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The brain-specific Ras guanine nucleotide exchange factor RasGRF1 is a protein harbouring a complex array of structural motifs. It contains a pleckstrin homology (PH1) domain, a coiled coil region (CC) and an ilimaquinone (IQ) one in addition to the catalytic Ras and Rac exchange factor domains. In this study, we used the recombinant N-terminal PH1, CC and IQ region (PHCCIQ) fused to the chitin-binding domain to isolate interacting proteins from mouse brain extracts. The use of an advanced software tool, the Pep-Miner, allowed clustering similar spectra from multiple mass spectrometry analysis, simplifying and improving the analysis of the complex peptide mixture. The most representative classes of RasGRF1-interacting proteins were ribosomal and other RNA-binding proteins, cytoskeletal proteins and proteins involved in vesicular trafficking. We confirmed the interaction of some of the identified proteins using different experimental approaches. We also demonstrated an RNA-dependent association of the PHCCIQ moiety of RasGRF1 with ribosomal protein S6 and Ras-GTPase-activating protein SH3-domain binding protein 2. In addition, we found that purified total RNA binds to the PHCCIQ fusion protein and the recombinant protein associates with poly(A)-sepharose. These data indicate that RasGRF1 can interact with different protein categories and exhibits a potential RNA-binding property. © 2008 Humana Press.

Ras-GRF1 is a neuron-specific guanine nucleotide exchange factor for Ras proteins. Mice lacking Ras-GRF1 (-/-) are severely impaired in amygdala-dependent long-term synaptic plasticity and show higher basal synaptic activity at both amygdala and hippocampal synapses (Brambilla et al.,, 1997). In the present study we investigated the effects of Ras-GRF1 deletion on hippocampal neuronal excitability. Electrophysiological analysis of both primary cultured neurons and adult hippocampal slices indicated that Ras-GRF1-/- mice displayed neuronal hyperexcitability. Ras-GRF1-/- hippocampal neurons showed increased spontaneous activity and depolarized resting membrane potential, together with a higher firing rate in response to injected current. Changes in the intrinsic excitability of Ras-GRF1-/- neurons can entail these phenomena, suggesting that Ras-GRF1 deficiency might alter the balance between ionic conductances. In addition, we showed that mice lacking Ras-GRF1 displayed a higher seizure susceptibility following acute administration of convulsant drugs. Taken together, these results demonstrated a role for Ras-GRF1 in neuronal excitability.

The Ras-GRF1 exchange factor molecule contains in addition to the catalytic domain two pleckstrin homology (PH1 and PH2), one IQ and one Dbl homology (DH) domains. In this study we investigated the role of such additional domains. We found that a Ras-GRF1 mutant lacking PH1 and IQ domains is sufficient to activate c-fos promoter in response to lysophosphatidic acid (LPA). The same mutant did not increase external stimuli-regulated kinase (ERK) activity, suggesting an additional mechanism for the induction of gene transcription. Isolated DH-PH2 module activates c-Jun NH2-terminal kinase and the c-fos promoter in response to LPA, providing the basis for an ERK-independent mechanism. These results provide evidence that Ras-GRF1 acts as a bifunctional molecule on both ERK-dependent and independent pathways.