Ral inhibits ligand-independent Notch signaling in Drosophila.

We discovered recently that the Drosophila Ral GTPase regulates Notch signaling and thereby affects cell patterning in the eye. Although Ral functions in the ligand signaling cells, Ral does not stimulate ligand signaling directly. Rather, in cells that express both Notch receptor and ligand, Ral activity promotes a cell to become the signaler by inhibiting Notch receptor activation in that cell. Moreover, Ral inhibits a particular pathway of Notch activation-receptor activation that occurs independent of ligand binding. In this Commentary, we discuss the phenomenon of ligand-independent Notch receptor activation and how this event might be regulated by Ral.

The small GTPase Ral couples the angiotensin II type 1 receptor to the activation of phospholipase C-delta 1.

The angiotensin II type 1 receptor (AT(1)R) plays an important role in cardiovascular function and as such represents a primary target for therapeutic intervention. The AT(1)R has traditionally been considered to be coupled to the activation of phospholipase C (PLC) beta via its association with G alpha(q/11), leading to increases in intracellular inositol phosphate (IP) and release of calcium from intracellular stores. In the present study, we investigated whether the small GTPase RalA contributed to the regulation of AT(1)R endocytosis and signaling. We find that neither RalA nor RalB is required for the endocytosis of the AT(1)R, but that RalA expression is required for AT(1)R-stimulated IP formation but not 5-HT(2A) receptor-mediated IP formation. AT(1)R-activated IP formation is lost in the absence of Ral guanine nucleotide dissociation stimulator (RalGDS), and requires the beta-arrestin-dependent plasma membrane translocation of RalGDS. G alpha(q/11) small interfering RNA (siRNA) treatment also significantly attenuates both AT(1)R- and 5-HT(2A) receptor-stimulated IP formation after 30 min of agonist stimulation. PLC-delta1 has been reported to be activated by RalA, and we show that AT(1)R-stimulated IP formation is attenuated after PLC-delta 1 siRNA treatment. Taken together, our results provide evidence for a G protein-coupled recepto-activated and RalGDS/Ral-mediated mechanism for PLC-delta 1 stimulation.

Immunogenicity of Ra1A and its tissue-specific expression in hepatocellular carcinoma.

In order to understand the immunogenicity of a tumor-associated antigen (TAA), Ras family small GTP binding protein (Ra1A) in hepatocellular carcinoma (HCC), autoantibody responses to RalA were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and indirect immunofluorescence assay in sera from patients with HCC and sera from normal individuals. Immunohistochemistry (IHC) study with tissue array slides was also performed to analyze protein expression profiles of RalA in HCC and control tissues. This study demonstrated that RalA had a relative higher frequency of autoantibody response in HCC (20.1%) compared to liver cirrhosis (3.3%), chronic hepatitis (0%), and normal individuals sera (0%). RalA also showed a stepwise increased expression from normal liver tissues (26.7%), liver cirrhosis tissues (45.0%) to HCC tissues (63.3%). Sensitivity and specificity of anti-RalA antibody in detection of HCC was 20.1% and 99.3%, respectively. The data suggested that RalA might contribute to liver malignant transformation, and could be used as a potential tumor marker in HCC detection.

The Small GTPase RalA controls exocytosis of large dense core secretory granules by interacting with ARF6-dependent phospholipase D1.

RalA and RalB constitute a family of highly similar Ras-related GTPases widely distributed in different tissues. Recently, active forms of Ral proteins have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. Since RalA is present on the plasma membrane in neuroendocrine chromaffin and PC12 cells, we investigated the potential role of RalA in calcium-regulated exocytotic secretion. We show here that endogenous RalA is activated during exocytosis. Expression of the constitutively active RalA (G23V) mutant enhances secretagogue-evoked secretion from PC12 cells. Conversely, expression of the constitutively inactive GDP-bound RalA (G26A) or silencing of the RalA gene by RNA interference led to a strong impairment of the exocytotic response. RalA was found to co-localize with phospholipase D1 (PLD1) at the plasma membrane in PC12 cells. We demonstrate that cell stimulation triggers a direct interaction between RalA and ARF6-activated PLD1. Moreover, reduction of endogenous RalA expression level interfered with the activation of PLD1 observed in secretagogue-stimulated cells. Finally, using various RalA mutants selectively impaired in their ability to activate downstream effectors, we show that PLD1 activation is essential for the activation of secretion by GTP-loaded RalA. Together, these results provide evidence that RalA is a positive regulator of calcium-evoked exocytosis of large dense core secretory granules and suggest that stimulation of PLD1 and consequent changes in plasma membrane phospholipid composition is the major function RalA undertakes in calcium-regulated exocytosis.

Ral and phospholipase D2-dependent pathway for constitutive metabotropic glutamate receptor endocytosis.

G-protein-coupled receptors play a central role in the regulation of neuronal cell communication. Class 1 metabotropic glutamate receptors (mGluRs) mGluR1a and mGluR5a, which are coupled with the hydrolysis of phosphoinositides, are essential for modulating excitatory neurotransmission at glutamatergic synapses. These receptors are constitutively internalized in heterologous cell cultures, neuronal cultures, and intact neuronal tissues. We show here that the small GTP-binding protein Ral, its guanine nucleotide exchange factor RalGDS (Ral GDP dissociation stimulator), and phospholipase D2 (PLD2) are constitutively associated with class 1 mGluRs and regulate constitutive mGluR endocytosis. Moreover, both Ral and PLD2 are colocalized with mGluRs in endocytic vesicles in both human embryonic kidney 293 (HEK 293) cells and neurons. Ral and PLD2 activity is required for the internalization of class 1 mGluRs but is not required for the internalization of the beta2-adrenergic receptor. Constitutive class 1 mGluR internalization is not dependent on the downstream Ral effector proteins Ral-binding protein 1 and PLD1 or either ADP-ribosylation factors ARF1 or ARF6. The treatment of HEK 293 cells and neurons with small interfering RNA both downregulates PLD2 expression and blocks mGluR1a and mGluR5a endocytosis. The constitutive internalization of mGluR1a and mGluR5a is also attenuated by the treatment of cells with 1-butanol to prevent PLD2-mediated phosphatidic acid formation. We propose that the formation of a mGluR-scaffolded RalGDS/Ral/PLD2 protein complex provides a novel alternative mechanism to beta-arrestins for the constitutive endocytosis of class 1 mGluRs.