| 1. |
- Tutzauer, Julia, et al.
(författare)
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G protein-coupled estrogen receptor (GPER)/GPR30 forms a complex with the β1-adrenergic receptor, a membrane-associated guanylate kinase (MAGUK) scaffold protein, and protein kinase A anchoring protein (AKAP) 5 in MCF7 breast cancer cells
- 2024
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Ingår i: Archives of Biochemistry and Biophysics. - 0003-9861. ; 752
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Tidskriftsartikel (refereegranskat)abstract
- G protein-coupled receptor 30 (GPR30), also named G protein-coupled estrogen receptor (GPER), and the β1-adrenergic receptor (β1AR) are G protein-coupled receptors (GPCR) that are implicated in breast cancer progression. Both receptors contain PSD-95/Discs-large/ZO-1 homology (PDZ) motifs in their C-terminal tails through which they interact in the plasma membrane with membrane-associated guanylate kinase (MAGUK) scaffold proteins, and in turn protein kinase A anchoring protein (AKAP) 5. GPR30 constitutively and PDZ-dependently inhibits β1AR-mediated cAMP production. We hypothesized that this inhibition is a consequence of a plasma membrane complex of these receptors. Using co-immunoprecipitation, confocal immunofluorescence microscopy, and bioluminescence resonance energy transfer (BRET), we show that GPR30 and β1AR reside in close proximity in a plasma membrane complex when transiently expressed in HEK293. Deleting the GPR30 C-terminal PDZ motif (-SSAV) does not interfere with the receptor complex, indicating that the complex is not PDZ-dependent. MCF7 breast cancer cells express GPR30, β1AR, MAGUKs, and AKAP5 in the plasma membrane, and co-immunoprecipitation revealed that these proteins exist in close proximity also under native conditions. Furthermore, expression of GPR30 in MCF7 cells constitutively and PDZ-dependently inhibits β1AR-mediated cAMP production. AKAP5 also inhibits β1AR-mediated cAMP production, which is not additive with GPR30-promoted inhibition. These results argue that GPR30 and β1AR form a PDZ-independent complex in MCF7 cells through which GPR30 constitutively and PDZ-dependently inhibits β1AR signaling via receptor interaction with MAGUKs and AKAP5.
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| 2. |
- de Valdivia, Ernesto Gonzalez, et al.
(författare)
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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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Ingår i: FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 32:1 Suppl, s. 6-685
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Konferensbidrag (refereegranskat)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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| 3. |
- Gonzalez de Valdivia, Ernesto, et al.
(författare)
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Human G protein-coupled Receptor 30 (GPR30) is N -glycosylated and N-terminal Domain Asparagine 44 is Required for Receptor Structure and Activity
- 2019
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Ingår i: Bioscience Reports. - 0144-8463. ; 39:2
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Tidskriftsartikel (refereegranskat)abstract
- GPR30, or G protein-coupled estrogen receptor (GPER), is a G protein-coupled receptor (GPCR) that is currently attracting considerable attention in breast cancer and cardiometabolic regulation. The receptor was reported to be a novel membrane estrogen receptor mediating rapid non-genomic responses. However, questions remain about both the cognate ligand and the subcellular localization of receptor activity. Here, we used HEK293 cells ectopically expressing N-terminally FLAG-tagged human GPR30 and three unique antibodies (Ab) specifically targeting the receptor N-terminal domain (N-domain) to investigate the role of N -glycosylation in receptor maturation and activity, the latter assayed by constitutive receptor-stimulated ERK1/2 activity. GPR30 expression was complex with receptor species spanning from about 40 kDa to higher molecular masses and localized in the endoplasmatic reticulum (ER), the plasma membrane (PM), and endocytic vesicles. The receptor contains three conserved asparagines, Asn25, Asn32, and Asn44, in consensus N -glycosylation motifs, all in the N-domain, and PNGase F treatment showed that at least one of them is N -glycosylated. Mutating Asn44 to isoleucine inactivated the receptor, yielding a unique receptor species at about 20 kDa that was recognized by Ab only in a denatured state. On the other hand, mutating Asn25 or Asn32 either individually or in combination, or truncating successively N-domain residues 1-42, had no significant effect either on receptor structure, maturation, or activity. Thus, Asn44 in the GPR30 N-domain is required for receptor structure and activity, whereas N-domain residues 1-42, including specifically Asn25 and Asn32, do not play any major structural or functional roles.
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| 4. |
- Narbe, Ulrik, et al.
(författare)
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The estrogen receptor coactivator AIB1 is a new putative prognostic biomarker in ER-positive/HER2-negative invasive lobular carcinoma of the breast
- 2019
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Ingår i: Breast Cancer Research and Treatment. - : Springer Science and Business Media LLC. - 0167-6806 .- 1573-7217. ; 175:2, s. 305-316
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: According to the 2017 St Gallen surrogate definitions of the intrinsic subtypes, Ki67, progesterone receptor (PR) and Nottingham histological grade (NHG) are used for prognostic classification of estrogen receptor (ER) positive/HER2-negative breast cancer into luminal A- or luminal B-like. The aim of the present study was to investigate if additional biomarkers, related to endocrine signaling pathways, e.g., amplified in breast cancer 1 (AIB1), androgen receptor (AR), and G protein-coupled estrogen receptor (GPER), can provide complementary prognostic information in a subset of ER-positive/HER-negative invasive lobular carcinoma (ILC). Methods: Biomarkers from 224 patients were analyzed immunohistochemically on tissue microarray. The primary endpoint was breast cancer mortality (BCM), analyzed with 10- and 25-year follow-up (FU). In addition, the prognostic value of gene expression data for these biomarkers was analyzed in three publicly available ILC datasets. Results: AIB1 (high vs. low) was associated to BCM in multivariable analysis (adjusted for age, tumor size, nodal status, NHG, Ki67, luminal-like classification, and adjuvant systemic therapy) with 10-year FU (HR 6.8, 95% CI 2.3–20, P = 0.001) and 25-year FU (HR 3.0, 95% CI 1.1–7.8, P = 0.03). The evidence of a prognostic effect of AIB1 could be confirmed by linking gene expression data to outcome in independent publicly available ILC datasets. AR and GPER were neither associated to BCM with 10-year nor with 25-year FU (P > 0.33). Furthermore, Ki67 and NHG were prognostic for BCM at both 10-year and 25-year FU, whereas PR was not. Conclusions: AIB1 is a new putative prognostic biomarker in ER-positive/HER2-negative ILC.
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| 5. |
- Tutzauer, Julia, et al.
(författare)
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Breast cancer hypoxia in relation to prognosis and benefit from radiotherapy after breast-conserving surgery in a large, randomised trial with long-term follow-up
- 2022
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Ingår i: British Journal of Cancer. - : Springer Science and Business Media LLC. - 1532-1827 .- 0007-0920. ; 126:8, s. 1145-1156
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Breast-conserving surgery followed by radiotherapy is part of standard treatment for early-stage breast cancer. Hypoxia is common in cancer and may affect the benefit of radiotherapy. Cells adapt to hypoxic stress largely via the transcriptional activity of hypoxia-inducible factor (HIF)-1α. Here, we aim to determine whether tumour HIF-1α-positivity and hypoxic gene-expression signatures associated with the benefit of radiotherapy, and outcome.METHODS: Tumour HIF-1α-status and expression of hypoxic gene signatures were retrospectively analysed in a clinical trial where 1178 women with primary T1-2N0M0 breast cancer were randomised to receive postoperative radiotherapy or not and followed 15 years for recurrence and 20 years for breast cancer death.RESULTS: The benefit from radiotherapy was similar in patients with HIF-1α-positive and -negative primary tumours. Both ipsilateral and any breast cancer recurrence were more frequent in women with HIF-1α-positive primary tumours (hazard ratio, HR0-5 yrs1.9 [1.3-2.9], p = 0.003 and HR0-5 yrs = 2.0 [1.5-2.8], p < 0.0001). Tumour HIF-1α-positivity is also associated with increased breast cancer death (HR0-10 years 1.9 [1.2-2.9], p = 0.004). Ten of the 11 investigated hypoxic gene signatures correlated positively to HIF-1α-positivity, and 5 to increased rate/risk of recurrence.CONCLUSIONS: The benefit of postoperative radiotherapy persisted in patients with hypoxic primary tumours. Patients with hypoxic primary breast tumours had an increased risk of recurrence and breast cancer death.
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| 6. |
- Tutzauer, Julia, et al.
(författare)
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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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Ingår i: Molecular Pharmacology. - 0026-895X. ; 100:3, s. 271-282
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Tidskriftsartikel (refereegranskat)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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| 7. |
- Tutzauer, Julia, et al.
(författare)
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Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer
- 2020
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 15:4
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Tidskriftsartikel (refereegranskat)abstract
- Background G protein-coupled estrogen receptor (GPER), or G protein-coupled receptor 30 (GPR30), is reported to mediate non-genomic estrogen signaling. GPR30 associates with breast cancer (BC) outcome and may contribute to tamoxifen resistance. We investigated the expression and prognostic significance of GPR30 in metachronous contralateral breast cancer (CBC) as a model of tamoxifen resistance. Methods Total GPR30 expression (GPR30TOT) and plasma membrane-localized GPR30 expression (GPR30PM) were analyzed by immunohistochemistry in primary (BC1; nBC1 = 559) and contralateral BC (BC2; nBC2 = 595), and in lymph node metastases (LGL; nLGL1 = 213; nLGL2 = 196). Death from BC (BCD), including BC death or death after documented distant metastasis, was used as primary end-point. Results GPR30PM in BC2 and LGL2 were associated with increased risk of BCD (HRBC2 = 1.7, p = 0.03; HRLGL2 = 2.0; p = 0.02). In BC1 and BC2, GPR30PM associated with estrogen receptor (ER)-negativity (pBC1<0.0001; pBC2<0.0001) and progesterone receptor (PR)-negativity (pBC1 = 0.0007; pBC2<0.0001). The highest GPR30TOT and GPR30PM were observed in triple-negative BC. GPR30PM associated with high Ki67 staining in BC1 (p<0.0001) and BC2 (p<0.0001). GPR30TOT in BC2 did not associate with tamoxifen treatment for BC1. However, BC2 that were diagnosed during tamoxifen treatment were more likely to express GPR30PM than BC2 diagnosed after treatment completion (p = 0.01). Furthermore, a trend was observed that patients with GPR30PM in an ER-positive BC2 had greater benefit from tamoxifen treatment. Conclusion PM-localized GPR30 staining is associated with increased risk of BC death when expressed in BC2 and LGL2. Additionally, PM-localized GPR30 correlates with prognostic markers of worse outcome, such as high Ki67 and a triple-negative subtype. Therefore, PM-localized GPR30 may be an interesting new target for therapeutic exploitation. We found no clear evidence that total GPR30 expression is affected by tamoxifen exposure during development of metachronous CBC, or that GPR30 contributes to tamoxifen resistance.
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