| 1. |
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| 2. |
- Broselid, Stefan, et al.
(author)
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G Protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex With Membrane-associated Guanylate Kinases (MAGUKs) and AKAP5 That Constitutively Inhibits cAMP Production.
- 2014
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In: Journal of Biological Chemistry. - 1083-351X. ; 289:32, s. 22117-22127
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Journal article (peer-reviewed)abstract
- GPR30, or G protein-coupled estrogen receptor (GPER), is a GPCR reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PSD-95/Discs-large/ZO-1 homology (PDZ) motif at the receptor C-terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor, and in MDCK cells expressing native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor influenced receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases (MAGUKs), including SAP97 and PSD-95, and A-kinase anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with protein kinase A (PKA) RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Thus, GPR30 forms a plasma membrane complex with a MAGUK and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane.
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| 3. |
- Broselid, Stefan
(author)
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The G protein-coupled receptor GPR30 signalosome - A novel G protein-independent mechanism regulating cAMP signaling and receptor trafficking
- 2014
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Doctoral thesis (other academic/artistic)abstract
- The large protein family called G Protein-coupled receptors (GPCRs) has co-evolved with life throughout evolution; from single cell organisms all the way to complex beings such as us humans. The fact that GPCRs are involved in essentially every physiological event, and that ~50% of drugs on the current market are either directly or indirectly targeted towards the function of GPCRs, we can be certain of their considerable importance. This thesis is dedicated solely to one particular GPCR, GPR30. This receptor is shrouded in uncertainty with contradictory results and opposing views on effectors and subcellular localization. The aim of this thesis was to elucidate the signaling and membrane trafficking of GPR30 in addition to look for any binding partners. My primary findings were: (1) GPR30 constitutively internalizes without any need for ligand binding. (2) GPR30 associates with cytokeratin filaments (3) GPR30 expression in ER+ breast cancer is a favorable prognostic marker for distant-disease-free survival. (4) GPR30 confer some constitutive pro-apoptotic signaling but also readily sensitizes the cells to other apoptotic stimuli. (5) GPR30 directly associates with RAMP3 in-vivo and in-vitro and RAMP3 expression has an impact on GPR30 subcellular localization in the murine heart. (6) GPR30 constitutively form a signalosome with Membrane associated guanylate kinase proteins (MAGUKs) and A Kinase Anchoring Protein 5 (AKAP5) through its C-terminal PDZ-motif. PKA-RII, which directly binds to AKAP5, is responsible for the attenuation of cAMP in response to cAMP- elevating agents.
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| 4. |
- de Valdivia, Ernesto Gonzalez, et al.
(author)
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Roles of PDZ-dependent Interactions and N-glycosylation in G Protein-coupled Estrogen Receptor 1 (GPER1)/GPR30-mediated Stimulation of ERK1/2 Activity
- 2018
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In: FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 32:1 Suppl, s. 6-685
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Conference paper (peer-reviewed)abstract
- G protein-coupled receptor 30 (GPR30) is a G protein-coupled receptor (GPCR) that is attracting considerable attention in breast cancer and cardiometabolic regulation. Following reports that GPR30 is required for some rapid estrogen responses, e.g. increased cAMP production and ERK1/2 activity, in estrogen receptor (ER)-negative cells, GPR30 was renamed G protein-coupled estrogen receptor 1 (GPER1). However, many questions remain about the identity of the cognate receptor ligand, receptor-effector coupling, and receptor membrane trafficking. To address the mechanism by which human GPR30 activates ERK1/2, we used HEK293 cells with and without ectopic expression of GPR30. Specifically, we investigated the role of the type I PSD-95/Discs-large/ZO-1 homology (PDZ) motif at the receptor C terminus (-SSAV) and three consensus sites for N glycosylation (N-X-S/T) in the receptor N-terminal domain (N25, N32, N44). We found previously that the C-terminal PDZ motif enables the receptor to interact with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, and this interaction is necessary for retaining the receptor in the plasma membrane and mediating a constitutive decrease in cAMP production that is not inhibited by pertussis toxin, thus independent of Gi/o. Here, we found that the receptor also constitutively increases ERK1/2 activity. Interestingly, this increase was inhibited by PTX as well as by wortmannin, but not by AG1478, indicating it is mediated by Gi/o and phosphoinositide 3-kinase (PI3K) but not epidermal growth factor receptor (EGFR) transactivation. Deleting the receptor PDZ motif or knocking down AKAP5 also inhibited the increase, showing that the PDZ interaction is also necessary for this response. Interestingly, the proposed GPR30 agonist G-1 increased ERK1/2 activity in a GPR30-dependent manner, but this increase was only observed at very low levels of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif, which completely inhibited the constitutive increase in ERK1/2 activity, did not inhibit the G-1-stimulated increase. Mutating the potential N-glycosylation residues N25 or N32 to I in the GPR30 N-terminal domain did not prevent receptor plasma membrane expression or ERK1/2 activation. On the other hand, mutating N44 to I completely prevented both plasma membrane expression and ERK1/2 activation, and caused receptor degradation. Thus, the PDZ-dependent receptor interaction with SAP97 and AKAP5, and therefore plasma membrane retention, is necessary for constitutive GPR30-mediated stimulation of ERK1/2 activation, whereas G-1-stimulated ERK1/2 activation may remain following constitutive internalization. On the other hand, N-glycosylation of N44 appears to be necessary for maturation of the receptor to the plasma membrane. Support or Funding Information Swedish Research Council and Swedish Cancer Foundation This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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| 5. |
- Gonzalez, Ernesto, et al.
(author)
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G protein-coupled Estrogen Receptor 1 (GPER1)/GPR30 Increases ERK1/2 Activity Through PDZ-dependent and -independent Mechanisms
- 2017
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In: Journal of Biological Chemistry. - 1083-351X. ; , s. 9932-9943
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Journal article (peer-reviewed)abstract
- G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of Gi/o. Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin (PTX) as well as by wortmannin, but not by AG1478, indicating that Gi/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor (EGFR) transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two Gi/o-mediated mechanisms; a PDZ-dependent apparently constitutive mechanism, and a PDZ-independent G-1-stimulated mechanism.
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| 6. |
- Lenhart, P.M., et al.
(author)
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G-protein-coupled receptor 30 interacts with receptor activity-modifying protein 3 and confers sex-dependent cardioprotection
- 2013
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In: Journal of Molecular Endocrinology. - 1479-6813. ; 51:1, s. 191-202
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Journal article (peer-reviewed)abstract
- Abstract Receptor activity-modifying protein 3 (RAMP3) is a single-pass transmembrane protein known to interact with and affect the trafficking of several G-protein-coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with GPR30, also known as G-protein-coupled estrogen receptor 1. GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Using bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3(-/-) mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3(+)(/)(+) and Ramp3(-/-) mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3 and sex dependent. Our results demonstrate that GPR30-RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo and that RAMP3 is required for GPR30-mediated cardioprotection.
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| 7. |
- Olde, Bjorn, et al.
(author)
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G protein-coupled Receptor 30 (GPR30) PDZ-dependently and Constitutively Increases ERK1/2 Signaling Through Calcineurin and Kinase Suppressor of Ras 2 (KSR2)
- 2016
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In: FASEB Journal. - 0892-6638. ; 30:1 Suppl, s. 518-518
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Conference paper (peer-reviewed)abstract
- The objective of the present study was to map the mechanism whereby G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor (GPER), stimulates extracellular signal-regulated kinase (ERK)1/2 signaling. GPR30 plays important roles in cancer and cardiometabolic regulation. We showed recently that GPR30 forms a plasma membrane complex through its C-terminal type I PSD-95/Discs-large/ZO-1 homology (PDZ) motif with a membrane-associated guanylate kinase (MAGUK) and protein kinase A (PKA)-anchoring protein 5 (AKAP5), and AKAP5-anchored PKA regulatory subunit RII suppresses receptor endocytosis and enables the receptor to constitutively inhibit cAMP production. Here, we investigated if this PDZ-dependent GPR30 complex also regulates ERK1/2 signaling. To do so, human and mouse GPR30 were ectopically expressed in HEK293 cells and MDCK cells, and receptors and effectors were monitored by immunoblotting, immunoprecipitation, confocal immunofluorescence microscopy, split luciferase reporter techniques, and reporter gene assays. We found that GPR30 constitutively increased extracellular signal-regulated kinase (ERK) 1/2 activity in several cell systems. The response was dependent on an intact receptor PDZ motif. Furthermore, knocking down AKAP5 or inhibiting calcineurin with FK506 inhibited the receptor response. GPR30 PDZ-dependently inhibited basal NFAT signaling, consistent with the receptor favoring AKAP5-anchoring of calcineurin. The calcineurin substrate kinase suppressor of Ras 2 (KSR2), a mitogen-activated protein kinase (MAPK) scaffold, enhanced the GPR30-promoted response, also dependently on the receptor PDZ motif. GPR30 also PDZ-dependently favored a membrane KSR2 complex at the expense of the monomeric form. On the other hand, disrupting AKAP5-PKA RII interaction with St-Ht31, or inhibiting protein kinase C (PKC) with GF109203X or epidermal growth factor receptor (EGFR) tyrosine kinase with AG1478 had no effect on the GPR30-stimulated response. FK506 also increased the amount of GPR30 in the plasma membrane, thus acting opposite to St-Ht31, which increased receptor endocytosis. We conclude that the PDZ-dependent GPR30 complex with AKAP5 includes calcineurin, which favors GPR30 endocytosis and enables the receptor to constitutively increase ERK1/2 signaling through KSR2. Support or Funding Information Swedish Cancer Foundation
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| 8. |
- Sandén, Caroline, et al.
(author)
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G Protein-Coupled Estrogen Receptor 1 (GPER1)/GPR30 Localizes in the Plasma Membrane and Trafficks Intracellularly on Cytokeratin Intermediate Filaments.
- 2011
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In: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 1521-0111 .- 0026-895X. ; 79:3, s. 400-410
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Journal article (peer-reviewed)abstract
- GPR30, or G protein-coupled estrogen receptor 1 (GPER1), was recently introduced as a membrane estrogen receptor and a candidate cancer biomarker and therapeutic target. However, several questions surround the subcellular localization and signaling of this receptor. In native cells, including mouse myoblast C(2)C(12) cells, Madine-Darby canine kidney (MDCK) epithelial cells, and human ductal breast epithelial tumor T47-D cells, G-1, a GPER1 agonist, and 17β-estradiol (E2) stimulated GPER1-dependent cAMP production, a defined plasma membrane (PM) event, and recruitment of β-arrestin2 to the PM. Staining of fixed and live cells showed that GPER1 was localized both in the PM and on intracellular structures. One such intracellular structure was identified as cytokeratin (CK) intermediate filaments, including those composed of CK7 and CK8, but apparently not endoplasmatic reticulum (ER), Golgi, or microtubules. Reciprocal co-immunoprecipitation of GPER1 and CKs confirmed an association of these proteins. Live staining also showed that the PM receptors constitutively internalize apparently to reach CK filaments. Receptor localization was supported using FLAG- and HA-tagged GPER1. We conclude that GPER1-mediated stimulation of cAMP production and β-arrestin2 recruitment occur in the PM. Furthermore, the PM receptors constitutively internalize and localize intracellularly on CK. This is the first observation that a G protein-coupled receptor (GPCR) is capable of associating with intermediate filaments, which may be important for GPER1 regulation in epithelial cells and the relationship of this receptor to cancer.
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| 9. |
- Tutzauer, Julia, et al.
(author)
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Ligand-independent G protein-coupled estrogen receptor/G protein-coupled receptor 30 activity : Lack of receptor-dependent effects of G-1 and 17b-estradiol
- 2021
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In: Molecular Pharmacology. - 0026-895X. ; 100:3, s. 271-282
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Journal article (peer-reviewed)abstract
- G protein-coupled receptor 30 (GPR30) is a membrane receptor reported to bind 17b-estradiol (E2) and mediate rapid nongenomic estrogen responses, hence also named G protein-coupled estrogen receptor. G-1 is a proposed GPR30-specific agonist that has been used to implicate the receptor in several pathophysiological events. However, controversy surrounds the role of GPR30 in G-1 and E2 responses. We investigated GPR30 activity in the absence and presence of G-1 and E2 in several eukaryotic systems ex vivo and in vitro in the absence and presence of the receptor. Ex vivo activity was addressed using the caudal artery from wild-type (WT) and GPR30 knockout (KO) mice, and in vitro activity was addressed using a HeLa cell line stably expressing a synthetic multifunctional promoter (nuclear factor jB, signal transducer and activator of transcription, activator protein 1)-luciferase construct (HFF11 cells) and a human GPR30-inducible T-REx system (T-REx HFF11 cells), HFF11 and human embryonic kidney 293 cells transiently expressing WT GPR30 and GPR30 lacking the C-terminal PDZ (postsynaptic density-95/discs-large/zonula occludens-1 homology) motif SSAV, and yeast Saccharomyces cerevisiae transformed to express GPR30. WT and KO arteries exhibited similar contractile responses to 60 mM KCl and 0.3 mM cirazoline, and G-1 relaxed both arteries with the same potency and efficacy. Furthermore, expression of GPR30 did not introduce any responses to 1 mM G-1 and 0.1 mM E2 in vitro. On the other hand, receptor expression caused considerable ligand-independent activity in vitro, which was receptor PDZ motif-dependent in mammalian cells. We conclude from these results that GPR30 exhibits ligand-independent activity in vitro but no G-1- or E2-stimulated activity in any of the systems used. SIGNIFICANCE STATEMENT Much controversy surrounds 17b-estradiol (E2) and G-1 as G protein-coupled receptor 30 (GPR30) agonists. We used several recombinant eukaryotic systems ex vivo and in vitro with and without GPR30 expression to address the role of this receptor in responses to these proposed agonists. Our results show that GPR30 exhibits considerable ligand-independent activity in vitro but no G-1- or E2-stimulated activity in any of the systems used. Thus, classifying GPR30 as an estrogen receptor and G-1 as a specific GPR30 agonist is unfounded.
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