Rad, Gem/Kir, and mRem (RGK) represent a unique GTPase family with largely unknown functions (Reynet, C., and C.R. Kahn. 1993. Science. 262:1441–1444; Cohen, L., R. Mohr, Y. Chen, M. Huang, R. Kato, D. Dorin, F. Tamanoi, A. Goga, D. Afar, N. Rosenberg, and O. Witte. Proc. Natl. Acad. Sci. USA. 1994. 91:12448–12452; Maguire, J., T. Santoro, P. Jensen, U. Siebenlist, J. Yewdell, and K. Kelly. 1994. Science. 265:241–244; Finlin, B.S., and D.A. Andres. 1997. J. Biol. Chem. 272:21982–21988). We report that Ges (GTPase regulating endothelial cell sprouting), a human RGK protein expressed in the endothelium, functions as a potent morphogenic switch in endothelial cells (ECs). Ges function is sufficient to substitute for angiogenic growth factor/extracellular matrix (ECM) signals in promoting EC sprouting, since overexpression of Ges in ECs cultured on glass leads to the development of long cytoplasmic extensions and reorganization of the actin cytoskeleton. Ges function is also necessary for Matrigel-induced EC sprouting, since this event is blocked by its dominant negative mutant, GesT94N, predicted to prevent the activation of endogenous Ges through sequestration of its guanine nucleotide exchange factor. Thus, Ges appears to be a key transducer linking extracellular signals to cytoskeleton/morphology changes in ECs.

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Rab proteins are a family of Ras-like small molecular weight GTPases that are localized to distinct subcellular compartments (1, 2) and believed to regulate specific steps of intracellular membrane trafficking (3-6). The functional cycle of Rab proteins involves the delivery of the GDP-bound forms to the target membrane by a GDP dissociation inhibitor (GDI) 1 (7-9), the exchange of GDP for GTP at membrane surface catalyzed by a guanine nucleotide exchange factor (GEF) (8, 9) and the retrieval of the GDP-bound forms from the membrane by GDI after GTP hydrolysis and membrane fusion (7). Localized on plasma membrane, clathrin-coated vesicles, and early endosomes (2), Rab5 has been shown to play an important role in early events of endocytosis (4, 5), although the exact mechanism of its function remains to be determined.It is known that Mg 2ϩ is essential for GTPase function and structure. Crystallographic studies of several GTP-binding proteins reveal a single Mg 2ϩ in the guanine nucleotide binding pocket, coordinating between the protein and guanine nucleotide in both GDP-and GTP analog-bound conformations (10 -15). Effects of Mg 2ϩ on guanine nucleotide binding, GTPase activity, and the structural integrity of GTP-binding proteins have been widely documented (16 -32). A key observation is that Mg 2ϩ inhibits GDP release from Ras-like GTP-binding proteins and therefore prevents binding of . However, the exact mechanism for this inhibitory effect and its physiological significance remains unknown.Important functional roles of the N-terminal domains of several Ras-like GTP-binding proteins also have been noted in studies of guanine nucleotide exchange (33-38). Myristoylation at the N terminus of ARF enhances its rate of GDP release (27), and N-terminal truncation of ARF results in loss of function by reducing its affinity for GDP and permitting GDP/GTP exchange in the absence of phospholipids (33). Moreover, deletion of the N terminus enables isolation of ARF in a nucleotide-free form (38). Finally, deletion of the N-terminal domain of Rab5 results in a loss of function (34 -37) and interferes with the protein's post-translational processing (37). These observations suggest that N-terminal domains of Ras-like GTP-binding proteins may participate in the regulation of guanine nucleotide exchange and represent crucial structural domains necessary for the function of the proteins.We have investigated mechanisms through which Mg 2ϩ and the N-terminal domain of Rab5 participate in its regulation of GDP release. . While the structure and function of Rab5 , a C-terminal truncation mutant, is influenced by Mg 2ϩ in the same fashion as Rab5WT , an N-and C-terminal truncation mutant, Rab5 , is resistant to the cation's effects. Thus, inhibition of GDP release by Mg 2ϩ appears to be exerted via chemical constraints due to the cation's coordination between GDP and Rab5, as well as conformational restraints involving the protein's N-terminal domain that are induced by Mg 2ϩ coordination with Ser 34 of Rab5. Based on the correl...

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To gain further insight into structural elements involved in Rab5 function, differences in the intrinsic tryptophan fluorescence of the GDP-and guanosine 5-O-(3-thiotriphosphate) (GTP␥S)-bound forms of the protein were examined. When excited at 290 nm, Rab5 displays emission maxima at 339.7 nm for the GDP-bound and 336.7 nm for the GTP␥S-bound forms. The tryptophan fluorescence intensity is quenched by ϳ25% in the GTP␥S-bound form relative to the GDP-bound conformation. Variant Rab5 molecules were created by sitedirected mutagenesis to convert the protein's two tryptophans to phenylalanine residues. Fluorescence studies reveal that the observed changes upon GDP/ GTP␥S exchange are due to a blue shift in the emission spectra for both Rab proteins are a family of Ras-like small molecular weight GTP-binding proteins localized to distinct subcellular compartments (1-6). Rabs have been shown to regulate specific steps of intracellular membrane trafficking (7-12). One member of the Rab family, Rab5, is localized on plasma membrane, clathrincoated vesicles, and early endosomes (2, 9, 10). Even though it has been shown to play an important role in early events of endocytosis (9 -12), the exact mechanism of Rab5 function remains to be determined. The current model for the GTPase cycle of Rab5 is that a GDP dissociation inhibitor (GDI) 1 delivers the GDP-bound protein to target membranes (13,14), where a guanine nucleotide exchange factor catalyzes GDP/ GTP exchange (14). After GTP hydrolysis, GDP-bound Rab5 is retrieved from the membrane by GDI (13, 15). Thus, membrane association and dissociation is correlated with the GTP-and GDP-bound states, respectively. The transition between GDP-and GTP-binding states is a universal molecular switch adopted by many GTP-binding proteins to regulate a diverse set of biological functions. An understanding of the structural differences between these guanine nucleotide binding conformations can help define the basis for interactions with regulatory molecules like GDI and guanine nucleotide exchange factor. Comparison of the two guanine nucleotide-bound conformations of Ras and EF-Tu indicates significant structural changes in two regions: switch I (residues 30 -38 in Ras and residues 41-62 in EF-Tu) and switch II (residues 60 -76 in Ras and residues 84 -96 in EF-Tu) (16,17). Both domains are on the surface of Ras and EF-Tu and are proposed to interact with the molecules' accessory proteins. In contrast, studies on G ␣t revealed three switch regions. Besides counterparts of switch I (residues 173-183) and switch II (residues 195-215), a third region or switch III (residues 227-238) was identified to undergo marked structural rearrangements and is thought to be unique to heterotrimeric GTPbinding proteins (18).Based on this crystallographic information, intrinsic tryptophan fluorescence measurements have proven to be a sensitive means to detect local conformational changes in the switch regions of G ␣o (19,20), G ␣t (21), and Ras (22,23). G ␣o and G ␣t both contain two tryptophans, one o...