title: Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras.
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.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
authors.forename: N P
authors.initials: NP
authors.lastname: Fam
authors.forename: W T
authors.initials: WT
authors.lastname: Fan
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.forename: L J
authors.initials: LJ
authors.lastname: Zhang
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: M F
authors.initials: MF
authors.lastname: Moran
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010749:Phosphoprotein Phosphatases / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1
chemical_list: D002147:Calmodulin; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D002118:Calcium