The relationship between bowling action classification and three-dimensional lower

The relationship between bowling action classification and three-dimensional lowe

GPR158, an Orphan Member of G Protein-Coupled Receptor Family C: Glucocorticoid-Stimulated Expression and Novel Nuclear Role

<div><p>Members of the large G protein-coupled receptor (GPCR) clan are implicated in many physiological and disease processes, making them important therapeutic drug targets. In the present study, we follow up on results of a pilot study suggesting a functional relationship between glucocorticoid (GC)-induced ocular hypertension and GPR158, one of three orphan members of the GPCR Family C. GC treatment increases levels of GPR158 mRNA and protein through transcriptional mechanisms, in cultured trabecular meshwork (TBM) cells derived from the eye's aqueous outflow pathway. Like treatment with GCs, transient overexpression of GPR158 stimulates cell proliferation, while siRNA knockdown of endogenous GPR158 has the opposite effect. Both endogenous and overexpressed GPR158 show an unusual subcellular localization pattern, being found almost entirely in the nucleus. However, when cells are treated with inhibitors of clathrin-mediated endocytosis, GPR158 is shifted to the plasma membrane. Mutation of a bipartite nuclear localization signal (NLS) in the 8^th helix also shifts GPR158 out of the nucleus, but in this case the protein is found in vesicles localized in the cytoplasm. These results suggest that newly synthesized GPR158 first traffics to the plasma membrane, where it rapidly undergoes endocytosis and translocation to the nucleus. Significantly, mutation of the NLS abrogates GPR158-mediated enhancement of cell proliferation, indicating a functional requirement for nuclear localization. GPR158 overexpression upregulates levels of the cell cycle regulator cyclin D1, but mutation of the NLS reverses this. Overexpression of GPR158 enhances the barrier function of a TBM cell monolayer, which is associated with an increase in the levels of tight junction proteins ZO-1 and occludin, similar to reported studies on GC treatment. Regulated paracellular permeability controls aqueous outflow facility <i>in vivo</i>. Since GCs stimulate GPR158 expression, the result is consistent with a role for elevation of GPR158 expression in GC-induced ocular hypertension.</p> </div

NucPred Analysis of the 22 Members of GPCR Family C.

<p>The PSIPRED program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057843#pone.0057843-Agrawal1" target="_blank">[20]</a> predicted the presence of 8^th helix and the NucPred score represents the threshold score for the fraction of proteins, correctly predicted as nuclear (specificity) versus the fraction of true nuclear proteins predicted (sensitivity) at or below given score <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057843#pone.0057843-Brameier1" target="_blank">[19]</a>.</p

The schematic representation of two-dimensional structure of GPR158.

<p>Three extracellular loops (ELs) and three intracellular loops (ILs) connect the seven TM (numbered I-VII). The arrow indicates putative PKC and PKA phosphorylation sites in the ILs. The cysteine residues in EL-1 and EL-2 involved in disulfide (S-S) bond formation are shown as a dotted grey double line. The eighth helix, bipartite NLS, c-Myc and PITX2 interaction motifs, and putative phosphorylation sites for kinases, such as CDK1, are indicated in the C-terminal tail. The leucine zipper domain, EGF like domain, N-glycosylation sites and putative CRD are shown in the N-terminal of GPR158. The conserved amino acid residues, KXXR and E, involved in G protein activation in class C GPCRs are marked in red color. CDK1, cyclin-dependent kinase 1; CRD, cysteine rich domain; EGF, epidermal growth factor; EL, extracellular loop; IL, intracellular loop; NLS, nuclear localization signal; PITX2, paired-like homeodomain transcription factor 2.</p

Overexpression of GPR158 protein.

<p>TBM-1 cells were transfected at 70–80% confluence using Lipofectamine LTX reagent with either indicated GPR158 expression plasmids or vector alone (<b>A, B and C</b>). Western blotting for the detection of GPR158 in whole cell lysates isolated from cells transfected with indicated plasmids using anti-C-terminal GPR158 antibody (<b>A and B</b>) and anti-GFP antibody (<b>B</b>). Total of 50 µg protein was subjected to SDS-PAGE analysis after boiling with 2-ME as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057843#s2" target="_blank">Materials and Methods</a>. The same membrane was striped and reprobed for β-actin for loading control. (<b>C</b>) The membrane pellet following cytosolic extract isolation from cells transfected with indicated plasmids was boiled in SDS-PAGE loading dye and loaded onto the gel. The western blotting was performed with anti-N-terminus GPR158 antibody. (<b>D</b>) Normal prostate tissue lysates from mice were isolated according to the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057843#s2" target="_blank">Materials and Methods</a>. The total lysates (30 µg) was subjected to western blotting using either anti-C-terminal GPR158 or anti-N-terminal GPR158 antibodies. The results are representative of three different transfection experiments. ***P<.001; **P<.01; *P<.05; ns, P>.05. (<b>E</b>) The illustration of the location of two cleavage sites and corresponding sizes of the fragments, which are detected using anti-C- and anti-N-terminal GPR158 antibodies. Arrows indicated cleavage sites, I and II, and arrowhead indicate N-glycosylation in N-terminal extracellular domain.</p

GPR158 promoter activity in TBM-1 cells following GC treatment.

<p>(<b>A</b>) Schematic illustration of GPR158 promoter (−1053/+25 bp, from transcription start site) indicating the location of three GREs. (<b>B and C</b>) TBM-1 cells were cotransfected with GPR158 promoter construct and β-galactosidase plasmid using Lipofectamine LTX reagent, followed by Dex (<b>B</b>) or TA (<b>C</b>) treatment for indicated time points. (<b>D</b>) TBM-1 cells were cotransfected with promoter-less pGL3 vector and β-galactosidase plasmid. Post-transfection, the cells were either left untreated or treated with vehicle control, or Dex or TA. Treatment was for 3 days for TBM-1 cells. (<b>B, C, and D</b>) The luciferase activity was normalized to that of the promoterless pGL3 basic vector. Data are expressed as mean ± SEM of three independent experiments ***P<.001; **P<.01; *P<.05; ns, P>.05.</p

Role of the bipartite NLS in nuclear localization and cell proliferation.

<p>(<b>A, B, C, D and E</b>) The fluorescent images were captured of cells transfected with either vector (<b>A</b>) or GPR158-GFP plasmid (<b>B</b>) or NLS-M1-GFP plasmid (<b>C</b>) or NLS-M2-GFP plasmid (<b>D</b>) or NLS-M1+2-GFP plasmid (<b>E</b>). The representative images from two independent transfection experiments are shown. 3 days post-transfection, the cells were fixed with 4% PFA, washed with PBS and the slides were mounted using VECTASHIELD with DAPI and viewed using Nikon Eclipse Ti-E fluorescence microscope. The merged images show GPR158-GFP as green and nuclear stain DAPI as blue. The zoom panel indicates the magnified image of an indicated cell. (<b>F</b>) After 3 days of transfection, the cells transfected with above indicated plasmids were trypsinized and counted using trypan blue dye in a hemocytometer chamber. The data represent mean ± SEM of two independent experiments. (<b>G</b>) Schematics showing amino acid sequence corresponding to the bipartite NLS, a part of 8^th helix of GPR158. The mutated amino acids are shown in color red in indicated NLS mutant constructs. (<b>H</b>) TBM-1 cells were transfected with either GPR158 wild type or NLS-M1+2 or GFP vector alone plasmid using Lipofectamine LTX reagent. After 3 days of transfection, the cell lysates were prepared using RIPA buffer and the western blotting for the detection of GPR158 and cyclin D1 was performed using appropriate antibodies. The same membrane was striped and reprobed for β-actin for loading control. The data represent two independent experiments performed in triplicate.</p

Oligonucleotide primers used in this study.

<p>The abbreviations are: GPR158, G protein-coupled receptor 158; Fra, fragment; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase–polymerase chain reaction; SDM, site-directed mutagenesis.</p

Nuclear trafficking of GPR158.

<p>(<b>A</b>) Indirect immunofluorescence microscopy was carried out using anti-C-terminal GPR158 antibody in vehicle treated (top panel) or Dex treated (middle panel) TBM-1 cells. The untreated cells labeled only with secondary and not with primary anti-GPR158 antibody, as negative control are indicated in bottom panel. Cell nucleus was labeled with propidium iodide (PI). All images were captured using Nikon Eclipse Ti-E fluorescence microscope at fixed acquisition parameters. Yellow indicates the colocalization of GPR158 with nuclear stain in merged images. (<b>B, top panel</b>) The image represents the <i>z</i>-stack projection of multiple confocal microscopy sections from the basal to the apical cell side, indicating the co-localization of GPR158 (green) with nuclear stain, PI (red). (<b>B, bottom panel</b>) The corrected total cell fluorescence (CTCF) from 50 individual cells in either vehicle or Dex treated sample was measured using NIH Image J software and normalized with negative control values. The bar diagram represent mean ± SEM of three independent experiments. The statistical analysis was carried out using unpaired Student's t tests (P<0.05). (<b>C</b>) The images were acquired using fluorescence microscopy of cells transfected with either vector or GPR158-GFP fusion plasmid. The representative image of single cell for both panels is shown from three independent transfection experiments. Post-transfection, the cells were fixed with 4% PFA, washed with PBS and the slides were mounted using VECTASHIELD with DAPI. The images were acquired on Nikon Eclipse Ti-E fluorescence microscope. (<b>D</b>) The nuclear extracts (25 µg) and cytosolic extracts (50 µg) isolated from cells overexpressing GPR158-GFP fusion protein were subjected to SDS-PAGE and western blotting to detect GPR158 using anti-C-terminal GPR158 antibody (dilution: 1∶1000), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057843#s2" target="_blank">Materials and Methods</a>. The purity of nuclear and cytosolic extracts in immunoblotting was confirmed using anti-beta actin and anti-histone H3 antibodies. Quantification of both fragments of GPR158 protein band intensities was measured by NIH Image J and expressed in terms of fold levels relative to levels in cytosolic extracts considered as 1.0. The statistical analysis was carried out using unpaired Student's t tests (P<0.05). The average fold and P values obtained from three different experiments are indicated in the figure. The vertical line indicates repositioned gel lanes. ***P<.001; **P<.01, *P<.05; ns, P>.05. (<b>E</b>) TBM-1 cells were either transfected with GFP vector alone (I, II and III) or GPR158-GFP plasmid (IV, V and VI). 3 days after transfection, the cells were either incubated in culture media (I and IV) or media containing ConA (II and V) (0.25 mg/mL) for 8 hrs or CPZ (III and VI) (10 µM) for 3 hrs. Following treatment, the cells were washed with PBS and fixed with 4% PFA for 20 mins at RT. The cells were washed again and mounted with VECTASHIELD containing DAPI and images were acquired with a Nikon Eclipse Ti-E fluorescence microscope. The images represent two independent experiments.</p

GPR158 regulates the proliferation of TBM-1 cells.

<p>TBM-1 cells were transfected at 70–80% confluence using Lipofectamine LTX reagent with either GPR158 expression plasmids or vector alone (<b>A</b>) OR either GPR158 siRNA or control scrambled siRNA as indicated (<b>B and C</b>) and incubated in growth medium for 3 days in a 6-well culture dishes. (<b>A and C</b>) After 3 days of transfection, the cells were trypsinized and counted using trypan blue dye in a hemocytometer chamber for the cells transfected with indicated plasmids. (<b>B</b>) Total RNA was isolated for analyzing the levels of GPR158 mRNA by qRT-PCR. β-actin was used as a reference gene. (<b>A, B and C</b>) The data represent mean ± SEM of three independent experiments. ***P<.001; **P<.01; *P<.05; ns, P>.05.</p