Ras-specific exchange factor GRF: Oligomerization through its Dbl homologydomain and calcium-dependent activation of Raf

The full-length versions of the Ras-specific exchange factors Ras-GRF1 (GRF 1) and Ras-GRF2 (GRF2), which are expressed in brain and a restricted numbe r of other organs, possess an ionomycin-dependent activation of Erk mitogen -activated protein kinase activity in 293T cells (C. L. Farnsworth et al., Nature 376:524-527, 1995; N. P. Fam et al., Mel. Cell. Biol. 17:1396-1406, 1996). Each GRF protein contains a Dbl homology (DH) domain. A yeast two hy brid screen was used to identify polypeptides that associate with the DH do main of GRF1. In this screen, a positive cDNA clone from a human brain cDNA library was isolated which consisted of the GRF2 DH domain and its adjacen t ilimaquinone domain. Deletion analysis verified that the two-hybrid inter action required only the DH domains, and mutation of Leu-263 to Gln (L263Q) in the N terminus of the GRF1 DH domain abolished the two-hybrid interacti on, while a cluster of more C-terminally located mutations in the DH domain did not eliminate the interaction. Oligomers between GRF1 and GRF2 were de tected in a rat brain extract, and forced expression of GRF1 and GRF2 in cu ltured mammalian cells formed homo- and hetero-oligomers. Introduction of t he L263Q mutation in GRF1 led to a protein that was deficient in oligomer f ormation, while GRF1 containing the DH cluster mutations formed homo-oligom ers with an efficiency similar to that of wild type. Compared to wild-type GRF1, the focus forming activity on NIH 3T3 cells of the GRF1 DH cluster mu tant was reduced, while the L263Q mutant was inactive. Both mutants were im paired in their ability to mediate ionomycin-dependent Erk activity in 293T cells. In the absence of ionomycin, 293T cells expressing,wild-type GRF1 c ontained much higher levels of Ras-GTP than control cells; the increase in Erk activity induced by ionomycin in the GRF1-expressing cells also induced a concomitant increase in Raf kinase activity, but without a further incre ase in the level Ras-GTP. We conclude that GRF1 and GRF2 can form homo- and hetero-oligomers via their DH domains, that mutational inactivation of oli gomer formation by GRF1 is associated with impaired biological and signalin g activities, and that in 293T cells GRF1 mediates at least two pathways fo r Raf activation: one a constitutive signal that is mainly Ras-dependent, a nd one an ionomycin-induced signal that cooperates with the constitutive si gnal without further augmenting the level of GTP-Ras.