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First published online April 23, 2007
doi: 10.1242/10.1242/jcs.03441

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The P2Y₂ nucleotide receptor requires interaction with v integrins to access and activate G₁₂

Department of Biochemistry, Life Sciences Center, University of Missouri-Columbia, Columbia, MO 65211, USA

View larger version (55K): [in this window] [in a new window]   Fig. 1. P2Y2R-mediated Rho activation and stress fiber formation require P2Y2R interaction with v integrins. (A) Cell surface expression of HA-tagged wild-type (WT) or RGE mutant (RGE) P2Y2Rs in 1321N1 cells was determined by flow cytometry. Cells stained with only secondary antibody were used as a negative control. (B) Cells expressing the WT or RGE mutant P2Y2R were treated with or without 1 mM UTP for 5 minutes prior to measuring ERK1/2 phosphorylation and Rho activity. Results shown are representative of three experiments. (C) Cells expressing the WT or RGE mutant P2Y2R were treated with or without 1 mM UTP or 20% FBS (positive control) for the indicated time and stress fibers were visualized by staining filamentous actin (shown in green) with Oregon-Green-labeled phalloidin. Texas-Red-labeled DNase I was used to label monomeric actin (shown in red). More than 100 cells were examined in three separate experiments, and representative images are shown. Bar, 50 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 2. ERK1/2 and Rho activation mediated by WT and AAA mutant P2Y2Rs. Human 1321N1 cells were transiently transfected with either WT (RGD) or AAA mutant P2Y2Rs in pcDNA3.1(–). Transfected cells were starved overnight and stimulated with the indicated concentration of UTP for 5 minutes prior to measuring ERK1/2 phosphorylation (A) or Rho activity (B). (A) UTP-induced with 1 mM UTP for 5 minutes before measuring ERK1/2 phosphorylation is expressed as fold increase over basal level in cells transfected with WT P2Y2R. Data points represent the mean ± s.e.m. of results from three experiments. (B) The Rho-GTP band density was normalized to total Rho and relative intensities are shown below each band as mean ± s.e.m. of results from four experiments.

View larger version (55K): [in this window] [in a new window]   Fig. 3. P2Y2R-mediated Rho activation and stress fiber formation require v integrin activity. Human 1321N1 cells expressing the WT P2Y2R were incubated overnight at 37°C in serum-free medium with 10 µg/ml anti-v5 or anti-3 antibody. Cells were then treated with or without 100 µM UTP for 5 minutes before analysis of Rho activity (A) or 30 minutes before analysis of stress fiber formation (B). (A) Representative blots from three experiments are shown. (B) More than 100 cells were examined in three separate experiments and representative images are shown. Bar, 50 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 4. P2Y2R-v integrin interaction regulates Rho-mediated signaling. (A,B) Human 1321N1 cells expressing the WT or RGE mutant P2Y2R were incubated with the indicated concentration of UTP for 5 minutes. Cell lysates were prepared and analyzed by immunoblotting with (A) anti-phospho-myosin light chain 2 (p-MLC-2) or (B) anti-phospho-cofilin antibodies. (C) Cells expressing the WT P2Y2R were incubated overnight with or without 10 µg/ml anti-v5 or anti-3 antibody, then stimulated with 100 µM UTP for 5 minutes. Cell lysates were prepared and analyzed by immunoblotting with anti-phospho-cofilin antibodies. (D) Cells expressing the WT P2Y2R were pretreated at 37°C in serum-free medium with the Rho-dependent kinase inhibitor Y-27632 (10 µM) for 1 hour or 200 ng/ml Bordetella pertussis toxin (PTX) overnight, then stimulated with 100 µM UTP for 5 minutes. Cell lysates were analyzed by immunoblotting with anti-phospho-cofilin antibodies. Protein loading in each lane was evaluated by stripping the membrane of antibodies and re-probing with anti-actin antibodies. Blots representative of 3-5 experiments are shown.

View larger version (37K): [in this window] [in a new window]   Fig. 5. P2Y2R-v integrin interaction is required for G12 coupling. (A,B) Membrane preparations from 1321N1 cells expressing the WT or RGE mutant P2Y2R or pLXSN vector-transfected cells (negative control) were used in [35S]GTPS binding assays in the presence or absence of 1 mM UTP. After termination of the assay, samples were immunoprecipitated with antiserum against (A) G12 or (B) Gq/11 and radioactivity in the immunoprecipitates was calculated. Data are the means ± s.e.m. of results from three separate experiments and are shown as fold increase over untreated cells. (C) Human 1321N1 cells expressing the WT or RGE mutant P2Y2R were incubated with 1 mM UTP for 2 minutes. G12 activation was detected by immunoprecipitation (IP) of G12 with anti-G12 antibody and immunoblotting (IB) of G12 with anti-phosphoserine/threonine antibody. Gq/11 activation was detected by IP with anti-phosphotyrosine antibody and IB with anti-Gq/11 antibody. Blots representative of three experiments are shown.

View larger version (29K): [in this window] [in a new window]   Fig. 6. P2Y2R-mediated G12 activation requires v integrin expression and activity. (A) Cells expressing the WT P2Y2R were incubated overnight at 37°C in serum-free medium with 10 µg/ml anti-v5 or anti-3 antibody, then stimulated with 100 µM UTP for 2 minutes. G12 phosphorylation was detected as described in Fig. 5. Relative intensities are shown below each band as mean ± s.e.m. of results from four experiments. (B) Cells expressing the WT P2Y2R were transfected with 5 µg of v antisense (AS) or sense (S) oligonucleotides, as described previously (Bagchi et al., 2005). Lipofectamine transfected cells served as a negative control. Expression of v integrin in these cells was determined by immunoblot analysis and a representative blot is shown. UTP-induced binding of [35S]GTPS to G12 was determined, as described in Fig. 5. Data are the mean ± s.e.m. of results from three separate experiments and are shown as fold increase over untreated cells.

View larger version (34K): [in this window] [in a new window]   Fig. 7. The P2Y2R accesses G12 in a complex with v integrins. (A) RGD-dependent association of the P2Y2R with v integrins and G12. Cells expressing the HA-tagged WT or RGE mutant P2Y2R and endogenous Gq/11, G12 and v integrins were treated with or without 1 mM UTP for 5 minutes and lysates were subjected to IP with mouse anti-HA antibody conjugated to agarose beads and IB with anti-G12, anti-Gq/11, anti-v integrin, or anti-HA antibody. Cells transfected with the pLXSN vector served as a negative control. (B) G12 associates with v integrins. Human 1321N1 cells expressing cDNA for the WT P2Y2R and G12 were treated with or without 100 µM UTP for 5 minutes and lysates were subjected to IP with IgG, anti-3 integrin, or anti-v integrin antibody followed by IB with anti-G12, anti-3 integrin or anti-v integrin antibody. Blots representative of three experiments are shown.

View larger version (55K): [in this window] [in a new window]   Fig. 8. Co-immunostaining of G12 and Go in cells exposed to a UTP gradient. Human 1321N1 cells expressing wild-type P2Y2Rs were plated on chambered coverslips and incubated overnight in 300 µl serum-free medium. UTP (1 µl of a 100 mM solution) was added at the bottom left and after 30 minutes, cells were washed, fixed, permeabilized and stained with rabbit anti-G12 and mouse anti-Go antibodies. Texas-Red-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgG were then used to localize G12 and Go, respectively. Images representative of three experiments are shown. Arrows indicate membrane protrusions indicative of lamellipodia formation. Bars, 50 µm (left column); 20 µm (enlarged images in right column).

View larger version (28K): [in this window] [in a new window]   Fig. 9. Effect of dominant-negative G12 (G12 DN) on signaling events mediated by the P2Y2R. (A-D) Human 1321N1 cells expressing the WT P2Y2R were cultured to 80% confluence and transiently transfected with the G12DN mutant in the pcDNA3.1+ vector. MOCK, vector transfected negative control. Transfected cells were incubated with 100 µM UTP for 5 minutes (A,B) or 30 minutes (C) and assayed for (A) Rho activity and cofilin phosphorylation, (B) ERK1/2 phosphorylation and (C) stress fiber formation, as described in the Materials and Methods. Total G12 was detected with anti-G12 antibody, as shown in A. Antibody-labeled G12 in (C) is shown in red and phalloidin-labeled filamentous actin is shown in green. (D) Cells (5x104) transfected with the indicated constructs were seeded into the upper chamber of Transwells. Lower chambers contained serum-free medium with or without 100 µM UTP. Cell migration was evaluated 16 hours after UTP stimulation and is expressed as the fold increase in the number of cells that moved across the Transwell membranes in response to UTP as compared to untreated controls. (A,B) Blots representative of three-five experiments are shown. (C) Images representative of three separate experiments are shown. Bar, 40 µm. (D) Data shown are the mean ± s.e.m. of results from five experiments.

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