Interplay between the heterotrimeric G-protein subunits Galphaq and Galphai2 sets the threshold for chemotaxis and TCR activation

Abstract

Background: TCR and CXCR4-mediated signaling appears to be reciprocally regulated pathways. TCR activation dampens the chemotactic response towards the CXCR4 ligand CXCL12, while T cells exposed to CXCL12 are less prone to subsequent TCR-activation. The heterotrimeric G proteins Galphaq and Galphai2 have been implicated in CXCR4-signaling and we have recently also reported the possible involvement of Galphaq in TCR-dependent activation of Lck (Ngai et al., Eur. J. Immunol., 2008, 38: 32083218). Here we examined the role of Galphaq in migration and TCR activation.

Results: Pre-treatment of T cells with CXCL12 led to significantly reduced Lck Y394 phosphorylation upon TCR triggering indicating heterologous desensitization. We show that knockdown of Galphaq significantly enhanced basal migration in T cells and reduced CXCL12-induced SHP-1 phosphorylation whereas Galphai2 knockdown inhibited CXCL12-induced migration.

Conclusion: Our data suggest that Galphai2 confers migration signals in the presence of CXCL12 whereas Galphaq exerts a tonic inhibition on both basal and stimulated migrational responses. This is compatible with the notion that the level of Galphaq activation contributes to determining the commitment of the T cell either to migration or activation through the TCR.

Figure 1

CXCL12-pretreatment attenuates TCR-induced Lck activation. (A) Jurkat TAg T cells were incubated with or without 10 &#956M PP2 for 30 minutes at 37°C prior to stimulation with CXCL12 (100 ng/ml) for the indicated time-points and stopped with lysis buffer. Cell lysates were subjected to immunoblotting with the indicated antibodies. (B) Jurkat TAg T cells were incubated with 100 ng/ml CXCL12 for 30 minutes at 37°C prior to stimulation with OKT3 (1.5 μg/ml) for the indicated time-points and stopped with lysis buffer. Cell lysates were subjected to immunoblotting with the indicated antibodies. Cells were also stained with anti-CXCR4 and surface expression of the receptor was analysed by flow cytometry. (C) Jurkat TAg T cells were transfected with G_αq-specific siRNA (GNAQ1103), G_αi2-specific siRNA (GNAI-2-1050) or control siRNA (GNAQ1103 M3). 48 hours post-transfection cells were stimulated with OKT3 (1.5 μg/ml) for the indicated time-points and stopped with lysis buffer. Cell lysates were subjected to immunoblotting with the indicated antibodies. All figures are representative of at least 3 independent experiments.

Figure 2

Gαq and Gαi2 have opposing roles in chemotaxis. (A) Jurkat TAg T cells were transfected with G_αq-specific siRNA (GNAQ1103), G_αi2-specific siRNA (GNAI-2-1050) or control siRNA (GNAQ1103 M3). 48 hours post-transfection cells were placed in Costar Transwell wells with/without 10 ng/ml CXCL12. After 2 hours incubation the numbers of migrated cells were counted by flow cytometry Migration index was calculated as the number of cells migrating divided by the number of basal migrating mock tx cells (n = 8). (B) Cells were subjected to transfection as in (A). 48 hours post-transfection cells were stimulated with CXCL12 for the indicated time points. Cells were stained with anti-CXCR4 and surface expression of the receptor was analysed by flow cytometry (n = 3). (C) Cells were subjected to transfection as in (A). 48 hours post-transfection cells were stained with Fluo-4 and Fura Red AM and calcium fluxes were measured by flow cytometry. Grey lines indicates CXCL12 stimulated cells, black line unstimulated cells. Representative of 3 independent experiments. (D) Cells were subjected to transfection as in (A). 48 hours post-transfection cells were stimulated with CXCL12 for the indicated time points, lysed and subjected to immunoblot analysis with the indicated antibodies. Levels of immunoreactive protein were quantified by densitometric scanning from 3 independent experiments and normalized against total protein (Mean ± S.E.M.).

Figure 3

CXCL12 stimulation primarily activates Gαi2.(A) Jurkat TAg T cells were tranfected with G-specific siRNA (GNAQ1103), G_αi2-specific siRNA (GNAI-2-1050) or control siRNA (GNAQ1103 M3). 48 hours post-transfection cells were pre-treated with Wortmannin for 30 minutes and analyzed for CXCL12 dependent and independent migratory responses. Migration index represents fold migration over basal in control cell wells. (B) Jurkat TAg T cells were transfected with Lck Y505F or empty vector migration was analysed in the absence and presence of CXCL12 (10 ng/ml). Migration index represents fold increase in migration over basal migration in wells with control cells. (C) Cells were subjected to tranfection as in (B) pre-treated with Wortmannin as in (A) and migration with CXCL12 was analysed. Migration index represents fold increase in migration over basal migration in wells with control cells.

Figure 4

Migration is inhibited by activation of SHP-1. (A) Jurkat TAg T cells were transfected with G_αq-specific siRNA (GNAQ1103), G_αi2-specific siRNA (GNAI-2-1050) or control siRNA (GNAQ1103 M3). 48 hours post-transfection cells were stimulated with CXCL12 for the indicated time periods and lysed in lysis buffer. SHP-1 immunoprecipitation was performed and immune complexes analysed by immunobloting with the indicated antibodies (n = 3). (B) Immunoblots from 3 independent experiments like in (A) were quantified by densiometric analysis. The graph show mean ± S.E.M. (C) Jurkat TAg T cells were tranfected with the phosphatase defective SHP-1 c/s (C453S) mutant or empty vector. 24 hours post-transfection cells were placed in Costar Transwell plates with or without CXCL12 (10 ng/ml) and migration analysed as above. Migration index represents fold increase in migration over basal migration in wells with vector tx cells (n = 3). (D) Activation of G_αi2 induces migration through a PI3K pathway whereas activation of G_αq inhibits migration through an Lck-SHP-1 pathway priming cell for activation through the TCR.