Tyrosine phosphorylation of RalGDS by c-Met receptor blocks its interaction with Ras.

RalGDS is a guanine nucleotide exchange factor that promotes the active GTP-bound form of Ral GTPases, RalA and RalB. GTP-bound Ras has the capacity to activate Ral GTPases at least in part by binding to the C-terminal Ras-binding domain (RBD) of RalGDS and directing the protein to Ral GTPases in the plasma membrane. In many cases, activation of Ral proteins complements other Ras effector pathways to carry out a cell function, but in others it opposes them. Moreover, in many cases activation of Ral proteins contributes to the oncogenic potential of Ras. However, in some cell types Ral proteins suppresses tumor formation, suggesting oncogenic stimuli that function through Ras may need to suppress Ral activation in order to transform cells. In this paper, we demonstrate a potential biochemical mechanism for such phenomena by showing that c-Met receptors promote the tyrosine phosphorylation of RalGDS at Y752 in its RBD, which blocks the binding of Ras to RalGDS.

Domain contributions to signaling specificity differences between Ras-guanine nucleotide releasing factor (Ras-GRF) 1 and Ras-GRF2.

Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2) constitute a family of similar calcium sensors that regulate synaptic plasticity. They are both guanine exchange factors that contain a very similar set of functional domains, including N-terminal pleckstrin homology, coiled-coil, and calmodulin-binding IQ domains and C-terminal Dbl homology Rac-activating domains, Ras-exchange motifs, and CDC25 Ras-activating domains. Nevertheless, they regulate different forms of synaptic plasticity. Although both GRF proteins transduce calcium signals emanating from NMDA-type glutamate receptors in the CA1 region of the hippocampus, GRF1 promotes LTD, whereas GRF2 promotes θ-burst stimulation-induced LTP (TBS-LTP). GRF1 can also mediate high frequency stimulation-induced LTP (HFS-LTP) in mice over 2-months of age, which involves calcium-permeable AMPA-type glutamate receptors. To add to our understanding of how proteins with similar domains can have different functions, WT and various chimeras between GRF1 and GRF2 proteins were tested for their abilities to reconstitute defective LTP and/or LTD in the CA1 hippocampus of Grf1/Grf2 double knock-out mice. These studies revealed a critical role for the GRF2 CDC25 domain in the induction of TBS-LTP by GRF proteins. In contrast, the N-terminal pleckstrin homology and/or coiled-coil domains of GRF1 are key to the induction of HFS-LTP by GRF proteins. Finally, the IQ motif of GRF1 determines whether a GRF protein can induce LTD. Overall, these findings show that for the three forms of synaptic plasticity that are regulated by GRF proteins in the CA1 hippocampus, specificity is encoded in only one or two domains, and a different set of domains for each form of synaptic plasticity.

Acquisition of contextual discrimination involves the appearance of a RAS-GRF1/p38 mitogen-activated protein (MAP) kinase-mediated signaling pathway that promotes long term potentiation (LTP).

RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

Ras-GRF2 mediates long-term potentiation, survival, and response to an enriched environment of newborn neurons in the hippocampus.

Hippocampal adult neurogenesis contributes to key functions of the dentate gyrus (DG), including contextual discrimination. This is due, at least in part, to the unique form of plasticity that new neurons display at a specific stage of their development when compared with the surrounding principal neurons. In addition, the contribution that newborn neurons make to dentate function can be enhanced by an increase in their numbers induced by a stimulating environment. However, signaling mechanisms that regulate these properties of newborn neurons are poorly understood. Here, we show that Ras-GRF2 (GRF2), a calcium-regulated exchange factor that can activate Ras and Rac GTPases, contributes to both of these properties of newborn neurons. Using Ras-GRF2 knockout mice and wild-type mice stereotactically injected with retrovirus containing shRNA against the exchange factor, we demonstrate that GRF2 promotes the survival of newborn neurons of the DG at approximately 1-2 weeks after their birth. GRF2 also controls the distinct form of long-term potentiation that is characteristic of new neurons of the hippocampus through its effector Erk MAP kinase. Moreover, the enhancement of neuron survival that occurs after mice are exposed to an enriched environment also involves GRF2 function. Consistent with these observations, GRF2 knockout mice display defective contextual discrimination. Overall, these findings indicate that GRF2 regulates both the basal level and environmentally induced increase of newborn neuron survival, as well as in the induction of a distinct form of synaptic plasticity of newborn neurons that contributes to distinct features of hippocampus-derived learning and memory.

Age-dependent role for Ras-GRF1 in the late stages of adult neurogenesis in the dentate gyrus.

The dentate gyrus of the hippocampus plays a pivotal role in pattern separation, a process required for the behavioral task of contextual discrimination. One unique feature of the dentate gyrus that contributes to pattern separation is adult neurogenesis, where newly born neurons play a distinct role in neuronal circuitry. Moreover,the function of neurogenesis in this brain region differs in adolescent and adult mice. The signaling mechanisms that differentially regulate the distinct steps of adult neurogenesis in adolescence and adulthood remain poorly understood. We used mice lacking RASGRF1(GRF1), a calcium-dependent exchange factor that regulates synaptic plasticity and participates in contextual discrimination performed by mice, to test whether GRF1 plays a role in adult neurogenesis.We show Grf1 knockout mice begin to display a defect in neurogenesis at the onset of adulthood (~2 months of age), when wild-type mice first acquire the ability to distinguish between closely related contexts. At this age, young hippocampal neurons in Grf1 knockout mice display severely reduced dendritic arborization. By 3 months of age, new neuron survival is also impaired. BrdU labeling of new neurons in 2-month-old Grf1 knockout mice shows they begin to display reduced survival between 2 and 3 weeks after birth, just as new neurons begin to develop complex dendritic morphology and transition into using glutamatergic excitatory input. Interestingly, GRF1 expression appears in new neurons at the developmental stage when GRF1 loss begins to effect neuronal function. In addition, we induced a similar loss of new hippocampal neurons by knocking down expression of GRF1 solely in new neurons by injecting retrovirus that express shRNA against GRF1 into the dentate gyrus. Together, these findings show that GRF1 expressed in new neurons promotes late stages of adult neurogenesis. Overall our findings show GRF1 to be an age-dependent regulator of adult hippocampal neurogenesis, which contributes to ability of mice to distinguish closely related contexts.

Transmission of reduced levels of miR-34/449 from sperm to preimplantation embryos is a key step in the transgenerational epigenetic inheritance of the effects of paternal chronic social instability stress.

The transgenerational effects of exposing male mice to chronic social instability (CSI) stress are associated with decreased sperm levels of multiple members of the miR-34/449 family that persist after their mating through preimplantation embryo (PIE) development. Here we demonstrate the importance of these miRNA changes by showing that restoring miR-34c levels in PIEs derived from CSI stressed males prevents elevated anxiety and defective sociability normally found specifically in their adult female offspring. It also restores, at least partially, levels of sperm miR-34/449 normally reduced in their male offspring who transmit these sex-specific traits to their offspring. Strikingly, these experiments also revealed that inducing miR-34c levels in PIEs enhances the expression of its own gene and that of miR-449 in these cells. The same induction of embryo miR-34/449 gene expression likely occurs after sperm-derived miR-34c is introduced into oocytes upon fertilization. Thus, suppression of this miRNA amplification system when sperm miR-34c levels are reduced in CSI stressed mice can explain how a comparable fold-suppression of miR-34/449 levels can be found in PIEs derived from them, despite sperm containing ~50-fold lower levels of these miRNAs than those already present in PIEs. We previously found that men exposed to early life trauma also display reduced sperm levels of miR-34/449. And here we show that miR-34c can also increase the expression of its own gene, and that of miR-449 in human embryonic stem cells, suggesting that human PIEs derived from men with low sperm miR-34/449 levels may also contain this potentially harmful defect.

Food, nutrition, and fertility: from soil to fork.

Food and nutrition-related factors, including foods and nutrients consumed, dietary patterns, use of dietary supplements, adiposity, and exposure to food-related environmental contaminants, have the potential to impact semen quality and male and female fertility; obstetric, fetal, and birth outcomes; and the health of future generations, but gaps in evidence remain. On 9 November 2022, Tufts University's Friedman School of Nutrition Science and Policy and the school's Food and Nutrition Innovation Institute hosted a 1-d meeting to explore the evidence and evidence gaps regarding the relationships between food, nutrition, and fertility. Topics addressed included male fertility, female fertility and gestation, and intergenerational effects. This meeting report summarizes the presentations and deliberations from the meeting. Regarding male fertility, a positive association exists with a healthy dietary pattern, with high-quality evidence for semen quality and lower quality evidence for clinical outcomes. Folic acid and zinc supplementation have been found to not impact male fertility. In females, body weight status and other nutrition-related factors are linked to nearly half of all ovulation disorders, a leading cause of female infertility. Females with obesity have worse fertility treatment, pregnancy-related, and birth outcomes. Environmental contaminants found in food, water, or its packaging, including lead, perfluorinated alkyl substances, phthalates, and phenols, adversely impact female reproductive outcomes. Epigenetic research has found that maternal and paternal dietary-related factors can impact outcomes for future generations. Priority evidence gaps identified by meeting participants relate to the effects of nutrition and dietary patterns on fertility, gaps in communication regarding fertility optimization through changes in nutritional and environmental exposures, and interventions impacting germ cell mechanisms through dietary effects. Participants developed research proposals to address the priority evidence gaps. The workshop findings serve as a foundation for future prioritization of scientific research to address evidence gaps related to food, nutrition, and fertility.

Activation of RalA is critical for Ras-induced tumorigenesis of human cells.

RalGEFs were recently shown to be critical for Ras-mediated transformed and tumorigenic growth of human cells. We now show that the oncogenic activity of these proteins is propagated by activation of one RalGEF substrate, RalA, but blunted by another closely related substrate, RalB, and that the oncogenic signaling requires binding of the RalBP1 and exocyst subunit effector proteins. Knockdown of RalA expression impeded, if not abolished, the ability of human cancer cells to form tumors. RalA was also commonly activated in a panel of cell lines from pancreatic cancers, a disease characterized by activation of Ras. Activation of RalA signaling thus appears to be a critical step in Ras-induced transformation and tumorigenesis of human cells.

Preimplantation Embryos Amplify Stress-Induced Changes in Sperm-Derived miRNA Content to Mediate Transgenerational Epigenetic Inheritance.

Chronically stressing male mice can alter the behavior of their offspring across generations. This effect is thought to be mediated by stress-induced changes in the content of specific sperm miRNAs that modify embryo development after their delivery to oocytes at fertilization. A major problem with this hypothesis is that the levels of mouse sperm miRNAs are much lower than those present in preimplantation embryos. This makes it unclear how embryos could be significantly impacted without an amplification system to magnify changes in sperm miRNA content, like those present in lower organisms where transgenerational epigenetic inheritance is well established. Here, we describe such a system for Chronic Social Instability (CSI) stress that can explain how it reduces the levels of the miR-34b,c/449a,b family of miRNAs not only in sperm of exposed males but also in preimplantation embryos ( PIEs ) derived from their mating, as well as in sperm of male offspring. Sperm-derived miR-34c normally positively regulates expression of its own gene and that of miR-449 in PIEs. This feed forward, auto-amplification process is suppressed when CSI stress reduces sperm miR-34c levels. Its suppression is important for the transmission of traits to offspring because restoring miR-34c levels in PIEs from CSI stressed males, which also restores levels of miR-449 in them, suppresses elements of elevated anxiety and defective sociability normally found specifically in their female offspring, as well as reduced sperm miR-34 and miR-449 levels normally found in male offspring, who pass on these traits to their offspring. We previously published that the content of sperm miR-34/449 is also reduced in men raised in highly abusive and/or dysfunctional families. We show here that a similar miRNA auto-amplification system functions in human embryonic stem cells. This raises the possibility that PIEs in offspring of these men also display reduced levels of miR-34/449, enhancing the potential translational significance of these studies.

Transgenerational rescue of a genetic defect in long-term potentiation and memory formation by juvenile enrichment.

The idea that qualities acquired from experience can be transmitted to future offspring has long been considered incompatible with current understanding of genetics. However, the recent documentation of non-Mendelian transgenerational inheritance makes such a "Lamarckian"-like phenomenon more plausible. Here, we demonstrate that exposure of 15-d-old mice to 2 weeks of an enriched environment (EE), that includes exposure to novel objects, elevated social interactions and voluntary exercise, enhances long-term potentiation (LTP) not only in these enriched mice but also in their future offspring through early adolescence, even if the offspring never experience EE. In both generations, LTP induction is augmented by a newly appearing cAMP/p38 MAP kinase-dependent signaling cascade. Strikingly, defective LTP and contextual fear conditioning memory normally associated with ras-grf knock-out mice are both masked in the offspring of enriched mutant parents. The transgenerational transmission of this effect occurs from the enriched mother to her offspring during embryogenesis. If a similar phenomenon occurs in humans, the effectiveness of one's memory during adolescence, particularly in those with defective cell signaling mechanisms that control memory, can be influenced by environmental stimulation experienced by one's mother during her youth.

RASGRF1 in CRF cells controls the early adolescent female response to repeated stress.

RASGRF1 (GRF1) is a calcium-stimulated guanine-nucleotide exchange factor that activates RAS and RAC GTPases. In hippocampus neurons, it mediates the action of NMDA and calcium-permeable AMPA glutamate receptors on specific forms of synaptic plasticity, learning, and memory in both male and female mice. Recently, we showed GRF1 also regulates the HPA axis response to restraint stress, but only in female mice before puberty. In particular, we found that after 7 days of restraint stress (7DRS) (30 min/day) both elevated serum CORT levels and induction of an anxiolytic phenotype normally observed in early adolescent (EA) female mice are blocked in GRF1-knockout mice. In contrast, no effects were observed in EA male or adult females. Here, we show this phenotype is due, at least in part, to GRF1 loss in CRF cells of the paraventricular nucleus of the hypothalamus, as GRF1 knockout specifically in these cells suppressed 7DRS-induced elevation of serum CORT levels specifically in EA females, but only down to levels found in comparably stressed EA males. Nevertheless, it still completely blocked the 7DRS-induced anxiolytic phenotype observed in EA females. Interestingly, loss of GRF1 in CRF cells had no effect after only three restraint stress exposures, implying a role for GRF1 in 7DRS stress-induced plasticity of CRF cells that appears to be specific to EA female mice. Overall, these findings indicate that GRF1 in CRF cells makes a key contribution to the distinct response EA females display to repeated stress.

Involvement of early embryonic miR-409-3p in the establishment of anxiety levels in female mice.

Small RNA molecules in early embryos, delivered from sperm to zygotes upon fertilization, are required for normal mouse embryonic development. Even modest changes in the levels of sperm-derived miRNAs appear to influence early embryos and subsequent development. For example, stress-associated behaviors develop in mice after injection into normal zygotes sets of sperm miRNAs elevated in stressed male mice. Here, we implicate early embryonic miR-409-3p in establishing anxiety levels in adult female, but not male mice. First, we found that exposure of male mice to chronic social instability stress, which leads to elevated anxiety in their female offspring across at least three generations through the paternal lineage, elevates sperm miR-409-3p levels not only in exposed males, but also in sperm of their F1 and F2 male offspring. Second, we observed that while injection of a mimic of miR-409-3p into zygotes from mating control males was incapable of mimicking this effect in offspring derived from them, injection of a specific inhibitor of this miRNA led to the opposite, anxiolytic effect in female, but not male, and offspring. These findings imply that baseline miR-409-3p activity in early female embryos is necessary for the expression of normal anxiety levels when they develop into adult females. In addition, elevated embryo miR-409-3p activity, possibly as a consequence of stress-induced elevation of its expression in sperm, may participate in, but may not be sufficient for, the induction of enhanced anxiety.

The environment versus genetics in controlling the contribution of MAP kinases to synaptic plasticity.

BACKGROUND: A challenge in biomedical research is to design experimental paradigms that reflect a natural setting. Even when freshly isolated tissues are used, they are almost always derived from animals housed in cages that poorly reflect the animal's native environment. This issue is highlighted by studies on brain function, where mice housed in a more natural "enriched environment" display enhanced learning and memory and delayed onset of symptoms of neurodegenerative diseases compared to mice housed conventionally. How the environment mediates its effects on brain function is poorly understood. RESULTS: We show that after exposure of adolescent mice to an "enriched environment," the induction of long-term potentiation (LTP), a form of synaptic plasticity that is thought to contribute to learning and memory, involves a novel signal transduction pathway that is nonfunctional in comparable mice housed conventionally. This environmentally gated signaling pathway, which rescues defective LTP induction in adolescent Ras-GRF knockout mice, consists of NMDA glutamate receptor activation of p38, a MAP kinase that does not contribute to LTP in mice housed conventionally. Interestingly, the same exposure to environmental enrichment does not have this effect in adult mice. CONCLUSIONS: This study reveals a new level of cell signaling control whereby environmental factors gate the efficacy of a specific signaling cascade to control how LTP is induced in adolescent animals. The suppression of this gating mechanism in mature animals represents a new form of age-dependent decline in brain plasticity.

Ral-GTPases: approaching their 15 minutes of fame.

Andy Warhol, the famous pop artist, once claimed that "in the future everyone will be famous for 15 minutes". The same, it seems, can be said of proteins, because at any given time some proteins become more "fashionable" to study than others. But most proteins have been highly conserved throughout millions of years of evolution, which implies that they all have essential roles in cell biology. Thus, each one will no doubt enter the limelight if the right experiment in the right cell type is done. A good example of this is the Ras-like GTPases (Ral-GTPases), which until recently existed in the shadow of their close cousins--the Ras proto-oncogenes. Recent studies have yielded insights into previously unappreciated roles for Ral-GTPases in intensively investigated disciplines such as vesicle trafficking, cell morphology, transcription and possibly even human oncogenesis.

The R-Ras GTPase mediates cross talk between estrogen and insulin signaling in breast cancer cells.

The signaling cascades activated by insulin and IGF-1 contribute to the control of multiple cellular functions, including glucose metabolism and cell proliferation. In most cases these effects are mediated, at least in part, by insulin receptor substrates (IRS), one of which is insulin receptor substrate 1 (IRS-1). R-Ras is a member of the Ras family of GTPases and is involved in a variety of biological processes, including integrin activation, cell migration, and control of cell proliferation. Here we demonstrate that both R-Ras and BCAR3, a regulator of R-Ras activity that has been implicated in breast cancer, regulate the level of IRS-1 protein in estrogen-dependent MCF-7 and ZR75 breast cancer cells. In particular, expression of a constitutively activated R-Ras mutant, R-Ras38V, or of BCAR3 accelerates the degradation of IRS-1, leading to the impairment of signaling through insulin but not epidermal growth factor receptors. Moreover, knockdown of endogenous R-Ras levels in MCF-7 cells inhibits IRS-1 degradation induced by estrogen signaling blockade but not by long-term insulin treatment. Consistent with these results, both R-Ras38V expression and estrogen signaling blockade lead to the degradation of IRS-1, at least in part, through calpain activity. These findings show that R-Ras activity mediates inhibition of insulin signaling associated with suppression of estrogen action, implicating this GTPase in a growth-inhibitory mechanism associated with antiestrogen treatment of breast cancer.

Tiam1-IRSp53 complex formation directs specificity of rac-mediated actin cytoskeleton regulation.

The exchange factor Tiam1 regulates multiple cellular functions by activating the Rac GTPase. Active Rac has various effects in cells, including alteration of actin cytoskeleton and gene expression, via binding to and modulating the activity of diverse effector proteins. How individual Rac effectors are selected for activation and regulated in response to upstream signals is not well understood. We find that Tiam1 contributes to both of these processes by binding to IRSp53, an adaptor protein that is an effector for both Rac and Cdc42. Tiam1 directs IRSp53 to Rac signaling by enhancing IRSp53 binding to both active Rac and the WAVE2 scaffold. Moreover, Tiam1 promotes IRSp53 localization to Rac-induced lamellipodia rather than Cdc42-induced filopodia. Finally, IRSp53 depletion from cells prevents Tiam1-dependent lamellipodia induced by Tiam1 overexpression or platelet-derived growth factor stimulation. These findings indicate that Tiam1 not only activates Rac but also contributes to Rac signaling specificity through binding to IRSp53.

Reduced levels of miRNAs 449 and 34 in sperm of mice and men exposed to early life stress.

Exposure of male mice to early life stress alters the levels of specific sperm miRNAs that promote stress-associated behaviors in their offspring. To begin to evaluate whether similar phenomena occur in men, we searched for sperm miRNA changes that occur in both mice and men exposed to early life stressors that have long-lasting effects. For men, we used the Adverse Childhood Experience (ACE) questionnaire. It reveals the degree of abusive and/or dysfunctional family experiences when young, which increases risks of developing future psychological and physical disorders. For male mice, we used adolescent chronic social instability (CSI) stress, which not only enhances sociability defects for >1 year, but also anxiety and defective sociability in female offspring for multiple generations through the male lineage. Here we found a statistically significant inverse correlation between levels of multiple miRNAs of the miR-449/34 family and ACE scores of Caucasian males. Remarkably, we found members of the same sperm miRNA family are also reduced in mice exposed to CSI stress. Thus, future studies should be designed to directly test whether reduced levels of these miRNAs could be used as unbiased indicators of current and/or early life exposure to severe stress. Moreover, after mating stressed male mice, these sperm miRNA reductions persist in both early embryos through at least the morula stage and in sperm of males derived from them, suggesting these miRNA changes contribute to transmission of stress phenotypes across generations. Since offspring of men exposed to early life trauma have elevated risks for psychological disorders, these findings raise the possibility that a portion of this risk may be derived from epigenetic regulation of these sperm miRNAs.

Distinct roles for Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) and Ras-GRF2 in the induction of long-term potentiation and long-term depression.

NMDA-type glutamate receptors (NMDARs) contribute to many forms of long-term potentiation (LTP) and long-term depression (LTD). NMDARs are heteromers containing calcium-permeating neuronal receptor 1 (NR1) subunits and a variety of NR2 subunits. Evidence suggests that, in the CA1 region of the hippocampus, NR2A-containing NMDARs promote LTP whereas NR2B-containing receptors promote LTD. However, the calcium sensors that distinguish between these signals to promote the appropriate form of synaptic plasticity are not known. Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) and Ras-GRF2 are highly similar calcium-stimulated exchange factors that activate Ras and Rac GTPases. Here, using a set of Ras-GRF knock-out mice, we show that Ras-GRF2 contributes predominantly to the induction of NMDAR-dependent LTP, whereas Ras-GRF1 contributes predominantly to the induction of NMDAR-dependent LTD in the CA1 region of the hippocampus of postpubescent mice (postnatal days 25-36). In contrast, neither Ras-GRF protein influences synaptic plasticity in prepubescent mice (postnatal days 14-18). Ras-GRF2 mediates signaling from (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl-phosphonic acid-sensitive (NVP-AAM077-sensitive) (NR2A-containing) NMDARs to the Ras effector extracellular signal-related protein kinase 1/2 (Erk1/2) mitogen-activated protein (MAP) kinase, a promoter of NMDAR-induced LTP at this site. In contrast, Ras-GRF1 mediates signaling from ifenprodil-sensitive (NR2B-containing) NMDARs to the Rac effector p38 MAP kinase, a promoter of LTD. These findings show that, despite their similar functional domain organization, Ras-GRF1 and Ras-GRF2 mediate opposing forms of synaptic plasticity by coupling different classes of NMDARs to distinct MAP kinase pathways. Moreover, the postnatal appearance of Ras-GRF-dependent LTP and LTD coincides with the emergence of hippocampal-dependent behavior, implying that Ras-GRF proteins contribute to forms of synaptic plasticity that are required specifically for mature hippocampal function.

Cell signaling: life or death decisions of ras proteins.

The discovery of a new downstream target for the Ras GTPases - a Nore1-Mst1 protein complex - reveals a mechanism by which Ras can promote apoptosis, and suggests that the influence of Ras on cell survival or death depends upon the relative level of activation of its various target proteins.

RalA but not RalB enhances polarized delivery of membrane proteins to the basolateral surface of epithelial cells.

RalA and RalB constitute a family of highly similar (85% identity) Ras-related GTPases. Recently, active forms of both RalA and RalB have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. However, we show here that only active RalA enhances the rate of delivery of E-cadherin and other proteins to their site in the basolateral membrane of MDCK cells, consistent with RalA being a regulator of exocyst function. One reason for this difference is that RalA binds more effectively to the exocyst complex than active RalB does both in vivo and in vitro. Another reason is that active RalA localizes to perinuclear recycling endosomes, where regulation of vesicle sorting is thought to take place, while active RalB does not. Strikingly, analysis of chimeras made between RalA and RalB reveals that high-affinity exocyst binding by RalA is due to unique amino acid sequences in RalA that are distal to the common effector-binding domains shared by RalA and RalB. Moreover, these chimeras show that the perinuclear localization of active RalA is due in part to its unique variable domain near the C terminus. This distinct localization appears to be important for RalA effects on secretion because all RalA mutants tested that failed to localize to the perinuclear region also failed to promote basolateral delivery of E-cadherin. Interestingly, one of these inactive mutants maintained binding to the exocyst complex, suggesting that RalA binding to the exocyst is necessary but not sufficient for RalA to promote basolateral delivery of membrane proteins.