Characterization of R-Ras3/M-Ras Null Mice Reveals a Potential Role in Trophic Factor Signaling

R-Ras3/M-Ras was initially isolated based on its sequence similarity to the prototypic Ras oncogene through PCR-based methodology (22), expressed sequence tag (EST) database search (14, 7, 32), and subtractive hybridization (20). It is closely related (∼60%) to TC21/R-RAS2, which was identified through low-stringency hybridization method (6) and as a mutated oncogene in a human ovarian tumor cell line (5). M-Ras belongs to the Ras subgroup of small G proteins which includes Ha-, N-, and Ki-Ras; R-Ras; Rap1A/Krev1; Rap1B; Rap2A; Rap2B; TC21/R-Ras2; RalA; RalB; Rheb; Rin; and Rit (reviewed in references 3 and 34). M-Ras possesses functional motifs shared by other Ras-related GTPases. For example, residues essential for guanine nucleotide-binding and catalysis are highly conserved. In addition, the canonical effector-binding switch I and switch II domains are identical to that of the prototypic Ras oncogenes. Thus, sequences that can confer M-Ras-specific biological properties are likely to be located in other regions of this unique GTPase. Indeed, M-Ras has a unique amino-terminal sequence, MATSAVPSDNLPT, that is not found in other GTPases. In the carboxyl-terminal region, M-Ras harbors a polybasic stretch (residue 187 to 191: KKKTK) that is reminiscent of that of K-Ras4B (4). Also, the membrane-targeting consensus sequence in the C terminus, CVIL, would predict M-Ras to be modified by the geranylgeranyl transferase. The biochemical properties of M-Ras resemble that of H-Ras more than other related members, such as R-Ras and TC21 (28). For example, both H-Ras and M-Ras share the same exchange factors, Sos, RasGRF, and CalDAG-GEFII (RasGRP) and GTPase activating protein (GAP) proteins, such as p120 GAP and NF-1 (28). Apart from the conventional Ras effectors, several downstream signaling molecules have been implicated in mediating M-Ras-dependent signaling (28, 32). Interestingly, guanine nucleotide exchange factors (GEFs) for Rap (MR-GEF/RA-GEF-2) (32) and Ral (RGL3) (8) have been shown to interact with M-Ras in a GTP-dependent manner. These findings suggest that M-Ras may coordinate biological events through cross talk with other GTPases. In fibroblasts, M-Ras has been shown to activate the phosphatidylinositol 3-kinase (PI3-K)-dependent signaling cascade while only weakly stimulating the mitogen-activated protein kinase (MAPK) pathway (15). On the contrary, M-Ras strongly stimulates MAPK in PC12 cells through binding and activation of B-Raf (16). By far, the highest level of M-Ras expression is found in the central nervous system (CNS). Indeed, by in situ hybridization, high levels of M-Ras are found in the mouse hippocampus and the Purkinje layer of the cerebellum (16). H-Ras also displays a similar pattern of expression in the CNS except that it is present in both the granular and Purkinje layers of the cerebellum (40). On the contrary, both K- and N-Ras show a significantly lower level of expression in the juvenile rat brain (40). The regional expression of R-Ras and TC21 in the CNS has not been reported. However, recent data suggest that R-Ras expression is more restricted to both endothelial and vascular smooth muscle cells (18). Apart from the CNS, M-Ras expression has also been reported in muscle cells (22) and in interleukin-9 (IL-9)-induced T-cell clones (20). Interestingly, M-Ras expression was also shown to be elevated after food search memory tests (35), as well as during thermal adaptation in newborn chicks (19). These findings implicate a role for M-Ras in synaptic plasticity in both the hippocampal and the hypothalamic regions of the CNS. The bona fide physiological functions of M-Ras during development and in somatic tissues are still largely unknown. M-Ras was initially referred to as Microspike-Ras based on its ability to induce protrusions in the cell periphery of Swiss 3T3 fibroblasts (22). Ectopic expression of a constitutively active M-Ras in PC12 cells leads to neurite outgrowth (16). In mammary epithelial cell system, M-Ras expression has been implicated in the process of transformation and epithelial-to-mesenchymal transition (39). M-Ras null mice have not yet been reported. Prior studies involving the generation of knockout (KO) mice targeting different members of the Ras family revealed potential functional overlap among GTPases. For example, H-Ras or N-Ras null mice are viable with no apparent phenotypes. Surprisingly, mice lacking both H- and N-Ras also display no prominent defects (10). However, K-Ras null mice are embryonic lethal due to defects in the fetal liver (13, 17). In addition, mice heterozygous for K-Ras in a N-Ras null background display hematopoietic defects and die at embryonic day 10 (E10) to E12 (13). The high level of M-Ras expression in the CNS raises the possibility of a unique role for this G protein in this system. In the present study, we report the generation of an M-Ras KO mouse and provide an initial characterization of these animals at the cellular and biochemical levels.