Similar to Ras,2
activated Ral-GTP interacts with multiple, functionally divergent downstream effectors (). The first effector was identified in a yeast 2-hybrid library or cDNA expression library screens using RalA, leading to independent discovery of RalBP1 (Ral binding protein 1)/RLIP76 (76-kDa Ral-interacting protein 1)/ RIP (Ral-interacting protein).38-40
In addition to a Ral-GTP binding domain,41
RalBP1 also contains a RhoGAP homology catalytic domain with activity for Rac1 and Cdc42 but not RhoA. GTP-bound RalA and RalB can interact with RalBP1 through a conserved Ral binding domain, regulating RalBP1 subcellular localization but not intrinsic RhoGAP activity.42
Rac1 activation promotes membrane ruffling at the leading edge of migrating cells, whereas Cdc42 promotes filopodia formation.
Figure 4. Ral effectors. RalA and/or RalB have been determined to interact with a diverse spectrum of downstream effectors. Most bind preferentially to the GTP-bound protein, whereas some are nucleotide independent. Ral effector networks can regulate endocytosis, (more ...)
RalBP1 can also serve as a scaffold and associate with a diversity of other proteins. Two independent yeast 2-hybrid library screening studies identified the closely related Reps1 (RalBP1-associated Eps homology (EH) domain protein 1) and Reps2/POB1 (partner of RalBP1) proteins that interact with RalBP1 C-terminal sequences distinct from the RhoGAP and Ral binding domains.43,44
Reps1 and Reps2 contain EH domains, which are found on proteins involved in endocytosis. Reps1 associates through its EH domain with Rab11-FIP2, a Rab11 binding protein implicated in endocytosis.45
Reps2/POB1, via its EH domain, interacts with Epsin and Eps15,46,47
proteins that regulate receptor-mediated endocytosis. Similarly, a second RalBP1 interaction, through N-terminal sequences, is with the µ2 subunit of the plasma membrane–associated AP-2 tetrameric complex.48
AP-2 promotes clathrin coat formation and specific recognition of membrane receptors for endocytosis. Epsin, Eps15, and Rab11-FIP2 can also interact directly with AP-2. These interactions support a role for RalBP1 in the regulation of receptor-mediated endocytosis.
RalBP1 also interacts with ARIP2 (activin receptor-interacting protein 2), which regulates endocytosis of activin type II receptors,49
HSF1 (heat shock factor 1),50
which regulates expression of heat shock genes in response to stress, cyclin B1 during mitosis,51
and PSD-1, a postsynaptic scaffolding protein implicated in the regulation of excitatory synaptic function.52
RalBP1 was also identified independently as a transporter activity, designated DNP-SG (S-(2, 4-dinitrophenyl)glutathione) ATPase, involved in the active transport of conjugated and unconjugated electrophiles out of cells.53
RalBP1 contains 2 ATP binding sites54
that allow it to function as an ATP-dependent transporter protein and efflux pump for small molecules, including anticancer drugs and endogenous metabolites.55
Inhibition of RalBP1 expression or function has been shown to cause regression of lung, kidney, melanoma, colon, and prostate cancer cell line xenografts, although the significance of these observations for Ral function is not clear.
Perhaps the best characterized of the Ral effectors are 2 components of the octameric exocyst complex (also called Sec6/8 complex), Sec5 and Exo84.56,57
The octameric exocyst complex is involved in the regulation of exocytosis.58,59
The exocyst facilitates the tethering of post-Golgi secretory vesicles to the plasma membrane prior to exocytic fusion, and exocyst function has been implicated in a variety of cellular processes including cell migration and tumor cell invasion. Ral regulates exocyst subcellular localization rather than assembly.60
Studies with Ral effector domain mutants and/or RNA interference have implicated exocyst function in several Ral-mediated functions.12
Other less characterized effectors of RalA include filamin, an actin filament crosslinking protein required for RalA-induced filopodia formation.61
RalA and RalB have also been shown to directly interact with and activate phospholipase C delta 1 (PLCδ1).62
PLCδ1 is not a conventional effector in that interaction with RalB was nucleotide independent and required the N-terminal 11 residues of RalB. Activation of various G protein–coupled receptor signaling pathways stimulates Ral-dependent PLC activation.
Another effector of Ral is phospholipase D (PLD1),63
which leads to the generation of lipid second messengers, including phosphatidic acid, lysophosphatidic acid, and diacylglycerol. However, PLD1 does not function classically as a Ral effector in that PLD1 association is not regulated by GDP/GTP cycling and interaction is through Ral N-terminal sequences distinct from the switch I and II sequences involved in GTP-dependent effector binding. Instead, RalA activation of PLD1 includes the additional association with the Arf6 small GTPase.64
Although RalA and RalB share 100% sequence identity in residues involved in effector interaction () and, where studied in vitro
, can interact with the same effectors, RalA and RalB can exhibit strikingly different roles in normal and neoplastic cell function. These functional differences are largely because of their distinct subcellular membrane locations. Whereas RalA is found at the plasma membrane and with endosomes, RalB is primarily endosome associated.65
However, Ral subcellular localization is dynamic and can be regulated by its activation state and by phosphorylation.66,67
Ral subcellular localization in turn influences specific effector interactions. Some examples of RalA and RalB differences in oncogenesis are summarized below.