G_q/11 and β-arrestin-dependent GPR54-coupled pathways regulate the spatial distribution of pERK.

<p>Immunofluorescence analysis of GPR54 expression and pERK levels in GPR54-EYFP overexpressing G_q/11 and β-Arr KO MEFs and their WT parents before and after 10 minutes of Kp-10 treatment. Each inset contains cells that express and do not express GPR54 (green; first and fourth columns). These cells were analyzed for pERK levels (red fluorescence) before (0 minutes) and after (10 minutes) Kp-10 treatment. Differential interference contrast (DIC) images of cells and DAPI-stained nuclei (with GPR54 and pERK) are shown to provide greater spatial information. In all cases, the same image acquisition settings were applied, thus receptor and pERK levels can be compared between cell lines and treatments. The images are representative of 3 independent experiments.</p

GPR54 activates ERK1/2 in a β-arrestin-dependent manner.

<p>Representative autoradiograph (A) and densitometric analysis (B) showing the expression of total and activated ERK1/2 in GPR54 overexpressing β-Arr1/2 double KO (β-Arr1/2 KO) and corresponding wild type parental (WT2) MEFs following stimulation with 100 nM Kp-10 (for the indicated time points). (C) Representative Western blot confirming absence and presence of expression of FLAG-GPR54 in non-electroporated (NT) and FLAG-GPR54 overexpressing β-Arr1/2 KO and WT2 MEF cells, respectively. Western blot analyses were done using monoclonal anti-ERK1/2, anti-phospho ERK1/2, and anti-DDK (FLAG) antibodies. The data represent the mean ± S. E. of 4 independent experiments. ^##<i>P</i><0.01 vs 0 min. control (within the specific cell line). **<i>P</i><0.01 vs respective wild-type control at the indicated time point.</p

β-arrestin-2 and G_q/11 regulate the GPR54-dependent ERK1/2 activation in a rapid and temporally overlapping manner.

<p>Representative autoradiographs and densitometric analyses showing the expression of total and activated ERK1/2 in GPR54 overexpressing (A) β-Arr1 KO and WT1 MEFs; (B) β-Arr2 KO and WT2 MEFs; and (C) G_q/11 KO and WT MEFs following stimulation with 100 nM Kp-10 (for the indicated time points). All western blot analyses were done using monoclonal anti-ERK1/2 and anti-phospho ERK1/2 antibodies. The data represent the mean ± S. E. of 3 independent experiments. # <i>P</i><0.05, ##<i>P</i><0.01 vs 0 min. control (within the specific cell line). *<i>P</i><0.05 vs respective wild-type control at the indicated time point.</p

L148S interacts with β-arrestin-1 and 2 while GPR54 continues to modulate gene expression in the absence of G_q/11.

<p>(A) Representative autoradiograph showing the co-immunoprecipitation of β-arrestin-1 and β-arrestin-2 with FLAG-L148S. HEK 293 cells were transfected with 10 µg of β-arrestin-1 or -2-GFP and 10 µg of FLAG-L148S or empty FLAG vector. Cells were left untreated or stimulated with 100 nM Kp-10 for 5 minutes. Lysates were prepared, immunoprecipitated with mouse anti-FLAG antibody, and immunoblotted with mouse anti-GFP antibody. The expression of β-arrestin-1 or -2-GFP in 50 µg of total protein from the corresponding HEK 293 cell lysates is also shown. The data represent the mean ± SE for three independent experiments. IB: Immunoblotting; IP: immunoprecipitation. (B) Gene microarray analysis of G_q/11 KO MEFs versus WT control MEFs that were treated with Kp-10 for six hours. The data is presented as a three-way Venn diagram and shows the overlap of genes changing greater than 2 fold in their expression (up or down) as a proportion of all the probesets on the Affymetrix Mouse Gene 1.0 ST Array. In total, 1,635 out of a total of 35,556 probesets are altered greater than 2-fold (4.6%) (<i>P</i><0.05).</p

GPR54-mediated gene expression in the hypothalamic cell line, GT1-7 is altered following reduced β-arrestin-1 and 2 expression.

<p>(A) GT1-7 cell lines stably expressing shRNAs against β-arrestin-1 (line 712) and β-arrestin-2 (line 153) were created and β-arrestin expression was determined by western blotting. This data revealed that lines 712 and 153 expressed about 50% less β-arrestin-1 and -2 than the WT parental line expressing scrambled shRNA sequences. (B) Unsupervised heirarchical clustering showing alterations of gene expression in the GT1-7 lines (712 and 153) relative to wild type. ANOVA was used to determine those probesets changing significantly (<i>P</i><0.05) at least 1.5 fold in 712 and/or 153 versus WT. This list was then subjected to heirarchical clustering using the average linkage algorithm and displayed as a heat map (representative samples shown). Red: high expressor, blue: low expressor. Color scale: log(BASE 2) normalized intensity.</p

GPR54 stimulation of ERK1/2 is enhanced in the absence of β-arrestin-1.

<p>Representative autoradiograph (A) and densitometric analysis (B) showing the expression of total and activated ERK1/2 in GPR54 overexpressing β-Arr1 KO and corresponding wild type parental (WT1) MEFs following stimulation with 100 nM Kp-10 (for the indicated time points). (C) Representative western blot confirming absence and presence of expression of FLAG-GPR54 in non-electroporated (NT) and FLAG-GPR54 overexpressing β-Arr1 KO and WT1 MEFs, respectively. Representative autoradiograph (D) and densitometric analysis (E) showing the expression of total and activated ERK1/2 following stimulation with 100 nM Kp-10 (for indicated time points) of FLAG-GPR54 overexpressing β-Arr1 KO MEFs co-transfected with either GFP vector (grey bars) or β-Arr1-GFP (white bars). All western blot analyses were done using monoclonal anti-ERK1/2 and anti-phospho ERK1/2 antibodies. Monoclonal anti-β-Arr1 and anti-DDK (FLAG) antibodies were also used. The data represent the mean ± S. E. of 4 independent experiments (B) or the mean ± S. E. of 3 independent experiments (E). ^#<i>P</i><0.05, ^##<i>P</i><0.01 vs 0 min. control (within the specific cell line). *<i>P</i><0.05, **<i>P</i><0.01 vs respective wild-type (or ‘add-back’) control at the indicated time point.</p

GPR54 positively regulates ERK1/2 activation in a β-arrestin-2-dependent manner.

<p>Representative autoradiograph (A) and densitometric analysis (B) showing the expression of total and activated ERK1/2 in GPR54 overexpressing β-Arr2 KO and corresponding wild type parental (WT2) MEFs following stimulation with 100 nM Kp-10 (for the indicated time points). (C) Representative Western blot confirming absence and presence of expression of FLAG-GPR54 in non-electroporated (NT) and FLAG-GPR54 overexpressing β-Arr2 KO and WT2 MEFs, respectively. (D) Representative western blot showing the expression of total and activated ERK 1/2 following treatment of the β-Arr2 KO and corresponding wild type parental (WT2) MEFs with 10 ng/ml EGF (for the indicated time point). This was used as a control to assess whether the ERK1/2 pathway was still functional in these cells. Representative autoradiograph (E) and densitometric analysis (F) showing the expression of total and activated ERK1/2 following stimulation with 100 nM Kp-10 (for indicated time points) of FLAG-GPR54 overexpressing β-Arr2 KO MEFs co-transfected with either GFP vector (grey bars) or β-Arr2-GFP (white bars). All western blot analyses were done using monoclonal anti-ERK1/2 and anti-phospho ERK1/2 antibodies. Monoclonal anti-β-Arr2 and anti-DDK (FLAG) antibodies were also used. The data represent the mean ± S. E. of 4 independent experiments (B) or the mean ± S. E. of 3 independent experiments (F). ^##<i>P</i><0.01 vs 0 min. control (within the specific cell line). *<i>P</i><0.05, ** <i>P</i><0.01vs respective wild-type (or ‘add-back’) control at the indicated time point.</p

GPR54 positively regulates ERK1/2 activation in a G_q/11-dependent manner.

<p>Representative autoradiograph (A) and densitometric analysis (B) showing the expression of total and activated ERK1/2 in GPR54 overexpressing G_q/11 KO and corresponding WT parental MEFs following stimulation with 100 nM Kp-10 (for the indicated time points). (C) Representative Western blot confirming absence and presence of expression of FLAG-GPR54 in non-electroporated (NT) and FLAG-GPR54 overexpressing G_q/11 KO and WT MEFs, respectively. (D) Representative western blot showing the expression of total and activated ERK 1/2 following treatment of the G_q/11 KO and corresponding WT parental MEFs with 10 ng/ml EGF (for the indicated time points). This was used as a control to assess whether the ERK1/2 pathway was still functional in these cells. Representative autoradiograph (E) and densitometric analysis (F) showing the expression of total and activated ERK1/2 following stimulation with 100 nM Kp-10 (for indicated time points) of GPR54 overexpressing G_q/11 KO MEFs co-transfected with either GFP vector (grey bars) or untagged G_q (white bars). All western blot analyses were done using monoclonal anti-ERK1/2 and anti-phospho ERK1/2 antibodies. Polyclonal anti-G_q and monoclonal anti-DDK (FLAG) antibodies were also used. The data represent the mean ± S. E. of 4 independent experiments (B) or the mean ± S. E. of 3 independent experiments (F). ^##<i>P</i><0.01 vs 0 min. control (within the specific cell line). *<i>P</i><0.05, ** <i>P</i><0.01 vs respective wild-type (or ‘add-back’) control at the indicated time point.</p

GPR54 is coupled to ERK MAPK pathway in WT MEFs.

<p>Representative autoradiographs (A and C) and densitometric analyses (B and D) showing the expression of total and activated ERK1/2 in GPR54 overexpressing WT1 (β-arrestin-1 KO parent) and WT2 (β-arrestin-2 and 1/2 KO parent) MEFs following 10-minute treatment with increasing concentrations of Kp-10 (0–1000 nM). Representative autoradiograph (E) and densitometric analysis (F) showing the expression of total and activated ERK1/2 in GPR54 overexpressing WT1 and WT2 parental MEF cell lines following 100 nM Kp-10 treatment (for the indicated time points: 0, 5, 10, 30 and 60 minutes). Western blot analyses were done using monoclonal anti-ERK1/2 and anti-phospho ERK1/2 antibodies. The data represent the mean ± S. E. of 4 independent experiments. *<i>P</i><0.05; **<i>P</i><0.01vs control (0 min.).</p

Concurrent IDO and TS downregulation sensitizes A549 cells to 5FUdR more than TS knockdown alone.

<p>A549 cells were transfected with control or anti-thymidylate synthase (anti-TS) siRNA, treated with IFNγ (25 ng/ml) for 48 h, with 5FUdR (40 nM) for 72 h, and then enumerated. Bars indicate mean proliferation relative to appropriate controls ± SD (n = 3). <b>A</b>: Proliferation of clonal A549 cell populations induced with IFNγ and then treated with 5FUdR, but untransfected with siRNA of any kind. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing non-targeting control shRNA. <b>B</b>: Proliferation of the same clonal A549 cell populations transfected with control non-targeting siRNA, TS siRNA #3, or TS siRNA #4, induced with IFNγ, and then treated with 5FUdR. Bars represent values normalized to values obtained from clones treated with IFNγ but untreated with pemetrexed or siRNA; those cells were considered to have a proliferation value of 100% after IFNγ treatment. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing non-targeting control shRNA. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. Data presented a representative experiment from two independent experiments. Results are normalized to control cells not treated with 5FUdR, but with treated with IFNγ. <b>Panel C</b>: shows the pooled results from panel A and B.</p