Domain Contributions to Signaling Specificity Differences Between Ras-Guanine Nucleotide Releasing Factor (Ras-GRF) 1 and Ras-GRF2

Abstract

Background: Ras-GRF1 and Ras-GRF2 are similar exchange factors with different functions in synaptic plasticity. Results: Chimeras between Ras-GRF proteins reveal that IQ, pleckstrin homology, coiled-coil, and CDC25 domains are most important for signaling specificity. Conclusion: Signaling specificity of GRF proteins is encoded in a surprisingly small number of their common domains. Significance: Domains of Ras-GRF proteins have been identified for future studies on signaling specificity.

R-Ras contributes to LTP and contextual discrimination

“…(PH- pleckstrin homology, CC- coiled-coil, IQ- calmodulin binding domain, DH- Dbl homology, REM- Ras exchange motif, CDC25- Ras and R-Ras activating domain) D. HFS-LTP in brain slices of DBKO mice is reconstituted in the CA1 with (PCQ) 1 GRF2. (DBKO, 111.63 ± 2.23%; WT-GRF1, 165 ± 9.4%; (PCQ) 1 GRF2, 150 ± 10.7%, Two-Way ANOVA, Virus effect, F 2,944 = 6.06, p = 0.011; Bonferroni post-hoc , WT-GRF1 vs. (PCQ) 1 GRF2, p > 0.05 at all time points, also see data as reported in (Jin et al, 2014, Fig. 3A) E. Hippocampal slices from mice previously injected with virus expressing R-Ras miRNA1 or 2 into the CA1 area of RasGRF1 KO mice were stimulated with HFS-LTP and processed for immunostaining of phosphorylated p38.…”

RasGRF1 regulates the hypothalamic–pituitary–adrenal axis specifically in early-adolescent female mice

“…Since the hippocampus is one of the key regulators of this mechanism and our lab showed previously that GRF1 regulates synaptic plasticity there, we focused first on the hippocampus. We previously published (Jin et al, 2014) that we can reconstitute all of the known synaptic plasticity functions of GRF1 in GRF1(−/−) mice by stereotactic injection of adenoviruses expressing GRF1 into the CA1 region of the hippocampus. We also described a GRF1 mutant, (PCQ 2 ) GRF1, that expresses at the same level as WT GRF1 but has none of the synaptic plasticity functions of GRF1 (see Fig 1 and Table 1 from (Jin et al, 2014)).…”

“…We previously published (Jin et al, 2014) that we can reconstitute all of the known synaptic plasticity functions of GRF1 in GRF1(−/−) mice by stereotactic injection of adenoviruses expressing GRF1 into the CA1 region of the hippocampus. We also described a GRF1 mutant, (PCQ 2 ) GRF1, that expresses at the same level as WT GRF1 but has none of the synaptic plasticity functions of GRF1 (see Fig 1 and Table 1 from (Jin et al, 2014)). Thus, we infected the ventral CA1 of GRF1(−/−) mice with virus expressing either WT GRF1 (GRF1-AV) or inactive GRF1 (PCQ 2 )GRF1 and waited for 7 days, a period we showed previously allowed both forms of GRF1 to restore GRF1 expression to levels found in WT mice ((Jin et al, 2014).…”

“…We also described a GRF1 mutant, (PCQ 2 ) GRF1, that expresses at the same level as WT GRF1 but has none of the synaptic plasticity functions of GRF1 (see Fig 1 and Table 1 from (Jin et al, 2014)). Thus, we infected the ventral CA1 of GRF1(−/−) mice with virus expressing either WT GRF1 (GRF1-AV) or inactive GRF1 (PCQ 2 )GRF1 and waited for 7 days, a period we showed previously allowed both forms of GRF1 to restore GRF1 expression to levels found in WT mice ((Jin et al, 2014). After expression, co-staining of GRF with either the neuronal marker, NeuN, or glia marker, GFAP, showed that GRF proteins were found overwhelmingly in neurons, not glia, consistent with the neuron-specific synapsin promoter used in these experiments and the known localization of endogenous GRF proteins (Zippel et al, 1997) (data not shown).…”

“…Injections were performed with a 10 µl Hamilton syringe fitted with a custom made blunt ended 30-gauge needle (Hamilton). Each injection consisted of 1 µl of Adenovirus expressing various GRF proteins (see (Jin et al, 2013; Jin et al, 2014) for description of viruses used and how they were generated) infused at a rate of 0.06 µl/min. An infusion pump controlling the plunger on the Hamilton syringe precisely regulated the rate of injection.…”