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. 2020 Sep 3;15(9):e0226056.
doi: 10.1371/journal.pone.0226056. eCollection 2020.

Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells

Julie Pratibha Singh  1 , Manisha Dagar  1 , Gunjan Dagar  1 , Sudhir Kumar  2 , Sudhir Rawal  3 , Ravi Datta Sharma  4 , Rakesh Kumar Tyagi  2 , Gargi Bagchi  1
Affiliations

Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells

Julie Pratibha Singh et al. PLoS One. .

Abstract

The androgen receptor (AR) is activated in patients with castration resistant prostate cancer (CRPC) despite low circulating levels of androgen, suggesting that intracellular signaling pathways and non-androgenic factors may contribute to AR activation. Many G-protein coupled receptors (GPCR) and their ligands are also activated in these cells indicating that they may play a role in development of Prostate Cancer (PCa) and CRPC. Although a cross talk has been suggested between the two pathways, yet, the identity of GPCRs which may play a role in androgen signaling, is not established yet. By using blast analysis of 826 GPCRs, we identified a GPCR, GPCR 205, which exhibited maximum similarity with the ligand binding domain of the AR. We demonstrate that adhesion GPCR 205, also known as GPR56, can be activated by androgens to stimulate the Rho signaling pathway, a pathway that plays an important role in prostate tumor cell metastasis. Testosterone stimulation of GPR56 also activates the cAMP/ Protein kinase A (PKA) pathway, that is necessary for AR signaling. Knocking down the expression of GPR56 using siRNA, disrupts nuclear translocation of AR and transcription of prototypic AR target genes such as PSA. GPR56 expression is higher in all twenty-five prostate tumor patient's samples tested and cells expressing GPR56 exhibit increased proliferation. These findings provide new insights about androgen signaling and identify GPR56 as a possible therapeutic target in advanced prostate cancer patients.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Sequence comparison of mammalian GPCRs and the ligand binding domain of AR.
A) The 3D structures of candidate GPCRs as predicted using I-TASSER. The 3D models of the target protein were deposited to the Protein Model Database (PMDB) and each model was assigned a unique PMDB model identifier ID. B) Example of Ramachandran plots for selection of best model of the GPCRs (i.e. for GPRC6A and GPCR 205 (Ramachandran plot for other GPCRs not shown). The models for the GPCRs were selected based on the maximum residues obtained in the most favored region (MFR) using PROCHECK software. C) In silico molecular docking of selected GPCRs with testosterone (ligand) using Schrodinger Glide Maestro. Using multiple sequence alignment (clustal W) the conserved regions in selected GPCRs were identified. Testosterone was docked at the conserved region of the target GPCR to have a specific region docking prediction. D) Residue interaction diagram using Schrodinger Maestro software showing residues surrounding testosterone. The critical residues capable of making covalent interactions with testosterone are I626, W623, W563, N574. The residues I626 and W623 were mutated into Proline and Arginine using site directed mutagenesis for mutation experiments.
Fig 2
Fig 2. GPR56 expression in cell lines and membrane androgen binding.
A) RT-PCR was performed using total RNA extracted from LNCaP, PC3, DU145, HEK293 and TM3 cell lines using internal primers for GPR56. Beta actin was used as a control B) GPR56 protein expression detection in prostate and non-prostate cell lines (LNCaP, PC3, DU145, HEK293 and TM3) through immunoblotting. Western blotting was done using 50 ug of protein from cells lysate using GPR56 antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control C) Reduction of GPR56 protein expression in LNCaP cells transfected with GPR56-siRNA was detected using immunoblotting. GAPDH was used as a loading control. D-J) Flow cytometry analysis for detection of membrane androgen binding sites in LNCaP and HEK293 cell lines. Cells were labelled with T-BSA-FITC and the percentage of cells that bind specifically are given by Gate P3 and % parent shown below the figure. Membrane testosterone binding sites on LNCaP cells and cells transfected with GPR56-siRNA or scrambled siRNA were analyzed. Flow cytometry analysis was also performed in GPR56 transfected and non-transfected HEK 293 cells, and GPR56 overexpressing HEK293 cells transfected with GPR56-siRNA or scrambled siRNA. Cells were labelled with BSA-FITC as a control. Figures are representative of three independent experiments.
Fig 3
Fig 3. Binding of GPR56 mutants to testosterone and AR transactivation.
A) Representative picture of 1–693 aa sequence of GPR56 showing important domains and sequences included in mutants. The N-terminal fragment (NTF) is linked to C-terminal fragment (CTF) through GAIN domain and 7 transmembrane domain B-F) Flow cytometry analysis of HEK293 cells transfected with wild type GPR56, GPR56 NT mutant, GPR56 CT mutant and GPR56 double mutant and treated with T-BSA-FITC to assess binding. Cells were labelled with T-BSA-FITC and the percentage of cells that binds specifically are given by gate P3 and % parent are also shown. Cells were labelled with BSA-FITC as a control. Figures are representative of three independent experiments. G) Inhibition of AR transcription by knocking down GPR56 and PKA expression. LNCaP cells transfected with ARE-Luc (1μg) reporter plasmid with scrambled siRNA(100nM) or siGPR56 (100nM) and siPKA (120nM). The cells were treated with 0.1 and 10nM testosterone 24 h after transfection period. Values are mean ±S.D. from three independent experiments. * p< 0.01, ** p<0.001, two-way anova test. H) PC3 cells transfected with ARE-Luc (1μg) reporter plasmid with scrambled siRNA (100nM) or siGPR56 (100nM) and siPKA (120nM). The cells were treated with 0.1 and 10nM testosterone 24 h after transfection period. Values are mean ±S.D. from three independent experiments. * p< 0.01, ** p<0.001, two-way anova test. I) AR transcription analysis of GPR56-NT, GPR56-CT, and GPR56 double mutant. HEK293 cells transfected with ARE-Luc (1μg) reporter plasmid with GPR56, GPR56 N terminus mutant, GPR56 C terminus mutant, GPR56 double mutant (SD2) plasmids. The cells were treated with 10nM testosterone 24 h after transfection period. Values are mean ±S.D. from three independent experiments. * p< 0.01, ** p<0.001, two-way anova test. J) GPR56 regulates expression of PSA & TMPRSS2. LNCaP cells transfected with scrambled siRNA (Sc GPR56) or siRNA against GPR56 (siGPR56), were treated or not treated with testosterone (10nM)for 24 hrs. Total RNA was isolated and expression of PSA & TMPRSS2, AR target genes was measured by RT-PCR, beta-actin was used as control.
Fig 4
Fig 4. Multiple Sequence Alignment (MSA) of full length GPR56 in six different species.
I626 (highlighted in yellow), W623 (highlighted in pink), N574 (highlighted in green), and W563 (highlighted in blue) of GPR56 are involved in making good interaction with Testosterone are highly conserved in 5 different species.
Fig 5
Fig 5. Multiple Sequence Alignment of full-length adhesion GPCRs-Group VIII through clustal omega.
I626 (highlighted in green), W623 (highlighted in red), N574 (highlighted in pink) and W563 (highlighted in yellow) of GPR56 involved in making good interaction with testosterone are highly conserved in all adhesion GPCRs of group VIII.
Fig 6
Fig 6. Androgen-stimulation of GPR56 activates the PKA pathway.
A) PKA activity in LNCaP cells. PKA activity of GPR56-siRNA or siPKA transfected LNCaP cells was determined and compared to that of untransfected cells as control. GAPDH was used for normalization of protein content. Isoproterenol (Iso) was used as a positive control. Bars indicate average OD450, error bars indicate SD. Values are mean ±S.D. from three independent experiments * p< 0.01, ** p<0.001, by two-way anova test. B) Relative PKA activity in HEK 293 cells transfected with wild type GPR56, GPR56 N-terminus mutant, GPR56 C-terminus mutant and GPR56 I626 and W623 double mutant. The OD450 values corresponding to PKA activity in transfected HEK293 cells. GAPDH was used for normalization of protein content. Isoproterenol (Iso) was used as positive control. Bars indicate average OD450, error bars indicate SD. Values are mean ±S.D. from three independent experiments * p< 0.01, ** p<0.001, by two-way anova test. C and D) Effect of testosterone on cAMP production by LNCaP and HEK 293 cells. LNCaP (c) and HEK 293(d) were treated with 10nM testosterone or isoproterenol (10 μM) for 10 min. Isoproterenol was used as positive control. cAMP levels were determined using cAMP Glo Assay. * p<0.01, **p<0.001 by two-way anova test. Mean values obtained from three independent experiments.
Fig 7
Fig 7. Androgen binding to GPR56 triggers Rho activation.
A) HEK 293 cells were transfected using GPR56 or both GPR56 and Gα13 siRNA or GPR56 & scrambled siRNA. The cells were treated with testosterone (10nM) for 24 hrs and positive control (GTPYS) for 15 mins. The GTP-bound form of RhoA protein was detected by a pulldown assay using Rhotekin-RBD beads. Expression of RhoA in the cell lysate was estimated by western blotting using Anti-Rho antibody. Total lysate was used as the loading control. B) HEK 293 cells were transfected using GPR56, GPR56 NT mutant, GPR56 CT mutant or GPR56 double mutant and cells were treated directly with testosterone (10nM) for 24 hrs and H89 (30 μM) for 40 mins after treatment with testosterone. The GTP-bound form of RhoA protein was detected by a pulldown assay using Rhotekin-RBD beads. Expression of RhoA in the cell lysate was estimated by western blotting using anti-Rho antibody. Total lysate was used as the loading control.
Fig 8
Fig 8. GPR56 regulates the subcellular localization of AR.
A-B) Knockdown of GPR56 expression causes inhibition of AR translocation: LNCaP cells were transfected using GFP-AR, or GFP-AR and SiGPR56 (100nM), and treated with 10 nM testosterone for 24 h after transfection. Fluorescence Images captured at various time intervals i.e. 5 mins, 15 mins, 30 mins, 1 hour, 4 hours, 24 hours (other time intervals are shown in S2 Fig). Nuclei were visualized by Hoechst staining. Scale bar, 50um (added using Image J). C) Cytoplasmic and nuclear fractionation of AR in unstimulated (vehicle treated) and testosterone stimulated cells in presence and absence of GPR56 siRNA (siGPR56). LNCaP cells transfected with siRNA against GPR56 (120nM) were treated using vehicle or testosterone (10nM) for 1 hours. Western blotting was done using 50 ug of protein from cells lysate using AR antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was used as a cytoplasmic fraction marker and Histone H3B antibody as a nuclear fraction marker.
Fig 9
Fig 9. GPR56 expression in tumor samples.
A B & C) MTT assay in LNCaP, HEK 293 and PC3 cells. Cell proliferation in LNCaP cells transfected with GPR56-siRNA. Cell proliferation in LNCaP cells and cells transfected with siGPR56 were examined at 24 h, 48 h, 72 h or 96 h after seeding, using MTT assay. The data represents the mean ±S.D. of three independent experiments. Values are mean ±S.D. from three independent experiments. * p< 0.01, ** p<0.001, *** p<0.0001, two-way anova test B) Cell proliferation in non-transfected and GPR56 transfected HEK cells was examined 1–4 days after seeding using MTT assay. The data represents the mean ±S.D. of three independent experiments. * p< 0.01, ** p<0.001, *** p<0.0001, two-way anova test. Cell proliferation in PC3 cells transfected with GPR56-siRNA. Cell proliferation in PC3 cells and cells transfected with siGPR56 were examined at 24 h, 48 h, 72 h or 96 h after seeding, using MTT assay. The data represents the mean ±S.D. of three independent experiments. Values are mean ±S.D. from three independent experiments. * p< 0.01, ** p<0.001, *** p<0.0001, two-way anova test D) Representative RT-PCR analysis of GPR56 in matched normal (N) vs prostate tumor (T) tissue from individual patient’s samples (P1-P10) (P11-P25) patient’s samples. RNA was isolated from prostate tumor (T) and matched normal (N) tissue from individual patients and reverse transcribed using gene specific primers for GPR56 (as described in materials and methods). The amplified products were resolved on 2% agarose gel. The bands corresponding to GPR56 (N- terminal)-143 bp and GPR56 (C-terminal)-110 bp were observed. Beta actin was used as control. Quantification of band densities for N-terminal or the C- terminal region of the GPR56 mRNA transcript in patient’s tissue samples has been shown (using Image J).
Fig 10
Fig 10
A) GPR56 protein expression in normal versus tumor tissue. Western blot analysis has been performed using GPR56 antibody. Western blotting was done using 50 ug of protein from tissue lysate using GPR56 antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. GPR56 protein band intensity quantification in normal versus tumor tissue was done using Image J software. B) Analysis of TCGA data of Membrane androgen receptors genes (namely GPR56, GPRC6A, ZIP9, OXER1) expression and comparison with PSA gene expression in Normal Prostate (45) Vs Prostate Tumor patient’s samples (450) (TCGA_Prad). The analysis is done using GDC portal for cancer genomics. C). Following the classical pathway, testosterone transverses the membrane to enter the cytosol and bind to AR. It also activates GPR56 present on the membrane and causes activation of PKA and Rho pathway by non-genomic mechanism. Activation of both pathways are necessary for the translocation of AR into the nucleus and transcription of AR target genes.
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This work was supported by Department of Biotechnology(DBT), India No.BT/Bio-CARe/01/668/2011-12 (2014-2018) (to GB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.