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Differential response of the Ras exchange factor, Ras-GRF to tyrosine kinase and G protein mediated signals
1Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Abstract:
Ras-GRF, a guanine-nucleotide exchange factor that activates Ras p21, was tested for its ability to couple to either tyrosine kinase or heterotrimeric G protein signal transduction pathways. Ras-GRF failed to bind the SH2 and SH3 containing adaptor protein Grb2, either in vitro or in vivo. Furthermore, Ras-GRF did not form a stable complex with activated EGF receptor. However, as has been shown previously (Cen et al., 1994), the presence of Ras-GRF in NIH3T3 cells enhanced the activation of Ras induced by serum stimulation. A similar effect was not observed with PDGF stimulation. Moreover, serum stimulation lead to the hyperphosphorylation of Ras-GRF. Both the serum induced super-activation of Ras, and the hyperphosphorylation of Ras-GRF were blocked by pretreatment of cells with the Gi,o inhibitor pertussis toxin, but not by pretreatment with the tyrosine kinase inhibitor genistein. These results suggest that Ras-GRF has the capacity to mediate Ras activation initiated by signals using heterotrimeric G proteins.
Insights
Ras-GRF, a guanine-nucleotide exchange factor, activates Ras p21. It couples to G protein pathways, not tyrosine kinases, mediating Ras activation via serum stimulation.
Area of Science:
- Cellular signaling
- Molecular biology
- Signal transduction
Background:
- Ras-GRF is a guanine-nucleotide exchange factor known to activate Ras p21.
- Understanding the specific signaling pathways Ras-GRF interacts with is crucial for deciphering cellular communication.
Purpose of the Study:
- To investigate whether Ras-GRF couples to tyrosine kinase or heterotrimeric G protein signal transduction pathways.
- To elucidate the mechanism of Ras activation mediated by Ras-GRF.
Main Methods:
- In vitro and in vivo binding assays with Grb2 and EGF receptor.
- Ras activation assays in NIH3T3 cells stimulated with serum or PDGF.
- Pertussis toxin and genistein pretreatment to block Gi,o and tyrosine kinase pathways, respectively.
- Analysis of Ras-GRF phosphorylation status following serum stimulation.
Main Results:
- Ras-GRF did not bind Grb2 or form a stable complex with activated EGF receptor, indicating no direct coupling to tyrosine kinase pathways.
- Ras-GRF enhanced Ras activation induced by serum stimulation but not PDGF.
- Serum stimulation led to hyperphosphorylation of Ras-GRF.
- Both serum-induced Ras super-activation and Ras-GRF hyperphosphorylation were blocked by pertussis toxin but not genistein.
Conclusions:
- Ras-GRF mediates Ras activation initiated by signals utilizing heterotrimeric G proteins.
- Ras-GRF's interaction with tyrosine kinase pathways is indirect or absent.
- The findings highlight Ras-GRF's role in G protein-coupled signaling cascades.