Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 23 (7), 1567-75

Developmentally Regulated Role for Ras-GRFs in Coupling NMDA Glutamate Receptors to Ras, Erk and CREB

Affiliations

Developmentally Regulated Role for Ras-GRFs in Coupling NMDA Glutamate Receptors to Ras, Erk and CREB

Xuejun Tian et al. EMBO J.

Abstract

p140 Ras-GRF1 and p130 Ras-GRF2 constitute a family of calcium/calmodulin-regulated guanine-nucleotide exchange factors that activate the Ras GTPases. Studies on mice lacking these exchange factors revealed that both p140 Ras-GRF1 and p130 Ras-GRF2 couple NMDA glutamate receptors (NMDARs) to the activation of the Ras/Erk signaling cascade and to the maintenance of CREB transcription factor activity in cortical neurons of adult mice. Consistent with this function for Ras-GRFs and the known neuroprotective effect of CREB activity, ischemia-induced CREB activation is reduced in the brains of adult Ras-GRF knockout mice and neuronal damage is enhanced. Interestingly, in cortical neurons of neonatal animals NMDARs signal through Sos rather than Ras-GRF exchange factors, implying that Ras-GRFs endow NMDARs with functions unique to mature neurons.

Figures

Figure 1
Figure 1
Ras-GRF and Sos expression in wild-type and knockout mice. (A) In all, 10 μg of cell lysates of brain from wild-type, double Ras-GRF knockout (DBKO), single Ras-GRF1 knockout (GRF1(−/−)) or single Ras-GRF2 knockout (GRF2(−/−)) adult mice were probed for Ras-GRF expression by immunoblotting with an antibody that preferentially recognizes Ras-GRF1(top panel) or Ras-GRF2 (middle panel). Double RasGRF knockout mice were generated by crossing Ras-GRF1(−/−) mice with Ras-GRF2 (−/−) mice until double knockout mice were obtained. (B) The expression levels of Ras-GRFs in primary cultures of cortical neurons isolated from newborn mice and cultured for 7 days in vitro, cortical brain slices from newborn (day 0), day 10, day 20 and day 30–40 (adult) mice are shown by immunoblotting 10 μg of cell lysates with antibodies that preferentially recognize Ras-GRF1. (C) The expression levels of Sos proteins in samples similar to those described in (B) using antibodies that recognize both Sos1 and Sos2 proteins, although lysates were from cortical brain slices from adult Ras-GRF double knockout mice, are also shown to indicate that loss of Ras-GRF proteins does not affect their expression.
Figure 2
Figure 2
NMDA-induced Erk, Ras and CREB activation in cortical brain slices isolated from wild-type and Ras-GRF knockout mice. (A) Erk activation. Cortical brain slices from adult wild-type and single and double Ras-GRF knockout mice were treated with NMDA (100 μM) for various amounts of time. The slices were then lysed in detergent and immunoblots of lysates were performed using activation-specific phospho-Erk antibodies or antibodies against total Erk 1 and 2. (B) Ras activation. Lysates from 10 min time points described above in (A) were exposed to sepharose beads containing the Ras-binding domain of the Ras target Raf to affinity purify active Ras-GTP. The affinity-purified form of Ras and total Ras in the brain slice in lysates was assayed by immunoblotting with anti-Ras antibodies. The doublet band of Ras-GTP is routinely found in these preparations, presumably due to removal of the C-terminus, which is sensitive to proteolytic cleavage during incubation with beads (unpublished observations). (C) CREB activation. Cortical brain slices were stimulated with either NMDA as described above, or BDNF (100 ng/ml). Lysates immunoblotted with phospho-CREB (ser133) specific antibodies or with total CREB antibodies. (D) BDNF stimulation of Erk. Experiments were performed as in (A), except that BDNF (100 ng/ml) was used to stimulate the brain slices. The results for each section are representative of at least three independent experiments.
Figure 3
Figure 3
Comparison of neuronal damage and CREB activation in wild-type and Ras-GRF double knockout mice after stroke-induced ischemia. (A) Ischemic lesion volumes were measured 24 h after transient focal cerebral ischemia was produced, by occluding the right internal carotid artery of wild-type (wt) and Ras-GRF double knockout mice (grf1/grf2(−/−)). Data represent the average of eight mice for each group±s.d., P<0.01. (B) Cross sections of a representative experiment used to generate data in (A), with an area of ischemia marked with an arrow. (C) CREB activation in the control half or occluded half of the brain was measured 30 min after the initiation of ischemia in either wild-type or double Ras-GRF knockout mice. The data are the average of three experiments±s.d.
Figure 4
Figure 4
NMDA-induced Erk and CREB activation in neurons from neonatal animals. (A) Primary cultures of cortical neurons isolated from newborn mice of either wild-type or Ras-GRF double knockout mice were cultured in vitro for 7 days and then stimulated with NMDA (100 μM) for various amounts of time. Cell lysates were then assayed for Erk activation as described previously. Some neurons were exposed to the NMDAR inhibitor APV, to show that the effect of NMDA was specific. (B) Wild-type and Ras-GRF double knockout neurons were stimulated as described in (A) and then assayed for CREB activation as described previously. (C) Cortical brain slices were prepared from newborn mice as described previously for brain slices from adult animals. The samples were then stimulated and assayed as described in Figure 2. (D) Cortical brain slices from day 10 or day 20 wild-type or Ras-GRF double knockout mice were prepared and stimulated and assayed as described in Figure 2.
Figure 5
Figure 5
Sos protein involvement in NMDA-induced Erk activation in neurons from neonatal mice. (A) Shc/Grb2 complex formation. Cortical brain slices from neonatal or adult mice were treated with buffer (−) or NMDA (100 μm) (+) for 5 min and then lysates were immunoprecipitated with anti-Shc antibodies and then immunoblotted with anti-Grb2 antibodies. Total Shc in cell lysates in samples was also assessed. The data are representative of two independent experiments. (B, C) PP1 inhibition. Buffer or various concentrations of the Src family inhibitor PP1 were preincubated for 30 min with brain slices from either neonatal mice (B) or adult mice (C). The samples were then stimulated with NMDA and processed as described previously for Erk (top panels) or CREB (bottom panels) activation. The data are representative of at least two independent experiments.

Similar articles

See all similar articles

Cited by 53 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Feedback