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Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras
1Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Abstract:
Conversion of Ras proteins into an activated GTP-bound state able to bind effector proteins is catalyzed by specific guanine nucleotide exchange factors in response to a large number of extracellular stimuli. Here we report the isolation of mouse cDNAs encoding Ras-GRF2, a multidomain 135-kDa protein containing a COOH-terminal Cdc25-related domain that stimulates release of GDP from Ras but not other GTPases in vitro. Ras-GRF2 bound specifically to immobilized Ras lacking bound nucleotides, suggesting stabilization of the nucleotide-free form of Ras as a mechanism of catalyzing nucleotide exchange. The NH2-terminal region of Ras-GRF2 is predicted to contain features common to various signaling proteins including two pleckstrin homology domains and a Dbl homology region. Ras-GRF2 also contains an IQ motif which was required for its apparent constitutive association with calmodulin in epithelial cells ectopically expressing Ras-GRF2. Transient expression of Ras-GRF2 in kidney epithelial cells stimulated GTP binding by Ras and potentiated calcium ionophore-induced activation of mitogen-activated protein kinase (ERK1) dependent upon the IQ motif. Calcium influx caused Ras-GRF2 subcellular localization to change from cytosolic to peripheral, suggesting a possible mechanism for controlling Ras-GRF2 interactions with Ras at the plasma membrane. Epithelial cells overexpressing Ras-GRF2 are morphologically transformed and grow in a disorganized manner with minimal intercellular contacts. Northern analysis indicated a 9-kb GRF2 transcript in brain and lung, where p135 Ras-GRF2 is known to be expressed, and RNAs of 12 kb and 2.2 kb were detected in several tissues. Thus, Ras-GRF2 proteins with different domain structures may be widely expressed and couple diverse extracellular signals to Ras activation.
Insights
Researchers identified Ras-guanine nucleotide exchange factor 2 (Ras-GRF2), a protein that activates Ras proteins. This discovery sheds light on how extracellular signals are coupled to Ras activation and cellular responses.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Ras proteins are key regulators of cellular signaling pathways.
- Guanine nucleotide exchange factors (GEFs) activate Ras proteins by catalyzing GDP/GTP exchange.
- Understanding novel GEFs is crucial for deciphering complex signaling networks.
Purpose of the Study:
- To isolate and characterize a novel Ras-GEF, designated Ras-GRF2.
- To elucidate the mechanism by which Ras-GRF2 activates Ras proteins.
- To investigate the role of Ras-GRF2 in cellular signaling and transformation.
Main Methods:
- Isolation of mouse cDNAs encoding Ras-GRF2.
- In vitro assays to assess GDP release from Ras proteins.
- Binding studies using immobilized Ras.
- Ectopic expression in kidney epithelial cells to study protein localization and function.
- Analysis of downstream signaling pathways, including ERK1 activation.
- Northern blot analysis to determine transcript distribution.
Main Results:
- Ras-GRF2, a 135-kDa protein, stimulates GDP release from Ras.
- Ras-GRF2 binds to nucleotide-free Ras, stabilizing this form.
- The NH2-terminal region contains pleckstrin homology and Dbl homology domains.
- An IQ motif mediates calmodulin association and is essential for Ras-GRF2 function.
- Ras-GRF2 activates Ras, potentiates ERK1 activation, and alters subcellular localization upon calcium influx.
- Overexpression of Ras-GRF2 leads to cellular transformation and disorganized growth.
- Diverse Ras-GRF2 transcripts are detected in various tissues.
Conclusions:
- Ras-GRF2 is a novel GEF that activates Ras proteins through stabilization of the nucleotide-free state.
- Ras-GRF2 couples extracellular signals, such as calcium influx, to Ras activation.
- The IQ motif and calmodulin interaction are important for Ras-GRF2 activity and localization.
- Ras-GRF2 plays a role in cellular transformation and may be widely expressed, suggesting diverse roles in signaling.