SGEF has been classified as a putative exchange factor for Rho GTPases based on the presence of a Dbl homology (DH) domain, the conserved catalytic core of most RhoGEF proteins. a catalytically active DH domain. Finally, activated RhoG was found to be more effective than activated Rac, and comparable to SGEF, in its ability to trigger dextran uptake. Together, this work establishes SGEF as a RhoG exchange factor and provides evidence that both SGEF and RhoG regulate membrane dynamics in promotion of macropinocytosis. INTRODUCTION Huzhangoside D The Rho proteins are low-molecular-weight GTP-binding proteins that cycle between GDP and GTP bound states. Binding of GTP activates Rho GTPases by inducing structural shifts that support association of effector molecules that transmit downstream signals. Rho GTPases are negatively regulated primarily by the sequestering activity of Rho guanine dissociation inhibitors (RhoGDIs; Olofsson, 1999 ) and by the Rho GTPase-activating proteins (RhoGAPs), which trigger GTP hydrolysis upon binding (Moon and Zheng, 2003 ). Conversely, Rho GTPases are positively regulated by Rho guanine nucleotide exchange factors (RhoGEFs) that catalyze the exchange of bound GDP for GTP (Schmidt and Hall, 2002 ). Rho GTPases are modified by the addition of a prenyl moiety to their carboxyl-terminal cysteine residue. This posttranslational event is necessary for GTPase function (Ando 1992 , Ohya 1993 ), presumably by allowing interactions with phospholipid membranes and other proteins. There are currently 22 identified Rho family members that synergistically govern a number of cell functions, such as gene expression, cell adhesion, and cytoskeletal dynamics (Burridge and Wennerberg, 2004 ). Emerging goals of this field are to assign temporal and spatial activation patterns of the GTPases and understand how these enzymes engage in cross-talk with each other to effectively regulate cell function. For example, Gauthier-Rouviere (1998 ) discovered that GTP-bound RhoG was an upstream activator of both Rac1, a major regulator of KRIT1 membrane protrusion, and Cdc42, a protein linked with membrane protrusion, cell polarity, and vesicular trafficking. RhoG, an ubiquitously expressed GTPase, was isolated from a screen for mRNA molecules upregulated in G1 phase (Vincent 1992 ). RhoG shares significant homology with Rac and binds to a number of the same effector proteins, with the notable exception of p21-activated kinase (PAK; Gauthier-Rouviere 1998 ; Wennerberg 2002 ). Functionally, RhoG activity is sufficient to drive neurite outgrowth in PC12 cells and is required for NGF stimulation of outgrowth (Katoh 2000 ). Rac and Cdc42 signaling is also necessary; however, neither GTPase is sufficient to drive outgrowth when overexpressed alone (Katoh 2000 ), indicating that additional RhoG signaling and/or RhoG’s ability to coordinate Rac and Cdc42 activation is needed in the formation of neurite extensions. Katoh and Negishi (2003 ) discovered that GTP-RhoG binds to ELMO and forms a ternary complex with ELMO and DOCK180. Formation of this ternary complex is postulated to advance DOCK180 exchange factor activity against Rac, thereby providing a molecular connection between RhoG and downstream Rac activation. SGEF (SH3-containing Guanine Nucleotide Exchange Huzhangoside D Factor) was initially found in a screen for androgen-responsive genes in human prostate (Qi 2003 ). Two alternatively transcribed forms of SGEF gene were isolated, one which encodes the full-length molecule of 871 amino acids and the other an androgen-inducible splice-variant of 139 amino acids that contains only a small segment of the pleckstrin homology (PH) domain and the entire carboxyl Src homology 3 (SH3) domain. Northern blot analysis of SGEF mRNA revealed an ubiquitous low-level expression in a variety of tissues, with pronounced expression in liver. The 139-amino acid splice variant was restricted to liver and prostate. SGEF has been classified as a putative exchange factor for Rho GTPases based on the presence of a Dbl homology (DH) domain, the conserved catalytic core of most RhoGEF proteins. However, the expression, specificity, and function of the SGEF protein are unknown. We report here that SGEF is a RhoG guanine nucleotide exchange factor that stimulates macropinocytosis. Huzhangoside D MATERIALS AND METHODS Reagents Bovine serum.