Differential G protein subunit expression by prostate cancer cells and their interaction with CXCR5

BACKGROUND: Prostate cancer (PCa) cell lines and tissues differentially express CXCR5, which positively correlate with PCa progression, and mediate PCa cell migration and invasion following interaction with CXCL13. However, the differential expression of G protein α, β, and γ subunits by PCa cell lines and the precise combination of these proteins with CXCR5 has not been elucidated. METHODS: We examined differences in G protein expression of normal prostate (RWPE-1) and PCa cell lines (LNCaP, C4-2B, and PC3) by western blot analysis. Further, we immunoprecipitated CXCR5 with different G protein subunits, and CXCR4, following CXCL13 stimulation. To investigate constitutive coupling of CXCR5 with CXCR4 and PAR-1 we performed invasion assay in PCa cells transfected with G(αq/i2) or G(α13) siRNA, following CXCL13 treatment. We also investigated Rac and RhoA activity by G-LISA activation assay in PCa cells following CXCL13/thrombin stimulation. RESULT: Of the 22 G proteins studied, G(αi1-3), G(β1-4), G(γ5), G(γ7), and G(γ10) were expressed by both normal and PCa cell lines. G(αs) was moderately expressed in C4-2B and PC3 cell lines, G(αq/11) was only present in RWPE-1 and LNCaP cell lines, while G(α12) and G(α13) were expressed in C4-2B and PC3 cell lines. G(γ9) was expressed only in PCa cell lines. G(α16), G(β5), G(γ1-4), and G(γ13) were not detected in any of the cell lines studied. Surprisingly, CXCR4 co-immunoprecipitated with CXCR5 in PCa cell lines irrespective of CXCL13 treatment. We also identified specific G protein isoforms coupled to CXCR5 in its resting and active states. G(αq/11/)G(β3/)G(γ9) in LNCaP and G(αi2/)G(β3/)G(γ9) in C4-2B and PC3 cell lines, were coupled to CXCR5 and disassociated following CXCL13 stimulation. Interestingly, G(α13) co-immunoprecipitated with CXCR5 in CXCL13-treated, but not in untreated PCa cell lines. Inhibition of G(αq/i2) significantly decreased the ability of cells to invade, whereas silencing G(α13) did not affect CXCL13-dependent cell invasion. Finally, CXCL13 treatment significantly increased Rac activity in G(αq/i2) dependent manner, but not RhoA activity, in PCa cell lines. CONCLUSIONS: These findings offer insight into molecular mechanisms of PCa progression and can help to design some therapeutic strategies involving CXCR5 and/or CXCL13 blockade and specific G protein inhibition to abrogate PCa metastasis.