Identification of guanine nucleotide exchange factors (GEFs) for the Rap1 GTPase - Regulation of MR-GEF by M-Ras-GTP interaction

Although the Ras subfamily of GTPases consists of similar to 20 members, on ly a limited number of guanine nucleotide exchange factors (GEFs) that coup le extracellular stimuli to Ras protein activation have been identified. Fu rthermore, no novel downstream effecters have been identified for the M-Ras /R-Ras3 GTPase. Here we report the identification and characterization of t hree Ras family GEFs that are most abundantly expressed in brain. Two of th ese GEFs, MR-GEF (M-Ras-regulated GEF, KIAA0277) and PDZ-GEF (KIAA0313) bou nd specifically to nucleotide-free Rap1 and Rap1/Rap2, respectively. Both p roteins functioned as Rap1 GEFs in vivo. A third GEF, GRP3 (KIAA0846), acti vated both Ras and Rap1 and shared significant sequence homology with the c alcium- and diacylglycerol-activated GEFs, GRP1 and GRP2, Similarly to prev iously identified Rap GEFs, C3G and Smg GDS, each of the newly identified e xchange factors promoted the activation of Elk-1 in the LNCaP prostate tumo r cell line where B-Raf can couple Rap1 to the extracellular receptor-activ ated kinase cascade. MR-GEF and PDZ-GEF both contain a region immediately N -terminal to their catalytic domains that share sequence homology with Ras- associating or Ral-GDS/AF6 homology (RA) domains. By searching for in vitro interaction with Ras-GTP proteins, PDZ-GEF specifically bound to Rap1A- an d Rap2B-GTP, whereas MR-GEF bound to M-Ras-GTP. C-terminally truncated MR-G EF, lacking the GEF catalytic domain, retained its ability to bind M-Ras-GT P, suggesting that the RA domain is important for this interaction. Co-immu noprecipitation studies confirmed the interaction of M-Ras-GTP with MR-GEF in vivo, In addition, a constitutively active M-Ras(71L) mutant inhibited t he ability of MR-GEF to promote Rap1A activation in a dose-dependent manner . These data suggest that M-Ras may inhibit Rap1 in order to elicit its bio logical effects.