Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3)

Abstract

Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus, and bone marrow-derived dendritic cells, and is up-regulated upon leukocyte activation. We explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in leukocytes from mice lacking AGS3. No obvious differences in lymphocyte subsets were observed. Interestingly, however, AGS3-null B and T lymphocytes and bone marrow-derived dendritic cells exhibited significant chemotactic defects as well as reductions in chemokine-stimulated calcium mobilization and altered ERK and Akt activation. These studies indicate a role for AGS3 in the regulation of G-protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.

Keywords: AGS3; Chemokines; Chemotaxis; Cxcr4; G Proteins; G-protein-coupled Receptors (GPCR); GPR Motif; Gpsm1; Gαi; Gβγ.

FIGURE 1.

Immunoblot of WT and Gpsm1^−/− mouse thymocyte and splenocyte lysates. Thymocytes and splenocytes were isolated following red blood cell lysis and filtering to remove cell and tissue aggregates as described under “Experimental Procedures.” Lysates were prepared with 1% Nonidet P-40 lysis buffer and subjected to SDS-PAGE (100 μg/lane) and immunoblotting with AGS3, LGN, Gα_i2, Gα_i3, and actin-specific antisera as described under “Experimental Procedures.”

FIGURE 2.

AGS3 is up-regulated in lymphocytes and BMDCs upon stimulation. A, purified B and T lymphocytes from C57BL/6J mice were isolated as described under “Experimental Procedures.” Purified B cells were stimulated with 20 μg/ml anti-IgM F(ab′)2 fragment or 1 mg/ml LPS for 12 h. Purified T cells were stimulated with 0.1 μg/ml anti-CD3 and 20% IL-2 for 24–48 h. After treatment, cells were washed and lysed in SDS sample buffer and subjected to SDS-PAGE and immunoblotting with AGS3 and actin-specific antisera. con, control. B, BMDCs were prepared as described under “Experimental Procedures.” After 8 days, immature dendritic cells were cultured in the absence (−) or presence of 200 ng/ml LPS for the times indicated in the figure. After treatment, cells were washed and lysed with 1% Nonidet P-40 buffer and subjected to SDS-PAGE and immunoblotting with AGS3 and actin-specific antisera as described under “Experimental Procedures.”

FIGURE 3.

Chemotaxis of immune cells from WT and Gpsm1^−/− mice to chemokines CXCL12 and CCL19. A and B, B and T cells were separately isolated from freshly harvested splenocytes of WT and Gpsm1^−/− mice. Cells were loaded in Transwell migration chambers with the bottom chamber containing serum-free RPMI supplemented with 0.05 μm β-mercaptoethanol in the absence and presence of 300 ng/ml CXCL12 or CCL19 as indicated. After 5 h at 37 °C, cells in the bottom chamber were counted, and the percentage of cells migrating as compared with input was determined by subtracting the number of cells migrating to the bottom chamber in the absence of chemokine. Data are represented as the mean ± S.E. of three independent experiments with at least triplicate determinations. **, p < 0.01; ***, p < 0.001. C, bone marrow cells were harvested from WT and Gpsm1^−/− mice and cultured to iDCs or mDCs as described under “Experimental Procedures.” Dendritic cells were loaded in Transwell migration chambers with the bottom chamber containing serum-free RPMI in the absence and presence of 250 ng/ml CXCL12 or CCL19 as indicated. After 20 h at 37 °C, cells in the bottom chamber were counted, and the percentage of cells migrating as compared with input was determined by subtracting the number of cells migrating to the bottom chamber in the absence of chemokine. Data are represented as the mean ± S.E. of three independent experiments with at least triplicate determinations. **, p < 0.01. D, BMDCs were harvested and cultured as described above and pretreated with 10 μm gallein or vehicle for 30 min prior to measuring chemotaxis to CXCL12 as described under “Experimental Procedures.” **, p < 0.01 for gallein-treated as compared with vehicle control for each genotype. ##, p < 0.01 for Gpsm1^−/− vehicle control as compared with Gpsm1^+/+ vehicle control. Differences for gallein treatment groups between genotypes were not statistically significant.

FIGURE 4.

Gpsm1^−/− splenocytes and dendritic cells have impaired chemokine-stimulated calcium responses. A and B, freshly harvested splenocytes of WT and Gpsm1^−/− mice were seeded into clear bottom, black-walled 96-well plates at 1 × 10⁶ cells/well in 100 μl of serum-free, phenol red-free RPMI in the absence of serum for at least 1 h. Cells were incubated in Calcium 5 assay dye for 1 h at 37 °C and incubated at room temperature for 15 min prior to analysis. CXCL12 (A) or CCL19 (B) was added at a final concentration of 200 ng/ml by the FLIPRtetra, and measurements (relative light units, RLU) were taken every second for at least 300 s. C, bone marrow cells were harvested from WT and Gpsm1^−/− mice and cultured to BMDCs as described under “Experimental Procedures.” BMDCs were seeded at 250,000 cells/well in the absence of serum for ∼1 h. BMDCs were incubated in Calcium 5 dye for 1 h at 37 °C and incubated at room temperature for 15 min prior to analysis. CXCL12 was added at a final concentration of 200 ng/ml by the FLIPRtetra, and measurements (relative light units RLU) were taken every second for at least 300 s. Data are representative of three independent experiments with triplicate determinations.

FIGURE 5.

Gpsm1^−/− dendritic cells exhibit defects in CXCL12-stimulated phosphorylation of ERK1/2 and Akt. Single cell suspensions of Gpsm1^+/+ and Gpsm1^−/− cultured dendritic cells were pretreated in the absence or presence of 100 ng/ml pertussis toxin (PTX) for 18 h prior to the addition of 200 ng/ml CXCL12 as described under “Experimental Procedures.” At the indicated times, cells were lysed in 1% Nonidet P-40 lysis buffer containing protease and phosphatase inhibitors, and lysates (50 μg/lane) were subjected to SDS-PAGE, transferred to PVDF, and immunoblotted with anti-phospho-Akt (Ser⁴⁸³) (pAkt), anti-phospho-ERK (Tyr²⁰⁴) (pErk1/2), or total Akt- or total ERK-specific antibodies. Representative immunoblots are shown in the left panels, and densitometric analysis of at least three independent experiments (represented as means ± S.E.) are shown in the right panels. *, p < 0.05.

FIGURE 6.

Gpsm1^−/− splenocytes exhibit defects in CXCL12- and CCL19-stimulated phosphorylation of ERK1/2 and Akt. A and B, single cell suspensions of Gpsm1^+/+ and Gpsm1^−/− freshly isolated splenocytes were treated with 200 ng/ml CXCL12 (A) or CCL19 (B) as described under “Experimental Procedures.” At the indicated times, cells were lysed in 1% Nonidet P-40 lysis buffer containing protease and phosphatase inhibitors, and lysates (50 μg/lane) were subjected to SDS-PAGE, transferred to PVDF, and immunoblotted with anti-phospho-Akt (Ser⁴⁸³) (pAkt), anti-phospho-ERK (Tyr²⁰⁴) (pErk1/2), or total Akt- or total ERK-specific antibodies. Representative immunoblots are shown in the left panels, and densitometric analysis of at least three independent experiments (represented as mean ± S.E.) are shown in the right panels. *, p < 0.05; **, p < 0.01.