CLONING AND CHARACTERIZATION OF RAS-GRF2, A NOVEL GUANINE-NUCLEOTIDE EXCHANGE FACTOR FOR RAS

Conversion of Ras proteins into an activated GTP-bound state able to b ind effector proteins is catalyzed by specific guanine nucleotide exch ange factors in response to a large number of extracellular stimuli, H ere we report the isolation of mouse cDNAs encoding Ras-GRF2, a multid omain 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 nucleot ides, 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 signali ng proteins including two pleckstrin homology domains and a Dbl homolo gy region, Ras-GRF2 also contains an IQ motif which was required for i ts apparent constitutive association,vith calmodulin in epithelial cel ls ectopically expressing RaS-GRF2. Transient expression of Ras-GRF2 i n kidney epithelial cells stimulated GTP binding by Ras and potentiate d calcium ionophore induced activation of mitogen-activated protein ki nase (ERK1) dependent upon the IQ motif. Calcium influx caused Ras-GRF 2 subcellular localization to change from cytosolic to peripheral, sug gesting a possible mechanism for controlling Ras-GRF2 interactions wit h Ras at the plasma membrane. Epithelial cells overexpressing Ras-GRF2 are morphologically transformed and grow in a disorganized manner wit h minimal intercellular contacts. Northern analysis indicated a 9-kb G RF2 transcript in brain and lung, where p135 Ras-GRF2 is known to be e xpressed, and RNAs of 12 kb and 2.2 kb were detected in several tissue s. Thus, Ras-GRF2 proteins with different domain structures may be wid ely expressed and couple diverse extracellular signals to Ras activati on.