The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b 559 and four cytosolic components as follows: p47 phox , p67 phox , p40 phox , and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b 559 and can be mimicked in vitro by mixtures of membrane and cytosolic components exposed to an anionic amphiphile, serving as activator. We reported that prenylation of Rac1 endows it with the ability to support oxidase activation in conjunction with p67 phox but in the absence of amphiphile and p47 phox . We now show the following 6 points. 1) The Rac guanine nucleotide exchange factor Trio markedly potentiates oxidase activation by prenylated Rac1-GDP. 2) This occurs in the absence of exogenous GTP or any other source of GTP generation, demonstrating that the effect of Trio does not involve GDP to GTP exchange on Rac1. 3) Trio does not potentiate oxidase activation by prenylated Rac1-GTP, by nonprenylated Rac1-GDP in the presence or absence of amphiphile, and by a prenylated [p67 phox -Rac1] chimera in GDP-bound form. 4) Rac1 mutants defective in the ability to bind Trio or to respond to Trio by nucleotide exchange fail to respond to Trio by enhanced oxidase activation. 5) A Trio mutant with conserved Rac1-binding ability but lacking nucleotide exchange activity fails to enhance oxidase activation. 6) The effect of Trio is mimicked by displacement of Mg2+ from Rac1-GDP. These results reveal the existence of a novel mechanism of Rac activation by a guanine nucleotide exchange factor and suggest that the induction by Trio of a conformational change in Rac1, in the absence of nucleotide exchange, is sufficient for enhancing its effector function.

Phagocytes utilize oxygen radicals for the killing of engulfed microorganisms (reviewed in Ref. 1). Oxygen radicals also serve as signal transduction messengers in a variety of nonphagocytic cells (reviewed in Ref. 2). In phagocytes, the primordial oxygen radical, superoxide (O 2 . ) 1 is produced by the NADPH-driven reduction of molecular oxygen catalyzed by a membrane-localized flavocytochrome (cytochrome b 559 ), comprising two subunits, gp91 phox and p22 phox (reviewed in Ref. 3). All redox stations involved in electron flow from NADPH to O 2 are found on gp91 phox , and it is assumed that the redox cascade is initiated by a conformational change induced in gp91 phox by interaction with one or more cytosolic proteins. These are p47 phox , p67 phox , p40 phox , and the small GTPase Rac (Rac1 or -2). Upon stimulation of the phagocyte, they translocate to the membrane, resulting in the assembly of what is known as the NADPH oxidase complex (referred to as "oxidase") (reviewed in Ref. 4). The identity of the cytosolic component(s) responsible for causing the conformational change in gp91 phox is controversial. The principal candidate is p67 phox , based on the identification of an "activation domain" in p67 phox , consisting of residues 199 -210 (5), and on direct evidence of binding of p67 phox to gp91 phox , an interaction enhanced by Rac1 (6). This hypothesis also implies that p47 phox and Rac serve either as "carriers" for p67 phox from the cytosol to the membrane or, following their own translocation, as membrane "anchors" for the correct positioning of p67 phox in the assembled complex. Oxidase assembly can be elicited in vitro in a cell-free system consisting of phagocyte membranes and the cytosolic components p47 phox , p67 phox , and Rac, exposed to an anionic amphiphile (7,8). The role of the amphiphile is to induce a conformational change in p47 phox , resulting in its binding to p22 phox and the passive translocation of p67 phox , by virtue of its affinity for p47 phox , to the vicinity of gp91 phox . The in vivo equivalent of amphiphile action is the phosphorylation of critical serines in p47 phox , a process representing the starting point of a "p47 phoxinitiated" pathway of oxidase activation.
Under certain conditions, oxidase activation in vitro is also possible in the absence of p47 phox . Originally, this was achieved in an amphiphile-dependent cell-free system by having recom-binant p67 phox and Rac present at micromolar concentrations (9,10). A characteristic of earlier versions of the amphiphileactivated cell-free system is the use of bacterially expressed recombinant Rac, which did not undergo C-terminal geranylgeranylation (prenylation). We recently described oxidase activation in vitro in a system consisting of phagocyte membranes, p67 phox and prenylated Rac1, in the absence of amphiphile and p47 phox (11). Amphiphile-and p47 phox -independent oxidase activation could also be achieved in mixtures of membrane and a chimeric [p67 phox -Rac1] construct prenylated at the C terminus of the Rac1 segment (12). These findings support the existence of a second "Rac-initiated" pathway of oxidase activation (13)(14)(15)(16), and evidence is accumulating in support of a guanine nucleotide exchange factor (GEF), responsible for GDP to GTP exchange on Rac, being the key link in this pathway. In the present report we show that a minimal functional module of Trio, a Rac-specific GEF (17), markedly potentiates oxidase activation by prenylated Rac1 in vitro in the absence of an activator and of p47 phox . Surprisingly, the effect of Trio on Rac does not involve GDP to GTP exchange.
Preparation of Phagocyte Membrane Vesicles-Phagocyte membranes were prepared from guinea pig peritoneal macrophages, as described (7). The membranes were solubilized by 40 mM n-octyl-␤-Dglucopyranoside, and membrane vesicles were prepared by extensive dialysis against detergent-free buffer, as described (18).
Identification of Nucleotides Present on Recombinant Rac1-Bound nucleotides were liberated from recombinant Rac1 preparations as described before (24). They were identified and quantified by HPLC on a Partisil 10 SAX anion exchange column (250 ϫ 4.6 mm) (Whatman), as described in Ref. 25. Briefly, nucleotide standards or nucleotides liberated from recombinant or other proteins were injected into the column in a volume of 0.5 ml. This was followed by delivering 0.007 M KH 2 PO 4 , pH 4.0, at 1.5 ml/min for 15 min, followed by a linear gradient from 0.007 M KH 2 PO 4 , pH 4.0, to 0.25 M KH 2 PO 4 and 0.5 M KCl, pH 4.5, at 1.5 ml/min over a period of 45 min, and continuing the high molarity buffer for an additional 40 min at the same flow rate. The column eluate was monitored by passage through a diode array detector, set at 220 -450 nm (MD-1510, Jasco, Easton, MD). The amounts of nucleotides were determined by peak integration (based on absorbance at 254 nm) and related to 5-nmol amounts of the following nucleotide standards: AMP, GMP, ADP, GDP, ATP, and GTP.
Nucleotide Exchange-Prenylated Rac1 was subjected to nucleotide exchange to GTP␥S, as described before (11).
Cell-free NADPH Oxidase Assay-Activation of oxidase in vitro supported by prenylated Rac1 was assessed by measuring NADPH-dependent O 2 . production in a semi-recombinant cell-free system, in the absence of an amphiphilic activator and of p47 phox , essentially as described earlier (11,26). The components of the assay were added to 96-well microplates, in the following order: TrioN or buffer, prenylated Rac1, a mixture of solubilized membrane and p67 phox , and assay buffer in a total volume of 200 l. The mixture was incubated for 90 s at 25°C, and 240 M NADPH was added to initiate O 2 . production. The concentration of membrane was constant throughout all the experiments and corresponded to 5 nM cytochrome b 559 heme; the concentrations of TrioN, prenylated Rac1, and p67 phox were varied and are indicated under "Results." Most experiments were performed in the assay buffer used by this laboratory before (18), which contains 1 mM MgCl 2 . In some experiments, assay buffers containing 0.4 M and 4 mM free Mg 2ϩ were used; these were prepared by adding EDTA and MgCl 2 to the basic assay buffer in amounts calculated as described in Ref. 27.
Guanine Nucleotide Exchange Assay-The ability of TrioN to perform guanine nucleotide exchange on Rac1 and Rac1 mutants was assayed by the increase in fluorescence (excitation ϭ 361 nm; emission ϭ 440 nm), consequent to the uptake of the fluorescent GTP analog mant-GTP by Rac1, as described in Ref. 28. Briefly, 1 M Rac1 or Rac1 mutants were incubated with 0.375 M mant-GTP at 20°C in a magnetically stirred thermostated cuvette in a model FP-750 spectrofluorometer (Jasco, Easton, MD) in 20 mM Tris-HCl buffer, pH 7.4, also containing 150 mM NaCl and 4 mM MgCl 2 . This was followed by the addition of 0.5 M TrioN, and the kinetics of change in fluorescence was recorded over time.

RESULTS
Nucleotide Content of Native Rac1-It is generally assumed that recombinant small GTPases produced in E. coli are in the GDP-bound form. We put this hypothesis to a direct test by determining the nature and amount of nucleotide bound to native recombinant Rac1. As illustrated in Fig. 1B, the only nucleotide identified on Rac1 was GDP, and it was present at a ratio of 1.046 Ϯ 0.064 mol GDP/mol Rac1 (mean Ϯ S.E. of five experiments). We have shown before that the procedure employed to liberate the nucleotides bound to Rac did not lead to degradation of GTP to GDP (24). Further proof for the reliability of the procedure was offered by the finding that Rac1 mutant Q61L, known to hydrolyze GTP poorly (29), was found indeed to contain 0.8 mol of nucleotide/mol of Rac1, of which 83.5% was GTP and 16.5%, GDP (mean of two determinations) (Fig. 1C). The fact that a major part of mutant Q61L shall remain in the GTP-bound state throughout protein purification was predicted by Xu et al. (30) and is now proven unequivocally. All other recombinant Rac1 mutants, used in the experiments to be described, were found to contain exclusively bound GDP (results not shown).
Effect of Trio on Oxidase Activation by Prenylated Rac1-GDP-We found earlier that prenylated Rac1 exchanged to either GTP␥S or GDP␤S supported oxidase activation in the amphiphile-and p47 phox -independent cell-free system with equal potency (11). In those experiments, Rac1 was subject to nucleotide exchange at low Mg 2ϩ concentration achieved by metal chelation by EDTA. The original purpose of the experiments, having led to the findings described in this report, was to study the effect of "enzymatic" as opposed to "chemical" nucleotide exchange on prenylated Rac1 to GTP on its ability to support oxidase activation. We first examined the ability of prenylated native Rac1 (found to contain only GDP and, therefore, to be referred to as Rac1-GDP) to activate the oxidase in the amphiphile-and p47 phox -independent cell-free system. As seen in Fig.  2A, only a modest, dose-dependent, activation was found.
We next assessed the effect of inducing nucleotide exchange on Rac1 to GTP, in a manner closest to the physiological mechanism, by using a GEF. For this purpose, we chose Trio, a GEF containing two distinct guanine nucleotide exchange domains, specific for Rac1 and Rho (17,31). A recombinant N-terminal segment of Trio (residues 1225-1537), including the Rac1-specific N-terminal Dbl homology (DH) and pleckstrin homology (PH) domains, to be referred to as TrioN, was found to stimulate GDP to GTP exchange on Rac1 (21). The ability of TrioN to modulate oxidase activation by prenylated Rac1-GDP was assayed in the presence of exogenous GTP␥S and, as a control, in its absence. Surprisingly, TrioN exhibited a pronounced potentiating effect on oxidase activation by prenylated Rac1-GDP in the absence of added GTP␥S ( Fig. 2A  The enhancing effect of TrioN was seen only in the presence of both prenylated Rac1 and p67 phox (Table I) and was related to the relative concentrations of Rac1 and p67 phox present in the reaction. Thus, significantly higher activities were obtained when p67 phox was in excess over Rac1 than when the two components were present in equimolar amounts (results not shown). TrioN was inactive when heat-denatured and had no effect on prenylated Cdc42Hs-GDP, a Rho subfamily GTPase lacking oxidase-activating ability (Table I). p67 phox could be replaced by p67 phox truncated at residue 212, although both the basal and the TrioN-enhanced activity were lower than in the presence of non-truncated p67 phox (Fig. 2B). This suggests that the tetratricopeptide repeat region, involved in binding of p67 phox to Rac (32), and the activation domain (5) play important but not exclusive roles in TrioN-dependent activation.
TrioN exhibited no enhancing effect on oxidase activation by prenylated Rac1, exchanged to GTP␥S and freed of unbound nucleotide on a desalting column (Fig. 2C). It is of interest that although the level of oxidase activation achieved with prenylated Rac1-GTP␥S was higher than that measured with native Rac1 (Rac1-GDP), it was considerably lower than that found with Rac1-GDP combined with TrioN, in the absence of free GTP␥S. This suggests that the conformational change(s) occurring in Rac1 as a consequence of nucleotide exchange to GTP␥S might, at least in part, be different from those taking place under the influence of TrioN, although they both lead to an enhancement in the oxidase activating capacity of Rac1.
TrioN had only a minor enhancing effect on oxidase activation by nonprenylated Rac1-GDP in the canonical amphiphileand p47 phox -dependent system and did not convey an activating potential to nonprenylated Rac1-GDP, known to be inactive in the absence of amphiphile and p47 phox (both situations are shown in Fig. 2D). This was not due to a requirement for prenylation for interaction of Rac1 with TrioN to take place because nonprenylated Rac1 was found to bind TrioN (20).
We recently showed that a chimera consisting of the Nterminal 212 residues of p67 phox and full-length Rac1, when prenylated and in the GDP-bound form, was capable of moderate oxidase activation in the absence of an amphiphile and p47 phox (12). In marked contrast to its enhancing effect on a combination of prenylated Rac1-GDP and p67 phox -(1-212) (Fig.  2B), TrioN did not potentiate the activity of a [p67 phox -(1-212)-Rac1-(1-192)] chimera in GDP-bound form (Fig. 2E). It is possible that Rac1 chimerized to p67 phox is less accessible for interaction with TrioN or less "flexible" to respond to such an interaction by a change in conformation.
We performed dose-response experiments in which the concentration of either prenylated Rac1-GDP or TrioN was varied from 10 to 500 nM, although the concentration of the counterpart component was kept constant at 300 nM. As seen in Fig. 2, A and F, in both situations, plateaus were reached when the concentration of the component added in varying amounts was close to 300 nM. This is suggestive of the possibility that [Rac1-TrioN] complexes with a 1:1 stoichiometry are involved in activation, but more rigorous evidence is required to sustain such a proposal.

Enhancement of Oxidase Activation by TrioN Occurs in the Absence of Nucleoside
Triphosphates-Because of the finding that TrioN acts on Rac1 in the absence of exogenous GTP, it was essential to ensure that GTP was not introduced inadvertently into the assay as a contaminant of one of the components, or generated enzymatically by ATP-dependent conversion of GMP or GDP to GTP (33). Consequently, we examined the solubilized membrane preparation, recombinant baculovirusderived p67 phox , and recombinant TrioN for a possible content of ATP, GTP, GDP, or GMP. 10-mg amounts of solubilized and dialyzed membrane (corresponding to an amount 2000 times higher than that present in an individual oxidase assay mixture) and 20-nmol amounts of p67 phox and TrioN (corresponding to amounts 210 times higher than the maximal amounts present in the assay mixture) were subjected to the nucleotide extraction procedure applied to Rac1. The extracts were examined for the presence and quantity of nucleotides by chromatography on a Partisil 10 SAX anion exchange column, in relation to established nucleotide standards. No adenine or guanine nucleotides were detected in any of the samples examined. We conclude that, even if the presence of GTP-generating enzymes in the only non-recombinant component present in the assay (the membrane) is suspected, no nucleotides with the potential to serve as phosphate donors or acceptors were detected. This establishes with certainty that TrioN exerts it action on Rac1-GDP independently of nucleotide exchange to GTP.

Rac1-TrioN Interaction and a Catalytic Event
Are Required for Enhancement of Oxidase Activation by TrioN-It was demonstrated that specific residues on Rac1, located in the switch I and II regions, are essential for either binding of TrioN or the subsequent catalytic event leading to nucleotide dissociation  (20). We examined the importance of these residues in the oxidase activation enhancing effect of TrioN by using the Rac1 mutants Y32A (switch I) and W56F, Q61L, and Y64A (switch II). Rac1 Y32A was shown to retain the ability to bind TrioN but to lose its ability to respond to TrioN by GDP dissociation. The switch II mutants were found to be unable to bind TrioN; of special interest is mutant W56F because Trp 56 appears to be the critical residue determining the Rac specificity of GEFs, including Trio (20). Among switch II mutants, Rac1 Q61L stands out because it was found to be predominantly in the GTP-bound form (Fig. 1C), unlike all the other mutants that contained only GDP. It was also reported to have a higher affinity for a yet unidentified oxidase component, most likely p67 phox (29). We also examined the responsiveness of the negative dominant mutant T17N, known to possess a markedly lower affinity for GTP (34) but an unimpaired ability to bind GEFs (reviewed in Ref. 35).
As a preliminary to examining their responsiveness to TrioN in the oxidase assay, we assessed the ability of the Rac1 mutants to respond to TrioN by nucleotide exchange from GDP to mant-GTP. As seen in Fig. 3, wild type Rac1-GDP reacted to TrioN by a vigorous change in the slope of mant-GTP uptake. All Rac1 mutants, in GDP-bound form, were unresponsive to TrioN, as evident in the lack of change in the slope of mant-GTP uptake, following the addition of TrioN (Fig. 3). It is of interest that mutants T17N and Q61L also evidenced the lowest spontaneous uptake of mant-GTP, in accordance with the reported impairment in GTP binding of mutant T17N (34) and the slow nucleotide exchange rate of mutant Q61L (30).
We next examined the effect of the mutations on the capacity of nonprenylated Rac1 to support oxidase activation in the cell-free system. Mutants T17N, Y32A, W56F, Q61L, and Y64A (all at a concentration of 300 nM) were assayed in an amphiphiledependent cell-free system consisting of membrane (5 nM cytochrome b 559 heme), p47 phox (300 nM), p67 phox (300 nM), nonprenylated native (unexchanged) wild type or mutant Rac1 (300 nM), and LiDS (130 M). All Rac1 mutants, with the exception of T17N and Q61L, exhibited unchanged oxidase activating ability, in the range of 103-114% that of wild type Rac1. Rac1 T17N was only 41% as active as wild type Rac1, whereas Rac1 Q61L was more active (128%) than wild type Rac1 (results represent means of two experiments). Finally, the influence of mutations on the ability of the prenylated form of Rac1 to support oxidase activation in the amphiphile and p47 phox -independent cell-free system was examined. To achieve a maximal effect, prenylated wild type Rac1 and mutants were exchanged to GTP␥S and assayed in a system consisting of membrane (5 nM cytochrome b 559 heme), p67 phox (300 nM), and prenylated wild type or mutant Rac1-GTP␥S (300 nM) in the absence of LiDS. The activating abilities of the mutants were 39 (Y32A), 52 (W56F), and 72% (Y64A) that of wild type Rac1 (results represent means of two experiments). Mutant Q61L was 3.75 times more active than wild type Rac1, in support of the hypothesis that it has a higher affinity for p67 phox .
As seen in Fig. 4, A and B, TrioN was incapable of enhancing oxidase activation supported by prenylated Rac1 mutants T17N and Y32A. Because both mutants were expected to bind TrioN normally but did not respond to TrioN by mant-GTP uptake, it appears that an event, subsequent to binding and related to the catalytic action of TrioN, is required for the enhancement of oxidase activation. TrioN had little or no oxidase activation potentiating effect on Rac1 mutants W56, Q61L, and Y64A, known to be unable to bind TrioN (a minor effect was evident on mutant Y64A) (Fig. 4, C-E). It appears FIG. 3. TrioN stimulates uptake of mant-GTP by wild type Rac1 but not by several Rac1 mutants. Wild type Rac1-GDP or Rac1 mutants in the GDPbound form were added to cuvettes, containing mant-GTP, at the points of time indicated. After a few minutes, TrioN was added. Fluorescence was recorded continuously. The effect of TrioN is shown by the acute change in the slope of fluorescence increase or by the lack of such change. For methodological details, see "Experimental Procedures." The panels illustrate representative experiments out of three performed with each Rac1 preparation.
that the basal oxidase activating ability of the prenylated mutants, especially T17N and Y32A, was also lower than that of wild type Rac1 (with the notable exception of Q61L), as already noted in the course of preliminary testing of the mutants in GTP-bound form at a single concentration. The reason for this is not obvious.
A special case is mutant Q61L, which was found to contain predominantly GTP. Its basal oxidase activating ability (Fig.  4D) was found to exceed significantly that of wild type Rac1 in both native (GDP-bound) and GTP␥S-bound (exchanged to GTP␥S) forms (Fig. 2, A and C Recently, a double mutant of TrioN was described, the mutations being located at the C terminus of the DH domain (N1406A/D1407A). 2 This mutant binds normally to Rac1 but fails to stimulate nucleotide exchange. We tested the ability of TrioN mutant N1406A/D1407A to enhance oxidase activation by prenylated Rac1-GDP in the presence of p67 phox and in the absence of amphiphile and p47 phox . As seen in Table I, the TrioN mutant failed to enhance oxidase activation. This finding offers additional support to the conclusion derived from the results obtained with Rac1 mutants T17N and Y32A that enhancement of oxidase activation by prenylated Rac1 requires, in addition to Rac1-TrioN interaction, yet another step associated with the catalytic action of TrioN.
Effect of TrioN Is Mediated by Mg 2ϩ Displacement-It was shown that nucleotide exchange by Trio is effected principally through the displacement of bound Mg 2ϩ (21). Therefore, we reasoned that the effect of TrioN on Rac1 could be mimicked by removal of Mg 2ϩ from Rac1 by the divalent cation chelator EDTA. As shown in Fig. 5 and Table I

DISCUSSION
These results point toward a novel mechanism of Rac activation, the essence of which is the induction of a conformational change in Rac consequent to its interaction with a GEF. Based on findings made with Rac1 mutants W56F and Y64A, binding of TrioN to Rac1 is an absolute requirement for oxidase activation. The lack of effect of TrioN on Rac1 mutants T17N and Y32A demonstrates that binding to TrioN, although required, is not sufficient for activation and that an event normally related to the nucleotide exchange promoting effect of TrioN is involved. However, the fact that TrioN was fully effective when added to Rac1-GDP in the absence of exogenous GTP indicates that the effect of TrioN occurs independently of actual nucleotide exchange. Based on a model of GEF action (37), it is likely that, under these conditions, a [TrioN-Rac1-GDP] complex is formed. It was found that in the complex of the DH-PH module of the GEF Tiam1 with Rac1, the conformation of the switch I and II regions and their vicinities are altered (28). We suggest that TrioN induces a conformational change in Rac1-GDP, normally related to nucleotide exchange to GTP but now taking place in the absence of such an exchange. This conformational change appears to be related to the displacement by TrioN of Mg 2ϩ bound to Rac1 (21), in agreement with the TrioN-mimicking effect of Mg 2ϩ depletion by EDTA. It was, indeed, reported that a conformational change, represented by the opening of the switch I region, took place in RhoA-GDP as the direct result of Mg 2ϩ dissociation induced by Li 2 SO 4 (38). Less pronounced conformational changes were also found to be induced by Mg 2ϩ depletion in the switch II and insert regions. We propose that the conformational change in Rac1, consequent to interaction with TrioN, in the absence of exogenous GTP, results in an increased affinity of Rac1 for another oxidase component. The principal candidates are p67 phox (change in switch I) (39) or cytochrome b 559 (change in the insert region) (40). We favor the hypothesis that a [TrioN-Rac1-GDP-p67 phox ] complex is formed that translocates to the membrane. Once there, p67 phox interacts with cytochrome b 559 and activates the oxidase. Support for this proposal is offered by the finding that conditions leading to high affinity binding of Rac to p67 phox , such as a Q61L mutation in Rac (29) or chimerization of Rac with p67 phox (12,22), are also the situations in which TrioN is incapable of further enhancement of oxidase activation. Support for the proposal that a [TrioN-Rac1-GDP-p67 phox ] ternary complex is formed, is offered by the finding of a similar [GEF-GTPase-effector] ternary complex, consisting of the minimal functional domains of Dbl, Cdc42Hs, and p21-activated kinase 1 (PAK1), in which PAK1 was activated. 3 An alternative to the [TrioN-Rac1-GDP-p67 phox ] complex model is the causation by TrioN of a conformational change in Rac1, in the absence of complex formation with TrioN, leading to an increased affinity of Rac1 for another oxidase component. Further work is required for proving the veracity of one of the two models.
TrioN is a sequence module consisting of the N-terminal DH and PH domains of Trio. An issue to be clarified is the relative importance of the DH and PH domains in the effect of TrioN on Rac1. The finding that the TrioN mutant N1406A/D1407A is inactive indicates that the DH domain is involved in the oxidase activation enhancing activity of TrioN. It has been shown that the N-terminal PH domain of Trio binds to acidic phospholipids and might serve as a membrane localizing signal (41). We cannot yet establish the possible involvement of the PH domain in the potentiating effect of Trio, but it is likely that a [TrioN-Rac1-GDP] complex will express a high affinity for the membrane because of the presence of two groups binding to acidic phospholipids, the prenylated polybasic C terminus of Rac1 and the PH domain of Trio.
Finally, it remains to be established whether activation of Rac1 by Trio, in the absence of nucleotide exchange, represents but a particular example of a property shared by other Racspecific GEFs and whether such a mechanism is at work in the intact cell. Recently, a concept is emerging looking upon Rac GEFs not merely as mediators of nucleotide exchange on Rac but also as factors directing Rac toward specific effector pathways (42).