RasGRP, a Ras Guanyl Nucleotide- Releasing Protein with Calcium- and Diacylglycerol-Binding Motifs-Reference-Cited by-同舟云学术

RasGRP, a Ras Guanyl Nucleotide- Releasing Protein with Calcium- and Diacylglycerol-Binding Motifs

Published:1998-05-15 Issue:5366 Volume:280 Page:1082-1086
ISSN:0036-8075
Container-title:Science
language:en
Short-container-title:Science

Author:

Ebinu Julius O.¹²,Bottorff Drell A.¹²,Chan Edmond Y. W.¹²,Stang Stacey L.¹²,Dunn Robert J.¹²,Stone James C.¹²

Affiliation:

1. J. O. Ebinu, D. A. Bottorff, E. Y. W. Chan, S. L. Stang, J. C. Stone, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

2. R. J. Dunn, Center for Research in Neuroscience, Montreal General Hospital Research Institute, Montreal, Quebec, Canada H3G 1A4.

Abstract

RasGRP, a guanyl nucleotide–releasing protein for the small guanosine triphosphatase Ras, was characterized. Besides the catalytic domain, RasGRP has an atypical pair of “EF hands” that bind calcium and a diacylglycerol (DAG)-binding domain. RasGRP activated Ras and caused transformation in fibroblasts. A DAG analog caused sustained activation of Ras-Erk signaling and changes in cell morphology. Signaling was associated with partitioning of RasGRP protein into the membrane fraction. Sustained ligand-induced signaling and membrane partitioning were absent when the DAG-binding domain was deleted. RasGRP is expressed in the nervous system, where it may couple changes in DAG and possibly calcium concentrations to Ras activation.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

Reference33 articles.

1. Calcium Signaling in Neurons: Molecular Mechanisms and Cellular Consequences

2. RNA was isolated from the brain of an adult Sprague Dawley rat by the Trizol method (Gibco BRL), and polyadenylate RNA was selected with the Fasttrack system (Invitrogen). Synthesis of cDNAs was achieved with a combination of oligo(dT) and random DNA primers (Invitrogen). DNAs were cloned with Bst X1 adapters into the retrovirus vector pCVT1B [I. Whitehead, H. Kirk, R. Kay, Mol. Cell. Biol. 15, 704 (1995)]. After amplification of the library inE. coli, purified plasmids were converted to retrovirus form with a high-efficiency packaging system [W. Pear, et al., Proc. Natl. Acad. Sci. U.S.A. 90, 8392 (1993)]. To create a host cell line that was sensitive to weak transforming signals in the Ras pathway, we first engineered rat2 fibroblasts to express the v-H-ras Y32R (Tyr32 → Arg substitution) effector mutant using a helper-free, tkretrovirus vector (3). This mutation in v-H-rasis transformation-defective in rat2 cells, but it does transform the somatic mutant rv68BUR and has been used to select extragenic suppresser mutations in Mek1 (4). The encoded Ras protein interacts weakly with Raf in the yeast two-hybrid system [Stang S., et al., Mol. Cell. Biol. 17, 3047 (1997)]. Superinfection of Y32R-expressing rat2 cells with the viruses representing the cDNA library resulted in rare transformed foci. DNA extracted from focus-derived cell lines was used as a template in a PCR protocol, and recovered DNA fragments were cloned into pCTV3B, which expresses the selectable markerhygro. Individual plasmids were converted to virus form and tested for transformation. Transforming cDNAs were identified by sequence analysis.

3. Stone J. C., Blanchard R., Mol. Cell. Biol.11, 6158 (1991).

4. Bottorff D., Stang S., Agellon S., Stone J. C., ibid15, 5113 (1995).

5. A rat brain cDNA library in a phage vector was screened with an rbc7 probe and overlapping cDNA inserts representing the 5′ and 3′ ends of RasGRP were recovered. These inserts were sequenced to identify an in-frame initiator methionine preceded by a Kozak consensus sequence and the first downstream in-frame stop codon. The entire sequence of RasGRP was verified by sequence analysis of a DNA fragment recovered from rat brain RNA with a reverse transcription PCR (Fig. 1).

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