{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,6]],"date-time":"2026-03-06T21:37:29Z","timestamp":1772833049881,"version":"3.50.1"},"reference-count":33,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5366","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1998,5,15]]},"abstract":"<jats:p>RasGRP, a guanyl nucleotide\u2013releasing protein for the small guanosine triphosphatase Ras, was characterized. Besides the catalytic domain, RasGRP has an atypical pair of \u201cEF hands\u201d 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.<\/jats:p>","DOI":"10.1126\/science.280.5366.1082","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:37:56Z","timestamp":1027762676000},"page":"1082-1086","source":"Crossref","is-referenced-by-count":557,"title":["RasGRP, a Ras Guanyl Nucleotide- Releasing Protein with Calcium- and Diacylglycerol-Binding Motifs"],"prefix":"10.1126","volume":"280","author":[{"given":"Julius O.","family":"Ebinu","sequence":"first","affiliation":[{"name":"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."},{"name":"R. J. Dunn, Center for Research in Neuroscience, Montreal General Hospital Research Institute, Montreal, Quebec, Canada H3G 1A4."}]},{"given":"Drell A.","family":"Bottorff","sequence":"additional","affiliation":[{"name":"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."},{"name":"R. J. Dunn, Center for Research in Neuroscience, Montreal General Hospital Research Institute, Montreal, Quebec, Canada H3G 1A4."}]},{"given":"Edmond Y. W.","family":"Chan","sequence":"additional","affiliation":[{"name":"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."},{"name":"R. J. Dunn, Center for Research in Neuroscience, Montreal General Hospital Research Institute, Montreal, Quebec, Canada H3G 1A4."}]},{"given":"Stacey L.","family":"Stang","sequence":"additional","affiliation":[{"name":"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."},{"name":"R. J. Dunn, Center for Research in Neuroscience, Montreal General Hospital Research Institute, Montreal, Quebec, Canada H3G 1A4."}]},{"given":"Robert J.","family":"Dunn","sequence":"additional","affiliation":[{"name":"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."},{"name":"R. J. Dunn, Center for Research in Neuroscience, Montreal General Hospital Research Institute, Montreal, Quebec, Canada H3G 1A4."}]},{"given":"James C.","family":"Stone","sequence":"additional","affiliation":[{"name":"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."},{"name":"R. J. Dunn, Center for Research in Neuroscience, Montreal General Hospital Research Institute, Montreal, Quebec, Canada H3G 1A4."}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.7716515"},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","first-page":"3047","DOI":"10.1128\/MCB.17.6.3047","volume":"17","author":"Stang S.","year":"1997","unstructured":"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 \u2192 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.","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_4_2","first-page":"6158","volume":"11","author":"Stone J. C.","year":"1991","unstructured":"Stone J. C., Blanchard R., Mol. Cell. Biol.11, 6158 (1991).","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_5_2","first-page":"5113","volume":"15","author":"Bottorff D.","year":"1995","unstructured":"Bottorff D., Stang S., Agellon S., Stone J. C., ibid15, 5113 (1995).","journal-title":"ibid"},{"key":"e_1_3_1_6_2","unstructured":"A rat brain cDNA library in a phage vector was screened with an rbc7 probe and overlapping cDNA inserts representing the 5\u2032 and 3\u2032 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)."},{"key":"e_1_3_1_7_2","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1016\/0092-8674(87)90076-6","volume":"48","author":"Broek D.","year":"1987","unstructured":"Broek D., et al., Cell48, 789 (1987).","journal-title":"Cell"},{"key":"e_1_3_1_8_2","first-page":"1345","volume":"13","author":"Lai C.-C.","year":"1993","unstructured":"Lai C.-C., Boguski M., Broek D., Powers S., Mol. Cell. Biol.13, 1345 (1993).","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_9_2","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1038\/358351a0","volume":"358","author":"Shou C.","year":"1992","unstructured":"Shou C., Farnsworth C. L., Neel B. G., Feig L. A., Nature358, 351 (1992).","journal-title":"Nature"},{"key":"e_1_3_1_10_2","first-page":"524","volume":"376","author":"Farnsworth C. L.","year":"1995","unstructured":"Farnsworth C. L., et al., ibid376, 524 (1995).","journal-title":"ibid"},{"key":"e_1_3_1_11_2","doi-asserted-by":"crossref","first-page":"6511","DOI":"10.1073\/pnas.89.14.6511","volume":"89","author":"Bowtell D.","year":"1992","unstructured":"P. Chardin, et al., Science 260, 1338 (1993); Bowtell D., Fu P., Simon M., Senior P., Proc. Natl. Acad. Sci. U.S.A. 89, 6511 (1992).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_12_2","doi-asserted-by":"crossref","first-page":"781","DOI":"10.1038\/365781a0","volume":"365","author":"Egan S. E.","year":"1993","unstructured":"Egan S. E., Weinberg R. A., Nature365, 781 (1993).","journal-title":"Nature"},{"key":"e_1_3_1_13_2","doi-asserted-by":"crossref","first-page":"951","DOI":"10.1146\/annurev.bi.58.070189.004511","volume":"58","author":"Strynadka N. C. J.","year":"1989","unstructured":"Strynadka N. C. J., James M. N. G., Annu. Rev. Biochem.58, 951 (1989).","journal-title":"Annu. Rev. Biochem."},{"key":"e_1_3_1_14_2","first-page":"2177","volume":"13","author":"Leonardsen L.","year":"1996","unstructured":"Wild-type H-Ras and the p30 catalytic domain of RasGRF1 [Leonardsen L., DeClue J. E., Lybaek H., Lowy D. R., Willumsen B. M., Oncogene 13, 2177 (1996)] were expressed with His tags in Sf9 cells with the use of baculovirus vectors. The 51-kD RasGRP catalytic region (His6 fused to RasGRP residues 49 to 471) was expressed with the pET21a vector inE. coli. All three proteins were purified by nickel chromatography and dialyzed into buffer A [20 mM tris (pH 7.5), 100 mM NaCl, 1.0 mM MgCl2, 1.0 mM dithiothreitol, and 10% glycerol]. To follow GDP release, we incubated 1.0 \u03bcg of Ras complexed with [3H]GDP (33.5 Ci\/mmol) in the presence of 100 uM unlabeled GTP plus either buffer A (negative control), 3.0 \u03bcg of RasGRP, or 0.5 \u03bcg of p30GRF1 (positive control) in 0.05 ml at 30\u00b0C. The amount of radioactivity associated with Ras after 30 min was then determined by collecting Ras-GDP on beads coated with Y13-259 antibody to Ras, followed by washing and scintillation counting. Values shown in Fig. 2 A are the amount of radioactivity dissociated expressed as a percentage of that associated with Ras when the 30\u00b0C incubation was omitted. To monitor the association of Ras with GTP directly, we incubated 1.0 \u03bcg of Ras with 1.25 nmol of [\u03b132-P]GTP (8 Ci\/mmol) and either buffer A, 2.7 \u03bcg of RasGRP (catalytic region), or 0.44 \u03bcg of p30GRF1 in 0.05 ml for 10 min at 30\u00b0C. Complexes were recovered as above. The values in this case are expressed in Fig. 2 B as the percentage of maximal exchange, as observed when the control sample was adjusted to 2.0 mM EDTA and MgCl2 was added to 20 mM. The values obtained with buffer represent the spontaneous dissociation-association reactions. For both guanyl nucleotide exchange assays, the results are representative of three experiments.","journal-title":"Oncogene"},{"key":"e_1_3_1_15_2","unstructured":"J. O. Ebinu and J. C. Stone unpublished data."},{"key":"e_1_3_1_16_2","unstructured":"E. Y. W. Chan and J. C. Stone unpublished data."},{"key":"e_1_3_1_17_2","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1093\/oxfordjournals.jbchem.a134633","volume":"95","author":"Maruyama K.","year":"1984","unstructured":"E. coli strains were induced with isopropyl-\u03b2- d -thiogalactopyranoside to express GST-rbc7HA fusion proteins, and cells were lysed in SDS. Total cell lysates were resolved by SDS\u2013polyacrylamide gel electrophoresis (SDS-PAGE), blotted to nitrocellulose, and probed with [45Ca] as described [Maruyama K., Mikawa T., Ebashi S., J. Biochem.95, 511 (1984)]. The EF1\u2212 allele is a quadruple substitution converting each of the calcium-binding residues 483, 485, 487, and 493 to alanine. TheEF2\u2212 allele similarly substitutes residues 510, 512, 514, and 521 in the second EF hand.EF1\u2212EF2\u2212 contains all eight substitutions. Coomassie blue staining of a parallel gel demonstrated that equivalent amounts of fusion protein of each genotype were expressed. The experiment was duplicated.","journal-title":"J. Biochem."},{"key":"e_1_3_1_18_2","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1093\/oxfordjournals.jbchem.a135467","volume":"99","author":"Tanaka Y.","year":"1986","unstructured":"[3H]PDBu binding in the presence of phosphatidylserine was performed as described [Tanaka Y., Miyake R., Kikkawa U., Nishizuka Y., J. Biochem. 99, 257 (1986)]. The GST-DAG protein contains residues 538 to 598 of RasGRP. Mouse brain extracts, which contain substantial amounts of PKC, were used as a positive control. The experiment was duplicated.","journal-title":"J. Biochem."},{"key":"e_1_3_1_19_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/18.12.3587"},{"key":"e_1_3_1_20_2","doi-asserted-by":"crossref","first-page":"1621","DOI":"10.1016\/S0960-9822(02)70785-9","volume":"6","author":"Taylor S.","year":"1996","unstructured":"Ras-GTP levels in rat2 cells and rat2 cells expressing rbc7 were compared with a 32Pi-labeling method. After treatment with PMA (100 nM for 2 min), cells were lysed, and Ras-GDP and Ras-GTP were immunoprecipitated with antibody Y13-259. Ras-associated guanyl nucleotides were then separated by chromatography and quantified by phosphorimager analysis [J. C. Stone, M. Colleton, D. Bottorff, Mol. Cell. Biol. 13, 7311 (1993)]. The bars in Fig. 3 A represent the standard deviation of the mean of three separate experimental values. The amounts of Ras-GTP were compared in rat2 cells expressing the empty vector and rat cells expressing full-length RasGRP with a method that takes advantage of the ability of Ras-GTP to bind the Ras-binding domain of Raf [Taylor S., Shalloway D., Curr. Biol. 6, 1621 (1996)]. Cells were treated with endothelin-1 (100 nM for 10 min) and then lysed. The amount of Ras that associated with either GST-Raf (Raf-binding domain) or GST (negative control) was determined by immunoblotting with an antibody to Ras. Lysates contained very similar amounts of Ras.","journal-title":"Curr. Biol."},{"key":"e_1_3_1_21_2","doi-asserted-by":"crossref","first-page":"557","DOI":"10.1038\/379557a0","volume":"379","author":"Daub H.","year":"1996","unstructured":"Daub H., Weiss F. U., Wallasch C., Ullrich A., Nature379, 557 (1996).","journal-title":"Nature"},{"key":"e_1_3_1_22_2","doi-asserted-by":"crossref","unstructured":"I. Ambar and M. Sokolovsky Eur. J. Pharmacol. 245 31 (1993).","DOI":"10.1016\/0922-4106(93)90166-7"},{"key":"e_1_3_1_23_2","doi-asserted-by":"publisher","DOI":"10.1038\/364249a0"},{"key":"e_1_3_1_24_2","unstructured":"RNA was extracted from adult rat tissues with the Trizol method. Total RNA (10 \u03bcg) was resolved by electrophoresis in 1% agarose in borate buffer blotted onto Hybond nylon membrane (Amersham) hybridized with RasGRP cDNA probe and washed at high stringency."},{"key":"e_1_3_1_25_2","unstructured":"In situ hybridization was done on 10-\u03bcm sections of adult rat brain [D. M. Simmons et al. J. Histotechnol. 12 169 (1989)]. A 35 S-labeled RNA probe consisting of bases 1831 to 2014 of RasGRP was used for hybridization and the sections were dipped in NTB2 emulsion and exposed for 15 days. Control hybridizations to adjacent sections with a sense probe showed a low amount of diffuse uniform labeling."},{"key":"e_1_3_1_26_2","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1016\/S0092-8674(00)80226-3","volume":"89","author":"Rodriguez-Viciana P.","year":"1997","unstructured":"A. B. Vojtek, S. M. Hollenberg, J. A. Cooper, Cell 74, 205 (1993); L. Van Aelst, M. Barr, S. Marcus, A. Polverino, M. Wigler, Proc. Natl. Acad. Sci. U.S.A. 90, 6213 (1993); X. Zhang, et al., Nature 364, 308 (1993); P. H. Warne, P. Rodriguez-Viciana, J. Downward, ibid., p. 352; P. Rodriguez-Viciana et al., ibid. 370, 527 (1994); Rodriguez-Viciana P., et al., Cell 89, 527 (1997).","journal-title":"Cell"},{"key":"e_1_3_1_27_2","doi-asserted-by":"publisher","DOI":"10.1038\/36849"},{"key":"e_1_3_1_28_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.275.5300.661"},{"key":"e_1_3_1_29_2","doi-asserted-by":"publisher","DOI":"10.1038\/361315a0"},{"key":"e_1_3_1_30_2","first-page":"643","volume":"366","author":"Boguski M. S.","year":"1993","unstructured":"Boguski M. S., McCormick F., ibid366, 643 (1993).","journal-title":"ibid"},{"key":"e_1_3_1_31_2","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1111\/j.1432-1033.1984.tb08055.x","volume":"139","author":"Aitken A.","year":"1984","unstructured":"Aitken A., Klee C. B., Cohen P., Eur. J. Biochem.139, 663 (1984).","journal-title":"Eur. J. Biochem."},{"key":"e_1_3_1_32_2","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/0167-4781(93)90111-P","volume":"1174","author":"Aris J. P.","year":"1993","unstructured":"Aris J. P., et al., Biochim. Biophys. Acta1174, 171 (1993).","journal-title":"Biochim. Biophys. Acta"},{"key":"e_1_3_1_33_2","unstructured":"Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr."},{"key":"e_1_3_1_34_2","unstructured":"We thank R. Kay for advice on cDNA cloning and L. Agellon D. Brindley N. Dower M. James D. Lowy H. Ostergaard E. Shibuya and B. Sykes for useful discussions. Supported by grants to J.C.S. from the National Cancer Institute Canada."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.280.5366.1082","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T05:21:23Z","timestamp":1705123283000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.280.5366.1082"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1998,5,15]]},"references-count":33,"journal-issue":{"issue":"5366","published-print":{"date-parts":[[1998,5,15]]}},"alternative-id":["10.1126\/science.280.5366.1082"],"URL":"https:\/\/doi.org\/10.1126\/science.280.5366.1082","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1998,5,15]]}}}