Post-translational modifications of GTPases from the Ras superfamily enable them to associate with membrane compartments where they exert their biological activities. However, no protein acting like Rho and Rab dissociation inhibitor (GDI) that regulate the membrane association of Rho and Rab GTPases has been described for Ras and closely related proteins. We report here that the ␦ subunit of retinal rod phosphodiesterase (PDE␦) is able to interact with prenylated Ras and Rap proteins, and to solubilize them from membranes, independently of their nucleotide-bound (GDP or GTP) state. We show that PDE␦ exhibits striking structural similarities with RhoGDI, namely conservation of the Ig-like fold and presence of a series of hydrophobic residues which could act as in RhoGDI to sequester the prenyl group of its target proteins, thereby providing structural support for the biochemical activity of PDE␦. We observe that the overexpression of PDE␦ interferes with Ras trafficking and propose that it may play a role in the process that delivers prenylated proteins from endomembranes, once they have undergone proteolysis and carboxymethylation, to the structures that ensure trafficking to their respective resident compartments.

In search for effectors of the Ras-related GTPase Rap2, we used the yeast two-hybrid method and identified the C-terminal Ras/Rap interaction domain of the Ral exchange factors (RalGEFs) Ral GDP dissociation stimulator (RalGDS), RalGDS-like (RGL), and RalGDSlike factor (Rlf). These proteins, which also interact with activated Ras and Rap1, are effectors of Ras and mediate the activation of Ral in response to the activation of Ras. Here we show that the full-length RalGEFs interact with the GTP-bound form of Rap2 in the two-hybrid system as well as in vitro. When co-transfected in HeLa cells, an activated Rap2 mutant (Rap2Val-12) but not an inactive protein (Rap2Ala-35) co-immunoprecipitates with RalGDS and Rlf; moreover, Rap2-RalGEF complexes can be isolated from the particulate fraction of transfected cells and were localized by confocal microscopy to the resident compartment of Rap2, i.e. the endoplasmic reticulum. However, the overexpression of activated Rap2 neither leads to the activation of the Ral GTPase via RalGEFs nor inhibits Ras-dependent Ral activation in vivo. Several hypotheses that could explain these results, including compartmentalization of proteins involved in signal transduction, are discussed. Our results suggest that in cells, the interaction of Rap2 with RalGEFs might trigger other cellular responses than activation of the Ral GTPase.Ras proteins are monomeric GTPases that play a pivotal role in the control of cell proliferation; they function as binary switches by cycling between an inactive form bound to GDP and the active GTP-bound state (1). Activation, through the dissociation of bound GDP and subsequent binding of GTP, is catalyzed by GEFs, 1 such as CDC25/Ras-GRF and Sos (2-4). Return to the inactive state is ensured via stimulation of the low intrinsic GTPase activity of Ras by GTPase-activating proteins, such as p120-GTPase-activating protein, neurofibromin, and Gap IP4BP (2, 5). In the active GTP-bound state, Ras exerts its biological effects by turning on several effectors that activate downstream pathways. Soluble serine/threonine kinases B-Raf and c-Raf, once activated by Ras-GTP through a mechanism that is not fully understood, trigger a cascade of protein kinases that results in the activation of mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (6). Another target of Ras is the catalytic subunit of phosphatidylinositol 3-OH kinase (PI3K) (7); this pathway leads to the activation of the protein kinase Akt (8, 9), as well as the activation of Ras-related proteins of the Rho/Rac/Cdc42 family involved in controlling the polymerization state of the actin cytoskeleton, cell adhesion, and gene transcription (10, 11). Ras is also able to activate the related GTPase Ral through RalGEFs that constitute direct effectors of Ras (12-15); three such proteins, namely RalGDS (16), RGL (17), and Rlf (18) have been extensively characterized, and the isolation of another member of this family has been recently reported (19). Elegant genetic experiments have show...

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Leber's congenital amaurosis (LCA) is the earliest and most severe of all inherited retinal dystrophies. Recently, we mapped an LCA gene to chromosome 17p13.1 (LCA1) and ascribed the disease to mutations of the retinal guanylate cyclase (ret GC) gene in a subset of families of North African ancestry. Owing to the genetic heterogeneity of LCA and considering that LCA1 results from an impaired production of cGMP in the retina (with permanent closure of cGMP-gated cation channels), we hypothesized that the activation of the cGMP phosphodiesterase (PDE) could trigger the disease by lowering the intracellular cGMP level in the retina. The rod and cone cGMP-PDE inhibitory subunits were regarded therefore as candidate genes in LCA. Here, we report the exclusion of five rod and cone cGMP-PDE subunits in LCA families unlinked to chromosome 17p13.