Ras-Specific Exchange Factor GRF: Oligomerization through Its Dbl Homology Domain and Calcium-Dependent Activation of Raf

Ras GTPases, which play a pivotal role as transducers of various mitogenic and differentiation signals, function as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state (33). Ras is negatively regulated by GTPase-activating proteins (ras GAPs) that stimulate hydrolysis of GTP-Ras to GDP-Ras. The conversion of the GDP-bound form into the active form is stimulated by Ras-specific guanine nucleotide exchange factors (GNEFs), such as Ras-GRF (GRF, also known previously as CDC25Mm) (10, 44), Sos (45), and Ras-GRP (13, 48). GNEFs function by inducing release of bound GDP from Ras, which results in the rapid binding of GTP, because the concentration of free GTP is much higher than that of free GDP, and Ras has a greater affinity for GTP than for GDP (31). Ras contains several direct downstream targets, including Raf, which in turn activate the Mek and Erk mitogen-activated protein (MAP) kinases (7). Mammals contain two closely related sos genes, sos1 and sos2 (4), as well as two grf genes which encode homologous proteins, GRF1 and GRF2, respectively (17). While Sos1 and Sos2 are ubiquitously expressed, full-length GRF1 and GRF2 are primarily brain specific, although the full-length protein and various smaller forms have also been observed in other tissues (17, 21, 27, 43, 44, 50). Functionally, GRF1 has been implicated in synaptic transmission and the formation of long-term memory (5), in agreement with its presence in synaptic junctions (47). In mice grf1 has been shown to be imprinted, with only the paternal gene being expressed (40). Animals lacking detectable GRF1 protein are viable but grow more slowly than controls, presumably because of a hypothalamic defect, which is associated with low levels of circulating insulin-like growth factor 1 (30). Full-length GRF1 is a 140-kDa protein with many motifs common to other signaling molecules (Fig. ​(Fig.1).1). In addition to its C-terminally located Ras-catalytic domain, which is responsible for the stimulation of the guanine nucleotide exchange on Ras, GRF contains an N-terminal pleckstrin homology (PH) domain, a coiled-coil (CC) motif, an ilimaquinone (IQ) motif, a Dbl homology (DH) domain adjacent to a second PH domain, and a PEST motif. The N-terminal PH domain of GRF1 has been shown to bind the βγ subunit of heterotrimeric G proteins in vitro (49), and Mattingly and Macara have reported a phosphorylation-dependent activation of GRF1 by muscarinic receptors through the βγ subunit of a heterotrimeric G protein (37). The influx of calcium in human 293T cells, via the calcium ionophore ionomycin, has also been shown to activate GRF1, as measured primarily by an increased Erk1 activity that can be suppressed by a dominant inhibitory Ras mutant (19). The calcium-dependent activation is associated with the binding of calmodulin to the IQ motif, which acts cooperatively with the other motifs in the N terminus of GRF1, including both PH domains as well as the CC and DH domains (6, 24). A PEST-like region renders the protein susceptible in vitro to cleavage by calpain, although it is not clear if this cleavage also occurs in vivo (2). Ionomycin-dependent Erk1 activation has also been reported for GRF2, which is structurally similar to GRF1 (17). FIG. 1 Schematic representation of the domain structure of GRF1 and of the baits and preys used in the two-hybrid interactions. The various abbreviations are as defined in the text; PEST is the protein instability motif, and Catalytic refers to the Ras guanine ... The precise function of the GRF1 DH domain has not been determined. The c-Dbl oncoprotein is the prototype for signaling proteins that contain a DH domain (11). c-Dbl acts as a GNEF for two Rho family GTPases, Cdc42 and RhoA (39). Members of the Rho family of GTPases, which include Rac in addition to CDC42 and Rho, play important roles in mediating various cytoskeletal reorganizations in cells (52). The DH domain of c-Dbl is responsible for its exchange activity in vitro (28). It is believed that other Dbl family members also serve as GNEFs for one or more Rho family GTPases, although this ability has been shown for only a subset of DH domains (39). Mouse GRF2 has been recently shown to display in vitro exchange activity toward Rac (18). However, analogous attempts to show that GRF1 displays exchange activity towards any of the known Rho family GTPases have thus far yielded negative results (11a, 24). In this study, we included the DH domain of GRF1 as a bait in a yeast two-hybrid screen to identify protein(s) that may interact with this region. We report that the screen unexpectedly identified the DH domain of GRF2 as functionally interacting with the DH domain of GRF1 and that, in mammalian cells, GRF1 and GRF2 formed homo-oligomers as well as hetero-oligomers with each other. We also observed a calcium induced GRF1-dependent Erk activation that was associated with increased Ras activity but, surprisingly, was not correlated with a further increase in GTP-Ras. A point mutation in the DH domain reduced each of these GRF1-dependent activities, as well as the GRF1-dependent cell transformation.