RalA and RalB function as the critical GTP sensors for GTP-dependent exocytosis

Abstract

Although it has been established that the activation of GTPases by non-hydrolyzable GTP stimulates neurotransmitter release from many different secretory cell types, the underlying mechanisms remain unclear. In the present study we aimed to elucidate the functional role(s) for endogenous Ras-like protein A (RalA) and RalB GTPases in GTP-dependent exocytosis. For this purpose stable neuroendocrine pheochromocytoma 12 (PC12) cell lines were generated in which the expressions of both RalA and RalB were strongly downregulated. In these double knock-down cells GTP-dependent exocytosis was reduced severely and was restored after the expression of RalA or RalB was reintroduced by transfection. In contrast, Ca2+-dependent exocytosis and the docking of dense core vesicles analyzed by electron microscopy remained unchanged in the double knock-down cells. Furthermore, the transfected RalA and RalB appeared to be localized primarily on the dense core vesicles in undifferentiated and nerve growth factor-differentiated PC12 cells. Our results indicate that endogenous RalA and RalB function specifically as GTP sensors for the GTP-dependent exocytosis of dense core vesicles, but they are not required for the general secretory pathways, including tethering of vesicles to the plasma membrane and Ca2+-dependent exocytosis.

Figure 1.

Downregulation of RalA results in a limited level of reductions in GTP-dependent exocytosis. A, Immunoblot analysis of RalA knock-down cells. Total homogenates (15 μg) from the RalA knock-down (KD) and control (C) cells were analyzed by SDS-PAGE and immunoblotting, with the use of anti-RalA, anti-RalB, anti-Sec5, and anti-Exo84 antibodies. Numbers on the left indicate positions of molecular weight markers. B, NE release was stimulated by the indicated concentration of GppNHp from the permeabilized RalA knock-down cells (gray diamonds) and the paired control cells (black squares). Four pairs of RalA knock-down and control clones were examined. The error bars indicate SEM (n = 6); n represents the total number of samples tested in multiple experiments.

Figure 2.

Immunoblot analysis of the expression of various proteins in the RalA/RalB double knock-down cells. Total homogenates (15 μg) from the RalA/RalB double knock-down cells and the control RalA single knock-down cells were analyzed by SDS-PAGE and immunoblotting with the use of the antibodies listed on the right. Numbers on the left indicate positions of molecular weight markers.

Figure 3.

RalA/RalB double knock-down cells exhibit strong reductions in GTP-dependent exocytosis. Permeabilized RalA/RalB double knock-down cells (gray diamonds) and paired RalA single knock-down cells (black squares) were stimulated for NE release by using the indicated concentrations of GppNHp. Four pairs of the RalA/RalB double knock-down clones and the control RalA single knock-down clones were examined. The error bars indicate SEM (n=6–8).

Figure 4.

Transfection of RalA or RalB restores GTP-dependent exocytosis in the RalA/RalB double knock-down cells. A, Secretion of transfected NPY-hPLAP was stimulated with the indicated concentrations of GppNHp from wild-type PC12 cells (WT; gray diamonds) or RalA/RalB double knock-down (RalA^KD8/RalB^KD9) cells (DKD; black squares). The error bars indicate SEM (n=6). B, GppNHp-induced secretion of NPY-hPLAP from the double knock-down cells transfected with Myc-RalA (DKD plus Myc-RalA; black squares) or a control vector (DKD; gray diamonds). The transfected cells were permeabilized and stimulated with the indicated concen-trations of GppNHp. The error bars indicate SEM (n=8). C, GppNHp-induced secretion of NPY-hPLAP from the double knock-down cells transfected with pCMVmyc-rRalB(SNM) (DKD plus Myc-RalB; black squares) or a control vector (DKD; gray diamonds). The transfected cells were permeabilized and stimulated with the indicated concentrations of GppNHp. The error bars indicate SEM (n=6).

Figure 5.

Downregulation of RalA and RalB has no effect on High K⁺-stimulated Ca²⁺-dependent exocytosis. A, NE release from the RalA knock-down cells and the control cells was stimulated by a 15 min incubation with control K⁺ solution (5.6 mm; –) or high K⁺ solution (70 mm; +). Four pairs of the clones were examined. The error bars indicate SEM (n = 6–15). B, NE release from the RalA/RalB double knock-down cells and the RalA single knock-down cells was stimulated with a 15 min incubation with control K⁺ solution (5.6 mm; –) or high K⁺ solution (70 mm; +). Four pairs of the clones were examined. The error bars indicate SEM (n = 9–12).

Figure 6.

Downregulation of RalA and RalB has no effect on Ca²⁺-dependent exocytosis from permeabilized PC12 cells. A, The RalA/RalB double knock-down cells and the paired RalA single knock-down cells were permeabilized and incubated for NE release in the presence and absence of Ca²⁺ with and without brain cytosol. Four pairs of the clones were examined. The error bars indicate SEM (n = 6). B, Ca²⁺ titration of NE release from permeabilized wild-type PC12 cells (WT; open diamonds), RalA single knock-down (RalA^KD8/RalB^C7) cells (RalAKD; gray squares), and RalA/RalB double knock-down (RalA^KD8/RalB^KD9) cells (DKD; black squares). The error bars indicate SEM (n = 5).

Figure 7.

Electron microscopy of the RalA/RalB double knock-down cells does not reveal abnormalities in dense core vesicles. A, Electron micrograph of a single cell from RalA/RalB double knock-down (RalA^KD8/RalB^KD9) clones. Arrows indicate examples of dense core vesicles that are localized near the plasma membrane. Scale bar, 1 μm. B, The graph shows the mean number of vesicles present in each single-cell electron micrograph from the four different clones (two double knock-down clones and two RalA single knock-down clones). The error bars indicate SEM (n = 20–35). C, The graph shows the mean percentage distribution of dense core vesicles within individual PC12 cells, calculated from multiple single-cell electron micrographs from four different clones. Dense core vesicles were classed as being located within 50, 50–100, 100–200, 200–500, and 500–1000 nm as well as >1000 nm from the plasma membrane. The error bars indicate SEM.

Figure 8.

NGF-induced neurite outgrowth is not reduced by the downregulation of RalA/RalB. The wild-type PC12 cells (WT), the RalA single knock-down (RalA^KD8/RalB^C7) cells (RalAKD), and the RalA/RalB double knock-down (RalA^KD8/RalB^KD9) cells (DKD) were treated with NGF (100 ng/ml) for 5 d. Then, the cells were viewed by phase microscopy and photographed. Scale bars, 10 μm.

Figure 9.

Similar immunolocalization of transfected RalA and RalB in the double knock-down cells. Shown are confocal immunofluorescence images of the RalA/RalB double knock-down PC12 cells that were transfected with Myc-RalA (A–C, top panels) or Myc-RalB (D–F, bottom panels) expression plasmid. The transfected cells were stained with anti-Myc antibodies (Myc; clone 9E10) followed by Alexa 488-conjugated goat anti-mouse antibodies (A, D) and with anti-secretogranin II (SgII) antibodies followed by Alexa 568-conjugated goat anti-rabbit antibodies (B, E). The right panels (C, F) show merged images. Arrowheads in A–F indicate untransfected cells. Scale bars, 10 μm.

Figure 10.

Immunolocalization of transfected RalA in the double knock-down cells differentiated by NGF. Shown are confocal immunofluorescence images of the RalA/RalB double knock-down PC12 cells that were transfected with Myc-RalA expression plasmid and differentiated by NGF. The transfected cells were stained with anti-Myc antibodies (Myc; clone 9E10) followed by Alexa 488-conjugated goat anti-mouse antibodies (A, D, G) and with anti-secretogranin II (SgII) antibodies followed by Alexa 568-conjugated goat anti-rabbit antibodies (B, E, H). The right panels (C, F, I) show merged images. Arrows in A–F indicate the tips of the neurites of the transfected cells. Arrowheads in D–F indicate untransfected cells. Scale bars, 10 μm.