Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by catalyzing the exchange of bound GDP for GTP, thereby resulting in downstream effector recognition. Two metazoan families of GEFs have been described: Dbl-GEF family members that share conserved Dbl homology (DH) and Pleckstrin homology (PH) domains and the more recently described Dock180 family members that share little sequence homology with the Dbl family and are characterized by conserved Dock homology regions 1 and 2 (DHR-1 and -2). While extensive characterization of the Dbl family has been performed, less is known about how Dock180 family members act as GEFs, with only a single x-ray structure having recently been reported for the Dock9-Cdc42 complex. In order to learn more about the mechanisms used by the founding member of the family, Dock180, to act as a Rac-specific GEF, we set out to identify and characterize its limit functional GEF domain. A C-terminal portion of the DHR-2 domain, composed of approximately 300 residues (designated as Dock180 ), is shown to be necessary and sufficient for robust Rac-specific GEF activity both in vitro and in vivo. We further show that Dock180 binds to Rac in a manner distinct from Rac-GEFs of the Dbl family. Specifically, Ala 27 and Trp 56 of Rac appear to provide a bipartite binding site for the specific recognition of Dock180 , whereas, for Dbl family Rac-GEFs, Trp 56 of Rac is the sole primary determinant of GEF specificity. Based on our findings, we are able to define the core of Dock180 responsible for its Rac-GEF activity as well as highlight key recognition sites that distinguish different Dock180 family members and determine their corresponding GTPase specificities.Members of the Rho family of GTPases regulate a wide range of cellular activities including cell-cycle progression, gene transcription, cell migration, cell polarity, and vesicular trafficking through their abilities to bind to multiple downstream effectors (1-4). Rho GTPases switch between two states, the GDP-bound inactive state and the GTP-bound active state. Tight regulation of Rho GTPases and their nucleotide-bound state is important for mediating their different cellular functions (5). Three main classes of regulatory proteins for Rho GTPases have been identified and characterized. Guanine nucleotide exchange factors (GEFs) include ~70 mammalian/human proteins that promote the exchange of GDP for GTP on Rho GTPases. GTPase-activating proteins (GAPs) catalyze the hydrolysis of the bound GTP to GDP, and Rho GDP-dissociation inhibitors (GDIs) slow nucleotide exchange while serving to sequester Rho GTPases in the cytoplasm (6-8).Two families of GEFs have been discovered for Rho GTPases, which we refer to here as the Dbl (9) and Dock180 superfamilies (10-12). The Dbl family members all possess two † This work was supported by National Institutes of Health R01 Grants GM40654 and GM47458.* To whom correspondence should be addressed: Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Itha...

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