Representative blots are shown for the time courses. Summarishown. Data are expressed as the mean ± SEM of at least 3 observations and are expres

Protein 3 (RAMP3) in cardiac cells (Lenhart et al., 2013). It willbe of much interest to see if GPER1 interacts with other stabilizingproteins in other tissues and whether such interactions modify itspharmacological and signalling properties.

3.2. Pharmacology of GPER1

A considerable amount of evidence suggests that GPER1 is aGPCR that is preferentially activated by the steroid, 17b-estradiol(see Prossnitz and Barton, 2011; Filardo and Thomas, 2012). It isnot activated by estrone, 17a-estradiol or estriol. A putative spe-cific agonist, G1, and an antagonist, G15, have been developedfor GPER1, which have no binding to either of the nuclear estrogenreceptors ERa or ERb, and G1 was shown not to bind to 25 otherGPCRs (Bologa et al., 2006; Dennis et al., 2009; Blasko et al.,2009). These compounds have been used extensively to supportthe contention that many of the rapid, non-genomic actions of17b-estradiol in various tissues are mediated via the activation ofGPER1. However, the specificities of G1 and G15 have been calledinto question in some instances (Langer et al., 2010). Further, somerecent studies have even suggested that another steroid, aldoste-rone, may also be capable of activating GPER1 in some tissues, suchas rat vascular smooth muscle cells and rat vascular endothelialcells (Batenburg et al., 2012; Gros et al., 2011, 2013). To try todetermine if the effect of aldosterone on GPER1 is highly cell spe-cific, we have tested the pharmacology of this receptor when ex-pressed in a number of different expression systems.

When expressed in HEK293 cells GPER1 can couple to the stim-ulation of adenylyl cyclase activity and to calcium signalling (Filar-do et al., 2007). It can also couple to ERK 1/2 phosphorylation viaan integrin/EGFR pathway in various cancer cell lines (see Filardoand Thomas, 2012). In addition, when expressed in HEK 293 cellswe have shown (Evans et al., 2013; Bayliss and Evans, in prepara-tion) that GPER1 produced time dependent increases in ERK1/2phosphorylation in response to both 17b-estradiol and to G1(Fig. 2A and C). In both cases the responses peaked between 2

C G1 (100 nM)

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10 15 20 rone (1 nM)

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ERK 1/2 activation in GPER1 expressing HEK 293 cells. HEK 293 cells expressing HA-m starved for 3 days prior to stimulation with agonists for the times indicated andnse curves. Basal measurements in the absence of agonists at zero time are shown aspho-ERK (p-ERK) and total-ERK (t-ERK) by Western blotting as described previouslyes of the quantified blots for each of the time courses for the three agonists are alsosed as the percentage of zero time ERK 1/2 phosphorylation.

P.D. Evans et al. / General and Comparative Endocrinology 195 (2014) 157–163 161

and 5 min and were maximal for 17b-estradiol at 10 nM and for G1at 100 nM. Aldosterone was also able to produce increases in ERK1/2 phosphorylation in these cells with a similar time course butwith maximal effects observed at a lower concentration (1 nM)(Fig. 2B). Parallel experiments in wild type HEK 293 cells did notreveal any increases in ERK1/2 phosphorylation after exposure toany of the three agonists (Bayliss and Evans, unpublished). Furtherexperiments are required to determine if all three agonists are act-ing on the same binding site or differentially interacting with allo-steric sites on GPER1.

When expressed in Xenopus oocytes (Evans et al., 2013; Realeand Evans, in preparation) GPER1 could be activated by 2 minpulses of either 1 lM 17b-estradiol or aldosterone to induce smallinward currents in oocytes co-expressing G-protein coupled in-wardly rectifying potassium channels (GIRKs) (Fig. 3A). However,surprisingly, larger inward currents were observed in oocytes ex-posed to 1 lM dopamine. The responses to dopamine were dose-dependent with maximal effects occurring around 3 � 10�6 Mand threshold effects between 10�8 and 10�7 M (Fig. 3C). Uninject-ed oocytes, and oocytes expressing GIRKs alone, showed no re-sponses to equivalent pulses of any of the three agonists (Reale

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Fig. 3. The effect of dopamine, 17b-estradiol and aldosterone on currents generatedrectifying potassium channel (GIRKs) currents were induced in oocytes co-expressing GPestradiol or aldosterone. (B), Dose–response curve for the effects of dopamine on GIRKexpressing GPER1 (R) and the cyclic AMP cystic fibrosis gated chloride channel (CFTR1)hydroxytryptamine; and ADR = adrenaline. (D) Dose–response curve for the effects odeterminations. Xenopus oocytes were prepared, injected with cRNA for GPER1, CFTR1 aprocedures as described previously (Rogers et al., 2003).

and Evans, unpublished). Dopamine was also able to producedose-dependent increases in cyclic AMP levels in Xenopus oocytesexpressing GPER1, as measured by increases in the currents gener-ated by the co-expression of GPER1 with the cyclic AMP activatedcystic fibrosis chloride channel (CFTR1) (Fig. 3D). Control experi-ments (Fig. 3B) indicate that the effects produced by 2 min pulsesof 1 lM dopamine are mediated via interactions with the GPER1receptor. No responses were found in oocytes expressing thereceptor alone in the absence of the cystic fibrosis channel or whenthe cystic fibrosis channel was expressed in the absence of thereceptor. Preliminary pharmacological experiments indicate thatsmaller responses were observed after application of 2 min pulsesof noradrenaline (1 lM) but not to similar pulses of 5-HT. How-ever, equivalent size responses to those of dopamine were ob-served after exposure to 2 min pulses of 1 lM adrenaline inoocytes co-expressing both GPER1 and CFTR1. Similar to the dopa-mine experiments, the responses to adrenaline disappeared in oo-cytes lacking CFTR1 but, surprisingly, were still present in oocyteslacking GPER1 but expressing CFTR1. This suggests that adrenalineis likely to be producing its effects in these oocytes by a directaction on CFTR1.

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in Xenopus oocytes expressing human GPER1. (A), G-protein activated inwardlyER1 and GIRK 1 and GIRK 2, after exposure to 2 min pulses of 1 lM dopamine, 17b-currents. (C), Amine specificity of the Xenopus oocyte current responses in oocytes

to 2 min pulses of amines at 1 lM. DA = dopamine; NA = noradrenaline; 5HT = 5-f dopamine on CFTR1 chloride currents. Standard error bars = SEM of at least 3nd GIRKs and two-electrode voltage clamped recordings were made using standard

Fig. 4. Potential ‘‘agonist-specific coupling’’ (biased agonism) of different ligandsfor the GPER1 (GPR30) receptor. Evidence suggests that the receptor may beactivated by catecholamines and aldosterone, in addition to 17b-estradiol. Thereceptor can also interact with a number of different GPCRs, scaffolding proteins(such as PSD-95) and chaperone proteins (such as RAMP3), which may increase thetime it spends in the plasma membrane. It is not currently known if suchinteractions can alter the pharmacological and signalling profiles of the receptor.

162 P.D. Evans et al. / General and Comparative Endocrinology 195 (2014) 157–163

The later effect of dopamine on Xenopus oocytes expressingGPER1 is interesting in light of the observation that the rapid,non-genomic actions of 17b-estradiol on insulin release from pan-creatic b-cells and on glucagon release from pancreatic a-cells,have been suggested to be due to the activation of a ‘‘non-classicalmembrane estrogen receptor’’ (Nadal et al., 2000; Ropero et al.,2002) where the catecholamines, norepinephrine, dopamine andepinephrine were able to displace non-permeant estradiol-peroxi-dase (E-HRP) binding in these cells. It has recently been suggestedthat GPER1 can only be activated in pancreatic b-cells by high con-centrations of 17b-estradiol (100 nM–5 lM), whilst lower concen-trations (100 pM–50 nM) might activate the ERb pathway leadingto cyclic GMP production, the closure of KATP channels and calciumdependent release of insulin (Ropero et al., 2012). The above stud-ies raise the question of the relationship of GPER1 to the pancreaticislet estrogen sensitive ‘‘c-adrenergic receptor’’ (see Section 2.1above). It is not clear if GPER1 can bind catecholamines and/oraldosterone, in addition to, or in preference to, 17b-estradiol, inall tissues. Alternatively, the binding of these addition ligands toGPER1 may only be found in some tissues where GPER1 can eitherdimerise with other GPCRs or chaperone proteins, such as RAMP3,which can allosterically alter the pharmacological properties ofGPER1.

The above results are also interesting since dopamine is thoughtto be a non-membrane permeant agonist, in contrast to 17b-estra-diol, which is thought to be membrane permeant and to readilypass across lipid barriers, such as the plasma membrane. Thus,the effects of dopamine on GPER1 expressed in Xenopus oocytes,also provide evidence for the cell surface expression of GPER1and its accessibility to extracellular agonists.

4. Conclusions

The ecdysone activated GPCR from Drosophila, DmDopEcR, andthe 17b-estradiol activated GPCR from vertebrates, GPER1, appearto share a number of similarities in terms of their cellular location,signalling properties and pharmacology. Thus, they both appear tobelong to a group of GPCRs that are only expressed poorly in theplasma membrane, when expressed in clonal cell lines, and appearto be rapidly and constitutively internalized into endosomal com-partments and accumulate in a perinuclear location. Their

presence in the plasma membrane way well be stabilised in tissuesby interactions with a range of other GPCRs or with scaffolding orchaperone proteins (see Fig. 4). Such interactions may lead to alter-ations in their pharmacology, such that in certain cell types bothreceptors can interact with both steroids and catecholamines. Itis also possible that receptor interactions with these different li-gands can lead to various forms of agonist-specific coupling (orbiased agonism) (Evans et al., 1995; Kenakin, 1995; Kenakin andChristopoulos, 2013), whereby the different ligands induce differ-ent conformations of the receptors which can then preferentiallyinteract with different second messenger pathways. Futureresearch on these receptors will need to identify the specificmolecules they interact with in different cell types and therelationships between their binding sites for steroids andcatecholamines.

Acknowledgments

This work was supported by the BBSRC through the BabrahamInstitute and by a BBSRC Studentship to A.B. We thank ProfessorE.J. Filardo, Brown University, for kindly supplying us with theHEK 293 cell line expressing the HA-GPER1 receptor.

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