Fig. 4
AMPH-induced RhoA activation is mediated by G13. HEK293 cells in which various endogenous α-subunits of GPCRs were knocked out were co-transfected with DAT or DAT and EAAT3 (a). GS and Gq knockout cell lines exhibited similar sensitivity to AMPH as wild-type HEK293 cells with a decreased capacity to transport 3H-neurotransmitters following a 30-min pretreatment with AMPH (10 μM). Cells in which G12/13 were knocked out, however, lost sensitivity to AMPH pretreatment in regards to both DAT and EAAT3 trafficking. Wild-type HEK293 cells were co-transfected with DAT or DAT and EAAT3 along with mini-genes that interfere with the function of various α-subunits of GPCRs (b; Gilchrist et al. 1999 and 2001). The effect of AMPH on DAT and EAAT3 function were unaltered by the mini-genes that interfere with GS, G11 or G12. However, the effects of AMPH on DAT and EAAT3 were inhibited by co-expression of the G13 interfering minigene. We made cell-permeable versions of the alpha-interfering mini-genes by creating peptides of the interfering sequences with the addition of the TAT domain (YGRKKRRQRRR). HEK293 cells that were transiently transfected with DAT were treated with the TAT peptides for 30 min and then with AMPH (10 μM) for 30 min. Similar to the co-expression results, only the G13 interfering peptide selectively disrupted AMPH-mediated DAT internalization (c). Selectivity of the G13 and GS peptides was determined by recording responses of the Rho-FRET (d) or the AKAR4-FRET sensor (e) in HEK293 cells expressing DAT. AMPH-induced RhoA activation was prevented by pretreatment with the TAT-interfering peptide directed at G13 (red), while the GS interfering peptide (green) did not alter RhoA activation (d). AMPH-induced PKA activation detected by AKAR4 was blocked by the GS interfering peptide (green), but the G13 (red) response was similar to scrambled control (black, e). G13 expression (red and top panel) was detected in TH(+) cultured neurons (f, green and bottom panel). 3H-DA uptake in primary midbrain cultures was measured to reflect cell-surface localization of the DAT (g). With TAT-scrambled peptides, AMPH pretreatment (30 min, 10 μM) lead to a loss of DAT-mediated 3H-dopamine transport capacity but those treated with the TAT-13 interfering peptide, had no sensitivity to the AMPH pretreatment. (Asterisk indicates *p < 0.05, **p < 0.01, *** p < 0.001, and **** p < 0.0001 by two-way ANOVA with Sidak’s multiple comparisons test compared with vehicle control; n≥10 wells per condition)
