Rho family-specific guanine nucleotide dissociation inhibitors (RhoGDIs) decrease the rate of nucleotide dissociation and release Rho proteins such as RhoA, Rac and Cdc42 from membranes, forming tight complexes that shuttle between cytosol and membrane compartments. We have solved the crystal structure of a complex between the RhoGDI homolog LyGDI and GDP-bound Rac2, which are abundant in leukocytes, representing the cytosolic, resting pool of Rho species to be activated by extracellular signals. The N-terminal domain of LyGDI (LyN), which has been reported to be flexible in isolated RhoGDIs, becomes ordered upon complex formation and contributes more than 60% to the interface area. The structure is consistent with the C-terminus of Rac2 binding to a hydrophobic cavity previously proposed as isoprenyl binding site. An inner segment of LyN forms a helical hairpin that contacts mainly the switch regions of Rac2. The architecture of the complex interface suggests a mechanism for the inhibition of guanine nucleotide dissociation that is based on the stabilization of the magnesium (Mg2+) ion in the nucleotide binding pocket.

Neutrophils contain a soluble guanine-nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which was also observed for GTP[S]-activated recombinant Rac1, was independent of LyGDI, but required C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.

Phospholipase C-␤ (PLC␤) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLC␤ 2 isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42). However, the mechanism(s) of this stimulation are still unclear. Here, we employed fluorescence recovery after photobleaching to investigate the interaction of green fluorescent protein (GFP)-PLC␤ 2 with the plasma membrane. For either GFP-PLC␤ 2 or GFP-PLC␤ 2 ⌬, a C-terminal deletion mutant lacking the region required for stimulation by G␣ q , these interactions were characterized by a mixture of exchange with a cytoplasmic pool and lateral diffusion. Constitutively active Rac2(12V) stimulated the activity of both GFP-PLC␤ 2 and GFP-PLC␤ 2 ⌬ in live cells, and enhanced their membrane association as evidenced by the marked reduction in their fluorescence recovery rates. Both effects required the putative N-terminal pleckstrin homology (PH) domain of PLC␤ 2 . Importantly, Rac2(12V) dramatically increased the contribution of exchange to the fluorescence recovery of GFP-PLC␤ 2 , but had the opposite effect on GFP-PLC␤ 2 ⌬, where lateral diffusion became dominant. Our results demonstrate for the first time the regulation of membrane association of a PLC␤ isozyme by a GTP-binding protein and assign a novel function to the PLC␤ 2 C-terminal region, regulating its exchange between membrane-bound and cytosolic states.The activity of phospholipase C-␤ (PLC␤) 1 enzymes that hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) is stimulated with different orders of efficacy by G protein ␣ q subunits and by G protein ␤␥ dimers (1-3). In addition, the PLC␤ 2 isozyme is specifically activated in vitro by the Rho GTPases Rac and Cdc42, but not by RhoA (4 -6). As for all PLC␤ isozymes, activation by ␣ q requires the C-terminal region of PLC␤ 2 , and mutants carrying deletions in this region, such as the mutant PLC␤ 2 ⌬ that lacks the Phe 819 -Glu 1166 segment, are resistant to stimulation by ␣ q but are susceptible to activation by Rho GTPases and G protein ␤␥ subunits (3, 4, 7). Recent studies (4 -6) show that ␤␥ dimers and Rho GTPases activate PLC␤ 2 by interacting with different regions of the effector enzyme. Thus, the PLC␤ 2 catalytic subdomains X and Y are sufficient for efficient stimulation by ␤␥, whereas the putative pleckstrin homology (PH) domain of PLC␤ 2 is absolutely required for stimulation by Rho GTPases (6). Among the Rho GTPases, Rac1 and Rac2 are more potent stimulators than Cdc42 (6). Evidence for a tight connection between PLC␤ 2 and Rho GTPases in cells is provided by the chemoattractant receptor system, whose activation stimulates PLC␤ 2 and Rac1, Rac2 and Cdc42 (8 -11). Moreover, in accord with our in vitro studies on PLC␤ 2 activation by Rho GTPases (4, 6), a recent study conducted on myeloid-differentiated HL-60 cells demonstrated that dominant-negative Cdc42 disrupted the stimulation of inositol 1,4,5-trisphosphate formation mediated via the chemoattractant receptors whi...

Phospholipase C-␤ 2 (PLC␤ 2 ) is activated both by heterotrimeric G protein ␣-and ␤␥-subunits and by Rho GTPases. In this study, activated Rho GTPases are shown to stimulate PLC␤ isozymes with the rank order of PLC␤ 2 > PLC␤ 3 > PLC␤ 1 . The sensitivity of PLC␤ isozymes to Rho GTPases was clearly different from that observed for G protein ␤␥ dimers, which decreased in the following order: PLC␤ 3 > PLC␤ 2 > PLC␤ 1 for ␤ 1 ␥ 1/2 and PLC␤ 2 > PLC␤ 1 >>> PLC␤ 3 for ␤ 5 ␥ 2 . Rac1 and Rac2 were found to be more potent and efficacious activators of PLC␤ 2 than was Cdc42Hs. The stimulation of PLC␤ 2 by Rho GTPases and G protein ␤␥ dimers was additive, suggesting that PLC␤ 2 activation can be augmented by independent regulation of the enzyme by the two stimuli. Using chimeric PLC␤ 1 -PLC␤ 2 enzymes, ␤␥ dimers, and Rho GTPases are shown to require different regions of PLC␤ 2 to mediate efficient stimulation of the enzyme. Although the catalytic subdomains X and Y of PLC␤ 2 were sufficient for efficient stimulation by ␤␥, the presence of the putative pleckstrin homology domain of PLC␤ 2 was absolutely required for the stimulation of the enzyme by Rho GTPases. Taken together, these results identify Rho GTPases as novel PLC␤ regulators, which mediate PLC␤ isozyme-specific stimulation and are potentially involved in coordinating the activation of PLC␤ 2 by extracellular mediators in intact cells.Many extracellular signaling molecules elicit intracellular responses by activating inositol phospholipid-specific phospholipases C (PLCs), 1 which hydrolyze phosphatidylinositol 4,5-bisphosphate (PI-4,5-P 2 ) to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. These two second messengers modulate intracellular events through the regulation of intracellular free Ca 2ϩ and protein kinase C isozymes, respectively. The mammalian PLC isozymes can be divided into four major families: PLC␤, PLC␥, PLC␦, and PLC⑀ (1). The PLC␤ and PLC␥ subclasses have been shown to be regulated through G protein-coupled and protein-tyrosine kinase-linked receptors, respectively. The mechanisms by which PLC␦ isozymes and PLC⑀ are coupled to membrane receptors are less well understood (for recent reviews, see Refs. 1-4). Stimulation of PLC␤, of which four isozymes (PLC␤ 1 -PLC␤ 4 ) are known, is mediated by members of the ␣ q subfamily of G protein ␣ subunits and, excepting PLC␤ 4 , by G protein ␤␥ dimers (1-4). Activated ␣ q subunits stimulate PLC␤ in the rank order of efficacy of PLC␤ 1 Ն PLC␤ 3 Ͼ PLC␤ 2 . PLC␤ 4 is also activated by ␣ q subunits. The sensitivity of PLC␤ isozymes to ␤␥ dimers decreases in the order: PLC␤ 3

In this study, G␤ specificity in the regulation of G␤␥-sensitive phosphoinositide 3-kinases (PI3Ks) and phospholipase C␤ (PLC␤) isozymes was examined. Recombinant mammalian G␤ 1-3 ␥ 2 complexes purified from Sf9 membranes stimulated PI3K␥ lipid kinase activity with similar potency (10 -30 nM) and efficacy, whereas transducin G␤␥ was less potent. Functionally active G␤ 5 ␥ 2 dimers were purified from Sf9 cell membranes following coexpression of G␤ 5 and G␥ 2-His . This preparation as well as G␤ 1 ␥ 2-His supported pertussis toxin-mediated ADP-ribosylation of G␣ i1 . G␤ 1 ␥ 2-His stimulated PI3K␥ lipid and protein kinase activities at nanomolar concentrations, whereas G␤ 5 ␥ 2-His had no effect. Accordingly, G␤ 1 ␥ 2-His , but not G␤ 5 ␥ 2-His , significantly stimulated the lipid kinase activity of PI3K␤ in the presence or absence of tyrosine-phosphorylated peptides derived from the p85-binding domain of the platelet derived-growth factor receptor. Conversely, both preparations were able to stimulate PLC␤ 2 and PLC␤ 1 . However, G␤ 1 ␥ 2-His , but not G␤ 5 ␥ 2-His , activated PLC␤ 3 . Experimental evidence suggests that the mechanism of G␤ 5 -dependent effector selectivity may differ between PI3K and PLC␤. In conclusion, these data indicate that G␤ subunits are able to discriminate among effectors independently of G␣ due to selective protein-protein interaction.