The Ras-GRF1 exchange factor, which is regulated by increases in intracellular calcium and the release of G beta gamma subunits from heterotrimeric G proteins, plays a critical role in the activation of neuronal Ras. Activation of G protein-coupled receptors stimulates an increase in the phosphorylation of Ras-GRF1 at certain serine residues. The first of these sites to be identified, Ser(916) in the mouse sequence (equivalent to Ser(898) in the rat sequence), is required for full activation of the Ras exchange factor activity of Ras-GRF1 by muscarinic receptors. We demonstrate here that Ras-GRF1 is highly expressed in rat brain compared with the Sos exchange factor and that there is an increase in incorporation of (32)P into Ser(898) of brain Ras-GRF1 following activation of protein kinase A. Phosphorylation of Ras-GRF1 at Ser(916) is also required for maximal induction of Ras-dependent neurite outgrowth in PC12 cells. A novel antibody (termed 2152) that selectively recognizes Ras-GRF1 when it is phosphorylated at Ser(916/898) confirmed the regulated phosphorylation of Ras-GRF1 by Western blotting in both model systems of transfected COS-7 and PC12 cells and also of the endogenous protein in rat forebrain slices. Indirect confocal immunofluorescence of transfected PC12 cells using antibody 2152 demonstrated reactivity only under conditions in which Ras-GRF1 was phosphorylated at Ser(916/898). Confocal immunofluorescence of cortical slices of rat brain revealed widespread and selective phosphorylation of Ras-GRF1 at Ser(898). In the prefrontal cortex, there was striking phosphorylation of Ras-GRF1 in the dendritic tree, supporting a role for Ras activation and signal transduction in neurotransmission in this area.
"Our results link the activation of GRF1 by alcohol to the cAMP/PKA pathway. Specifically, alcohol exposure resulted in increased phosphorylation of GRF1 on a PKA site (serine 916), a post-translational modification that enhances the intrinsic GDP to GTP exchange activity of GRF1 (Mattingly, 1999; Yang et al., 2003). Although the increase in GRF1 activity can influence the function of other members of the p21Ras family, several studies suggest that GRF1 is the specific exchange factor for H-Ras but not N-Ras or K-Ras (Fernandez- Medarde and Santos, 2011). "
[Show abstract][Hide abstract] ABSTRACT: Uncontrolled consumption of alcohol is a hallmark of alcohol abuse disorders; however, the central molecular mechanisms underlying excessive alcohol consumption are still unclear. Here, we report that the GTP binding protein, H-Ras in the nucleus accumbens (NAc) plays a key role in neuroadaptations that underlie excessive alcohol-drinking behaviors. Specifically, acute (15 min) systemic administration of alcohol (2.5 g/kg) leads to the activation of H-Ras in the NAc of mice, which is observed even 24 h later. Similarly, rat operant self-administration of alcohol (20%) also results in the activation of H-Ras in the NAc. Using the same procedures, we provide evidence suggesting that the exchange factor GRF1 is upstream of H-Ras activation by alcohol. Importantly, we show that infection of mice NAc with lentivirus expressing a short hairpin RNA that targets the H-Ras gene produces a significant reduction of voluntary consumption of 20% alcohol. In contrast, knockdown of H-Ras in the NAc of mice did not alter water, quinine, and saccharin intake. Furthermore, using two-bottle choice and operant self-administration procedures, we show that inhibiting H-Ras activity by intra-NAc infusion of the farnesyltransferase inhibitor, FTI-276, produced a robust decrease of rats' alcohol drinking; however, sucrose consumption was unaltered. Finally, intra-NAc infusion of FTI-276 also resulted in an attenuation of seeking for alcohol. Together, these results position H-Ras as a central molecular mediator of alcohol's actions within the mesolimbic system and put forward the potential value of the enzyme as a novel target to treat alcohol use disorders.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 11/2012; 32(45):15849-58. DOI:10.1523/JNEUROSCI.2846-12.2012 · 6.34 Impact Factor
"Rasgrf1 is most highly expressed in central nervous system neurons (Sturani et al. 1997; Zippel et al. 2000), with lower expression levels in other somatic tissues (Font de Mora et al. 2003; Plass et al. 1996). Its product, RasGRF1 protein, is a guanine nucleotide exchange factor for Ras and Rac (Cen et al. 1993; Innocenti et al. 1999), activating these G-proteins in response to cellular calcium influx (Farnsworth et al. 1995) or serine phosphorylation (Mattingly et al. 1999; Yang et al. 2003) in pathways downstream of muscarinic receptor activity (Mattingly & Macara 1996), heterotrimeric G-protein subunit dissociation (Kiyono et al. 2000; Shou et al. 1995), and neurotrophin binding to TrkA, TrkB, and TrkC receptors (MacDonald et al. 1999; Robinson et al. 2005). "
[Show abstract][Hide abstract] ABSTRACT: Rasgrf1 is genomically imprinted; only the paternally inherited allele is expressed in the neonatal mouse brain until weaning, at which time expression becomes biallelic. Whereas Rasgrf1 has been implicated in learning and memory via knockout studies in adult mice, the effect of its normal imprinted expression on these phenotypes has not yet been examined. Neonatal mice with experimentally manipulated patterns of imprinted Rasgrf1 expression were assessed on an associative olfactory task. Neonates lacking the normally expressed wild-type paternal allele exhibited significant impairment in olfactory associative memory. Adult animals in which neonatal imprinting had been manipulated were also behaviorally assessed; while neonatal imprinting significantly affects body weight even into adulthood, no learning and memory phenotype attributable to imprinting was observed in adults. Additional analyses of neonates showed imprinted Rasgrf1 transcript selective to olfactory bulb even in mice that were null for Rasgrf1 in the rest of the brain and showed that Rasgrf1 affects Ras and Rac activation in the brain. Taken together, these results indicate that Rasgrf1 expression from the wild-type paternal allele contributes to learning and memory in neonatal mice.
"Recent studies have documented the functional contribution of Ras signaling pathways to various neural processes , including control of synaptic plasticity and connectivity (Arendt et al., 2004; Seeger et al., 2004), management of neuronal excitability (Tonini et al., 2001) or neuronal polarity and building of neuronal cytoarchitecture (Arendt et al., 2004; Yoshimura et al., 2006). In the same neuronal context, it is known that Ras-GRF1 may become phosphorylated in response to Ca 2ϩ , PKA, LPA/heterotrimeric G proteins and NGF stimulation (Mattingly and Macara, 1996; Mattingly et al., 1999; Yang et al., 2003; Norum et al., 2005; Robinson et al., 2005; Kesavapany et al., 2006), thus driving subsequent Ras activation. Ras- GRF1 is also known to couple NMDA glutamate receptors to Rac–p38, Ras–ERK and Ras–CREB pathways (Krapivinsky et al., 2003; Tian et al., 2004; Li et al., 2006a). "
[Show abstract][Hide abstract] ABSTRACT: We used manual macrodissection or laser capture microdissection (LCM) to isolate tissue sections of the hippocampus area of Ras-GRF1 wild type and knockout mice brains, and analyzed their transcriptional patterns using commercial oligonucleotide microarrays. Comparison between the transcriptomes of macrodissected and microdissected samples showed that the LCM samples allowed detection of significantly higher numbers of differentially expressed genes, with higher statistical rates of significance. These results validate LCM as a reliable technique for in vivo genomic studies in the brain hippocampus, where contamination by surrounding areas (not expressing Ras-GRF1) increases background noise and impairs identification of differentially expressed genes. Comparison between wild type and knockout LCM hippocampus samples revealed that Ras-GRF1 elimination caused significant gene expression changes, mostly affecting signal transduction and related neural processes. The list of 36 most differentially expressed genes included loci concerned mainly with Ras/G protein signaling and cytoskeletal organization (i.e. 14-3-3gamma/zeta, Kcnj6, Clasp2) or related, cross-talking pathways (i.e. jag2, decorin, strap). Consistent with the phenotypes shown by Ras-GRF1 knockout mice, many of these differentially expressed genes play functional roles in processes such as sensory development and function (i.e. Sptlc1, antiquitin, jag2) and/or neurological development/neurodegeneration processes affecting memory and learning. Indeed, potential links to neurodegenerative diseases such as Alzheimer disease (AD) or Creutzfeldt-Jacobs disease (CJD), have been reported for a number of differentially expressed genes identified in this study (Ptma, Aebp2, Clasp2, Hebp1, 14-3-3gamma/zeta, Csnk1delta, etc.). These data, together with the previously described role of IRS and insulin (known Ras-GRF1 activators) in AD, warrant further investigation of a potential functional link of Ras-GRF1 to neurodegenerative processes.
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