Specificity and Structural Requirements of Phospholipase C-beta Stimulation by Rho GTPases Versus G Protein beta gamma Dimers*

Daria IllenbergerDagger, Claudia Walliser, Bernd Nürnberg, Maria Diaz Lorente, and Peter Gierschik

From the Department of Pharmacology and Toxicology, University of Ulm, Albert-Einstein-Allee 11, Ulm D-89081, Germany

Received for publication, August 13, 2002, and in revised form, October 25, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phospholipase C-beta 2 (PLCbeta 2) is activated both by heterotrimeric G protein alpha - and beta gamma - subunits and by Rho GTPases. In this study, activated Rho GTPases are shown to stimulate PLCbeta isozymes with the rank order of PLCbeta 2 > PLCbeta 3 >=  PLCbeta 1. The sensitivity of PLCbeta isozymes to Rho GTPases was clearly different from that observed for G protein beta gamma dimers, which decreased in the following order: PLCbeta 3 > PLCbeta 2 > PLCbeta 1 for beta 1gamma 1/2 and PLCbeta 2 > PLCbeta 1 >>> PLCbeta 3 for beta 5gamma 2. Rac1 and Rac2 were found to be more potent and efficacious activators of PLCbeta 2 than was Cdc42Hs. The stimulation of PLCbeta 2 by Rho GTPases and G protein beta gamma dimers was additive, suggesting that PLCbeta 2 activation can be augmented by independent regulation of the enzyme by the two stimuli. Using chimeric PLCbeta 1-PLCbeta 2 enzymes, beta gamma dimers, and Rho GTPases are shown to require different regions of PLCbeta 2 to mediate efficient stimulation of the enzyme. Although the catalytic subdomains X and Y of PLCbeta 2 were sufficient for efficient stimulation by beta gamma , the presence of the putative pleckstrin homology domain of PLCbeta 2 was absolutely required for the stimulation of the enzyme by Rho GTPases. Taken together, these results identify Rho GTPases as novel PLCbeta regulators, which mediate PLCbeta isozyme-specific stimulation and are potentially involved in coordinating the activation of PLCbeta 2 by extracellular mediators in intact cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-P2) 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 Ca2+ and protein kinase C isozymes, respectively. The mammalian PLC isozymes can be divided into four major families: PLCbeta , PLCgamma , PLCdelta , and PLCepsilon (1). The PLCbeta and PLCgamma subclasses have been shown to be regulated through G protein-coupled and protein-tyrosine kinase-linked receptors, respectively. The mechanisms by which PLCdelta isozymes and PLCepsilon are coupled to membrane receptors are less well understood (for recent reviews, see Refs. 1-4). Stimulation of PLCbeta , of which four isozymes (PLCbeta 1-PLCbeta 4) are known, is mediated by members of the alpha q subfamily of G protein alpha  subunits and, excepting PLCbeta 4, by G protein beta gamma dimers (1-4). Activated alpha q subunits stimulate PLCbeta in the rank order of efficacy of PLCbeta 1 >=  PLCbeta 3 > PLCbeta 2. PLCbeta 4 is also activated by alpha q subunits. The sensitivity of PLCbeta isozymes to beta gamma dimers decreases in the order: PLCbeta 3 > PLCbeta 2 > PLCbeta 1 (3, 4).

Mammalian PLCbeta isoforms are differentially expressed in various tissues and cell types (5, 6). At the protein level, PLCbeta 1 is highly expressed in the central nervous system but is also present in several other tissues, e.g. adrenal gland, parotid gland, lung, and kidney (7-9). The PLCbeta 2 polypeptide is present at high levels in neutrophils and cultured myeloid cells but has also been detected in other cells types and tissues, including platelets (10), T lymphocytes (11), osteoblasts (12), vascular and tracheal smooth muscle cells (13, 14), cerebellum (12), spleen, and thymus (15). Myeloid cells have been found to contain both PLCbeta 2 and PLCbeta 3 (9, 16). However, PLCbeta 2 appears to be predominantly important in these cells, because inactivation of the PLCbeta 2 gene caused an almost complete loss of formyl peptide receptor-stimulated inositol phosphate formation in mouse neutrophils (15). Furthermore, PLCbeta 2, but not PLCbeta 3, was activated by complement C5a and formyl peptide receptors in transfected cells (17). The latter findings are intriguing in light of the fact that PLCbeta 3 has been shown to be stimulated to a remarkable extent by G protein beta gamma dimers in cell-free assays (9). In addition to myeloid cells, various other cell types and tissues contain the PLCbeta 3 isoform (8, 9). In contrast, the PLCbeta 4 protein shows a more limited tissue distribution and is primarily found in the retina and in specific regions of the brain (18, 19).

We have previously reported the identification of a PLCbeta 2-stimulating GTP-binding protein present in cytosolic fractions of bovine neutrophils (20). This cytosolic protein was shown to be a member of the Rho subfamily of GTPases, Cdc42Hs and/or Rac, associated with the Rho GDP dissociation inhibitor LyGDI (21). Rho GTPases form a subgroup of the Ras superfamily of GTP-binding proteins that have been shown to regulate a wide spectrum of cellular functions, including gene expression, cell cycle progression, and reorganization of the actin cytoskeleton (22-25). The activity of the Rho GTPases is determined by the ratio of their GTP/GDP-bound forms, which is regulated by at least three regulatory proteins: guanine nucleotide dissociation inhibitors, guanine nucleotide exchange factors, and GTPase-activating proteins (25).

Using purified proteins, we have previously demonstrated that PLCbeta 2 is activated by GTPgamma S-liganded Cdc42Hs and Rac1, but not by RhoA, through direct protein-protein interaction (21). This stimulation has been shown to be independent of LyGDI but to require both C-terminal processing of the Rho GTPases and the integrity of their effector regulating domain (21). Like G protein beta gamma dimers, activated Rho GTPases stimulated a deletion mutant of PLCbeta 2, PLCbeta 2Delta , lacking a C-terminal region necessary for stimulation by G protein alpha q subunits (21). The specificity of PLCbeta stimulation by Rho GTPases and the mechanisms of enzyme activation by Rho GTPases remained unknown. The goal of the study was to elucidate the sensitivity of PLCbeta isozymes to stimulation by Rho GTPases versus G protein beta gamma dimers, known activators of these enzymes, and to identify and to compare the structural requirements of PLCbeta stimulation by these two regulatory proteins. The results show that the three PLCbeta isozymes tested, PLCbeta 1, PLCbeta 2, and PLCbeta 3, are differentially sensitive to stimulation by activated Rho GTPases and beta gamma dimers. The specificity of Rho GTPase-mediated PLCbeta stimulation also differs from that reported for alpha q-mediated PLCbeta stimulation, with PLCbeta 2 being considerably less sensitive than PLCbeta 1 and PLCbeta 3 (1-4). This study identifies PLCbeta 2 as the PLCbeta isozyme most sensitive to stimulation by Rho GTPases, especially Rac1 and Rac2. Using PLCbeta 1-PLCbeta 2 chimeras constructed on the basis of structural domains predicted by the crystal structure of PLCdelta 1, we demonstrate that the presence of the catalytic subdomains X and Y of PLCbeta 2 is sufficient for beta gamma -dimer stimulation. In contrast, the presence of the putative pleckstrin homology (PH) domain of PLCbeta 2 is absolutely required for stimulation of the enzyme by activated Rho GTPases. Taken together, the results demonstrate, for the first time, the unique regulation of the activity of PLCbeta 2 by monomeric GTPases and G protein beta gamma dimers requiring different structural elements of this enzyme.

    EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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Recombinant PLCbeta Isoforms, C-terminally Deleted PLCbeta s Mutants, and PLCbeta 1-PLCbeta 2 Chimeras-- Construction of recombinant baculoviruses for expression of the bovine PLCbeta 1 and human PLCbeta 2 have been described (26, 20). The cDNA of human PLCbeta 3 (27) was cloned into the EcoRI site of the baculovirus transfer vector pVL1393 (Invitrogen, Carlsbad, CA).

The cDNAs of C-terminally deleted PLCbeta constructs were generated such that the proteins retained their C-terminal-most portions (GENPGKEFDTPL, PLCbeta 1; QDPLIAKADAQESRL, PLCbeta 2; and GADSESQEENTQL, PLCbeta 3), which had been used as synthetic peptides to generate PLCbeta subtype-specific antisera in rabbits (28). PLCbeta 2Delta , a deletion mutant of human PLCbeta 2, lacking a C-terminal region (Phe819-Glu1166) necessary for stimulation by alpha q, has been shown previously to be indistinguishable from wild-type PLCbeta 2 in terms of its interaction with PI-4,5-P2, Ca2+, and beta gamma dimers (29). The deletion mutants PLCbeta 1Delta and PLCbeta 3Delta , lacking the corresponding C-terminal regions, Val817-Leu1203 and Ala873-Ser1216, respectively, were generated by the PCR overlap extension method (30). Two PCR amplifications were performed using bovine PLCbeta 1 cDNA (GenBankTM accession number J03137) cloned into pVL1392 as template and the following two pairs of oligonucleotides as primers: 5'-CGTGGATTCATCTAACTATATGCC-3' (upstream, sense), 5'-GTTTTCTCCTTCATATCGGATTGGATTTGACAAAGC-3' (internal, antisense), 5'-TCCAATCCGATATGAAGGAGAAAACCCAGGAAAAGAG-3' (internal, sense), and 5'-CGCATGTTAACCCAAAGATATGG-3' (downstream, antisense). The two amplified fragments, flanking the sequence to be deleted, were re-annealed and re-amplified using the upstream and downstream primers. The final PCR product and the pVL1392-PLCbeta 1 were digested with NheI and BamHI, and the wild-type fragment was replaced by the corresponding mutant fragment. In the case of PLCbeta 3Delta , the two fragments flanking the upstream and downstream regions of the deletion were amplified using pVL1393-PLCbeta 3 as template and the following two pairs of oligonucleotides as primers: 5'-TGAGCGAGAGCTCCGCG-3' (upstream, sense), 5'-ACAGGTGCCCGCTCCTCTGGTCCATCAGGC-3' (internal, antisense), 5'-GATGGACCAGAGGAGCGGGCACCTGTCGG-3' (internal, sense), and 5'-GGTTCTTGCCGGGTCCCAGG-3' (downstream, antisense). The final PCR product and the vector pVL1393-PLCbeta 3 were digested with BlpI, and the wild-type fragment was replaced by the corresponding mutant fragment.

The PLCbeta 1-PLCbeta 2 chimeras were generated by the PCR overlap extension method. In chimera A, the N-terminal amino acids of PLCbeta 2 (residues 1-138) were replaced by the corresponding residues of PLCbeta 1 (residues 1-142), so that the putative PH domain was substituted. Substitution of the PH domain and the putative four EF-hand motives of PLCbeta 2 (residues 1-303) for the corresponding residues of PLCbeta 1 (residues 1-307) resulted in chimera B. Chimera A was constructed from the vectors pVL1392-PLCbeta 1 and pVL1393-PLCbeta 2Delta by PCR amplification using the following two pairs of primers: 5'-GAATTCATGGCCGGGGCACAGC-3' (upstream, sense), 5'-GGGAGGCGTTGGCCGTCAGCAGGTTTGTTGCCAAA-3' (internal, antisense), 5'-TTTGGCAACAAACCTGCTGACGGCCAACGCCTCCC-3' (internal, sense), 5'-GGATCCTCAGAGGCGGCTCTCCT-3' (downstream, antisense). The two amplified fragments were re-annealed and re-amplified using the upstream and the downstream primers, which introduced an EcoRI and a BamHI recognition site, respectively. Chimera B was constructed using the same upstream and downstream primers and the following two internal primers: 5'-GAAGAAAATGGAGTCGTTGCCCAGGACAAGCTGCT-3' (internal, antisense) and 5'-CAGCTTGTCCTGGGCAACGACTCCATTTTCTTCTC-3' (internal, sense). The final fragments were then ligated at their EcoRI and BamHI sites, and the resulting constructs were inserted into the baculovirus transfer vector pVL1392.

The entire PCR-amplified regions were sequenced and found to be identical to the expected sequences. Production of recombinant baculoviruses, expression, and isolation of PLCbeta isoforms were carried out according to published protocols (26).

Recombinant Rho GTPases-- The production of recombinant baculoviruses using BaculoGoldTM DNA (BD Pharmingen, San Diego, CA) and pVL1393 transfer vectors containing the respective Rho GTPase cDNA ligated into the BamHI/EcoRI site has been described previously (21). Isoprenylated membrane-bound Rho GTPases were solubilized by extracting the membranes with buffer containing 23 mM sodium cholate as described previously (21). Rac2-LyGDI heterodimers were purified from cytosolic fractions of baculovirus-infected insect cells (31).

[35S]GTPgamma S Binding-- Binding of [35S]GTPgamma S to Rho GTPases was assayed as described previously (21) with minor modifications. Briefly, samples (0.05-0.5 µg of protein extracted from membranes of baculovirus-infected insect cells with buffer containing sodium cholate) were incubated at 30 °C in an incubation mixture (40 µl) containing 25 mM Hepes-NaOH, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 20 mM MgCl2, 100 mM NaCl, 0.1% (v/v) GENAPOL® C-100 (Calbiochem, La Jolla, CA, HPLC grade), and 100 nM [35S]GTPgamma S (296 GBq/mmol). The incubation was terminated after 6 h as described earlier (31). We also used [35S]GTPgamma S binding to estimate the concentrations of activated GTPgamma S-bound Rho GTPases under conditions of the PLC assay (see below) in the presence of 100 µM [35S]GTPgamma S (115 GBq/mmol).

Phospholipase C Assay-- Phospholipase C activity was determined as described (21) with minor modifications. In brief, 5 µl of detergent-solubilized Rho GTPase and/or 5 µl of purified beta gamma were supplemented with 10 µl of soluble fraction of PLCbeta -baculovirus-infected insect cells and incubated at 25 °C for time periods as indicated in the figure legends in a volume of 60 µl containing 50 mM HEPES-NaOH, pH 7.2, 70 mM KCl, 3 mM EGTA, 2 mM dithiothreitol, 33 µM [3H]PI-4,5-P2 (185 GBq/mol), 536 µM phosphatidylethanolamine, and 150 nm free Ca2+. In all experiments comparing the effects of Rac2 and beta gamma , the final concentration of sodium cholate was 2 mM. Only beta gamma -mediated stimulation of wild-type versus C-terminally deleted PLCbeta isozymes (Fig. 6) was measured in the absence of sodium cholate. Results obtained in control experiments using purified PLCbeta 2Delta (31) were indistinguishable from that obtained with soluble fractions of PLCbeta 2Delta -baculovirus-infected insect cells.

Miscellaneous-- Purification of beta 1gamma 1 isolated from bovine retinal rod outer segment membranes has been described elsewhere (32). The method used to purify membrane-associated recombinant beta 1,5gamma 2-His dimers is described in a previous study (33). SDS-PAGE, immunoblotting, and determination of protein concentrations were performed as described previously (21). Polyclonal antibodies against Rho GTPases were from Santa Cruz Biotechnology (Santa Cruz, CA). Specific antisera against PLCbeta 1, PLCbeta 2, and PLCbeta 3 were a kind gift from Dr. P. J. Parker (28). The data are presented as means ± S.D. of triplicate determinations.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Stimulation of Recombinant PLCbeta Isozymes by beta gamma Dimers-- To determine the specificity of PLCbeta stimulation by G protein beta gamma dimers, the PLCbeta isozymes PLCbeta 1, PLCbeta 2, and PLCbeta 3 were produced as recombinant proteins in Sf9 insect cells. Fig. 1A shows that all three isozymes were expressed as soluble proteins in infected insect cells and migrated at molecular masses of ~150, 140, and 160 kDa, respectively, corresponding to the masses described for the native PLCbeta isozymes (34, 35, 28). When the amounts of the three PLCbeta isozymes were normalized according to their maximal activity at 1 mM free Ca2+ and 3.3 mM sodium deoxycholate (not shown) and then assayed at a more physiological Ca2+ concentration of 150 nM, recombinant PLCbeta 2 displayed an ~3-fold higher basal activity than recombinant PLCbeta 1 and PLCbeta 3 (Fig. 1B). In all experiments shown in this study, the amounts of the three recombinant PLCbeta preparations were adjusted to equal basal phospholipase C activity at 150 nM free Ca2+ in the presence of 2 mM sodium cholate. Under these conditions, purified beta gamma dimers of bovine retinal transducin (beta 1gamma 1, 300 nM) activated PLCbeta 1 and PLCbeta 2 only slightly (1.6-fold and 3.0-fold, respectively), but caused a marked (~20-fold) stimulation of PLCbeta 3 (Fig. 2).


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Fig. 1.   Expression of PLCbeta 1, PLCbeta 2, and PLCbeta 3 in baculovirus-infected insect cells. A, soluble fractions of Sf9 insect cells that had been infected with baculovirus-encoding bovine PLCbeta 1 (10 µg of protein) (lane 1), human PLCbeta 2 (30 µg of protein) (lane 2), and human PLCbeta 3 (60 µg of protein) (lane 3) were subjected to SDS-PAGE and immunoblotting using PLCbeta subtype-specific antisera. The apparent molecular weights of the marker proteins are indicated. The PLCbeta 1, PLCbeta 2, and PLCbeta 3 migrated at ~150, 140, and 160 kDa, respectively. Additional immunoreactive proteins of lower molecular masses were detected in all three preparations. Because soluble fractions of non-infected insect cells did not contain proteins reactive with the antisera used in this experiment (not shown), these proteins most likely correspond to proteolytic fragments of the full-length PLC isozymes. B, aliquots of the three soluble fractions were incubated in the presence of 150 nM free Ca2+ for the times indicated at the abscissa with phospholipid vesicles containing PI-4,5-P2. The reaction was terminated by the addition of chloroform/methanol/concentrated HCl, and the mixture was analyzed for inositol phosphates. See "Experimental Procedures" for details. Prior to this experiment, the amounts of the samples containing soluble PLCbeta 1, PLCbeta 2, and PLCbeta 3 were adjusted to give equal maximal PLC activities in the presence of 1 mM free Ca2+ and 3.3 mM sodium cholate (42) (not shown) (PLCbeta 1, 0.4 µg of protein/sample; PLCbeta 2, 1.5 µg of protein/sample; PLCbeta 3, 5.6 µg of protein/sample).


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Fig. 2.   Stimulation of PLCbeta 1, PLCbeta 2, and PLCbeta 3 by beta 1gamma 1. Aliquots of soluble fractions of insect cells expressing PLCbeta 1 (0.3 µg of protein/sample), PLCbeta 2 (0.5 µg of protein/sample), or PLCbeta 3 (4.0 µg/sample) were incubated for the times indicated at the abscissa in the absence (open symbols) or presence (closed symbols) of 300 nM beta 1gamma 1 purified from bovine retinal rod outer segments with phospholipid vesicles containing PI-4,5-P2. The incubation was performed at 150 nM free Ca2+.

Stimulation of Recombinant PLCbeta Isozymes by Rho GTPases-- To investigate the specificity of PLCbeta 2 stimulation by Rho family members, the recombinant Rho GTPases Rac1, Rac2, and Cdc42Hs were produced in baculovirus-infected insect cells, extracted from the membrane of infected cells with detergent-containing buffer, and reconstituted with a recombinant C-terminal deletion mutant of PLCbeta 2, PLCbeta 2Delta , in the presence of 100 µM GTPgamma S. Fig. 3 shows that both Rac1 and Rac2 caused a marked (~13-fold) stimulation of PLCbeta 2Delta . Rac2 was slightly more potent than Rac1. Thus, half-maximal stimulation was observed at ~40 nM Rac2 and 100 nM Rac1. Cdc42Hs was a less potent (EC50: 400 nM) and less efficacious (~8-fold) stimulator of PLCbeta 2Delta than Rac1 and Rac2. In additional experiments, we observed a similar rank order of potency of Rho GTPases (Rac2 >=  Rac1 > Cdc42Hs) to stimulate PLCbeta 2 when full-length enzyme rather than PLCbeta 2Delta was used (not shown). RhoA had no effect on full-length PLCbeta 2 or PLCbeta 2Delta when tested under the same conditions (not shown).


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Fig. 3.   Stimulation of PLCbeta 2Delta by the human Rho GTPases Cdc42Hs, Rac1, and Rac2. The recombinant Rho GTPases were extracted with buffer containing sodium cholate from membranes of baculovirus-infected insect cells and incubated at increasing concentrations with soluble proteins of insect cells expressing PLCbeta 2Delta (0.2 µg of protein/sample) and phospholipid vesicles containing PI-4,5-P2. PLC activity was measured for 1 h in the presence of 100 µM GTPgamma S. The concentrations of Rac1 (filled triangles), Rac2 (filled circles), and Cdc42Hs (filled squares) were estimated by determining the binding of [35S]GTPgamma S under the same buffer conditions. The Rho GTPases did not affect the activity of PLCbeta 2Delta in the presence of 100 µM GDP (not shown).

Next, the most potent Rho GTPase, Rac2, was incubated at increasing concentrations in the presence of either GDP or GTPgamma S with soluble preparations of recombinant wild-type PLCbeta 1, PLCbeta 2, and PLCbeta 3. Fig. 4 shows that PLCbeta 2 was clearly the PLCbeta isoform most sensitive to stimulation by GTPgamma S-activated Rac2. Thus, half-maximal and maximal (~4-fold) stimulation was observed at approximately 80 and 500 nM Rac2, respectively. Rac2 also appeared to stimulate PLCbeta 2 in the presence of GDP, albeit to a much lesser (~1.6-fold) extent. At high concentrations of Rac2, a reduction of PLCbeta 2 stimulation was observed both in the presence of GDP and GTPgamma S, suggesting an inhibitory effect of the membrane extracts on PLC activity. PLCbeta 1 and PLCbeta 3 were also activated by Rac2, but to a much lower extent and only at much higher concentrations of GTPgamma S-activated Rac2 (>= 1 µM). Additional measurements (not shown) of PLCbeta stimulation by C-terminally modified Cdc42Hs and Rac1 revealed an at least 10-fold higher potency of these GTPases toward PLCbeta 2 than toward PLCbeta 1 or PLCbeta 3. RhoA, a Rho GTPase incapable of stimulating PLCbeta 2 (21), affected neither PLCbeta 1 nor PLCbeta 3 activity under the same conditions. These data show that Rho GTPases stimulate PLCbeta 2 with a rank order of potency of Rac2 >=  Rac1 > Cdc42Hs and that, among the PLCbeta isoforms tested, PLCbeta 2 is most sensitive to stimulation by activated Rac2.


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Fig. 4.   Stimulation of PLCbeta 1, PLCbeta 2, and PLCbeta 3 by Rac2. Aliquots of soluble fractions of Sf9 cells expressing PLCbeta 1 (0.3 µg of protein/sample), PLCbeta 2 (0.5 µg of protein/sample), or PLCbeta 3 (4.0 µg of protein/sample) were incubated at increasing concentrations of Rac2 extracted from membranes of baculovirus-infected insect cells with buffer containing sodium cholate. Phospholipase C activity was measured with phospholipid vesicles containing PI-4,5-P2. The incubation was performed for 10 min at 150 nM free Ca2+ either in the presence of 100 µM GDP (empty circles) or in the presence of 100 µM GTPgamma S (filled circles).

Comparison of Full-length and C-terminally Deleted PLCbeta Isozymes-- A truncated PLCbeta isozyme related to PLCbeta 3 has previously been reported to be remarkably sensitive to activation by beta gamma dimers (36). Similarly, C-terminal truncation of PLCbeta 3 from human platelets by calpain has been shown to result in a marked augmentation of beta gamma stimulation (37). PLCbeta 1 has been shown to be cleaved by calpain between residues 880 and 881, generating two fragments of 100 and 45 kDa, respectively (38). The presence of ~45- to 50-kDa proteins in the preparations of recombinant PLCbeta isozymes, which are likely to represent C-terminal proteolytic fragments of the enzymes (cf. Fig. 1), raised the possibility that the observed order of PLCbeta stimulation by Rac2 was a consequence of C-terminal proteolysis of the PLCbeta isozymes. To challenge this hypothesis, we examined and compared the ability of Rac2 to stimulate the activity of C-terminal deletion mutants of PLCbeta 1, PLCbeta 2, and PLCbeta 3. A schematic representation of the wild-type and mutant PLCbeta isozymes is shown in Fig. 5A. As shown in Fig. 5B, all three mutants were expressed in baculovirus-infected insect cells as soluble proteins and migrated on SDS-polyacrylamide gels at the expected molecular weights. The deletion mutants displayed Ca2+ sensitivities indistinguishable from those of their wild-type counterparts (not shown). Reconstitution of the wild-type and mutant PLCbeta isoforms with GTPgamma S-activated Rac2 (500 nM) or beta 1gamma 1 (300 nM) showed that, surprisingly, the deletion of the C-terminal regions of each PLCbeta isozyme did not change the efficiency of beta 1gamma 1 to cause PLCbeta stimulation (Fig. 6). In agreement with earlier findings (20), the removal of the C-terminal part of PLCbeta 2 enhanced the extent of Rac2-mediated stimulation of the enzyme. Importantly, however, no enhancement of Rac2-mediated stimulation was observed for the C-terminally deleted variants of PLCbeta 1 and PLCbeta 3. Therefore, removal of the C-terminal regions of PLCbeta isozymes does not change the rank order of specificity of PLCbeta stimulation by both Rac2 and G protein beta gamma subunits.


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Fig. 5.   Schematic representation of wild-type phospholipases C-beta (PLCbeta ) and their C-terminally deletion mutants (PLCbeta Delta ) and expression of the deletion mutants in Sf9 cells. A, the structural organization of the PLCbeta isozymes, each made up of a putative pleckstrin homology domain (PH), four EF-hand motifs (4EF), the catalytic subdomains X and Y, and a C2 domain (C2), is based on the crystal structures of mammalian PLCdelta 1 (57, 58). The antibody recognition site (AB) and the position of the calpain cleavage site in PLCbeta 1 (arrow) are indicated. B, aliquots of soluble fractions (40 µg of protein/sample) of Sf9 cells expressing PLCbeta 1Delta (lane 1), PLCbeta 2Delta (lane 2), and PLCbeta 3Delta (lane 3) were subjected to SDS-PAGE and immunoblotting using specific antisera reactive against the respective PLCbeta isozymes.


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Fig. 6.   Stimulation of full-length PLCbeta enzymes and C-terminally deleted PLCbeta Delta enzymes by G protein beta gamma dimers and Rac2. Left panels, soluble fractions of insect cells expressing PLCbeta 1 (1.3 µg of protein/sample), PLCbeta 1Delta (0.25 µg of protein/sample), PLCbeta 2 (0.5 µg of protein/sample), PLCbeta 2Delta (1.4 µg of protein/sample), PLCbeta 3 (1.5 µg of protein/sample), or PLCbeta 3Delta (1.0 µg of protein/sample) were incubated with Rac2 (500 nM) extracted from membranes of baculovirus-infected insect cells with buffer containing sodium cholate. The incubations were performed for 8 min (upper left panel), 10 min (middle left panel), and 30 min (lower left panel) in the absence (empty bars) or in the presence (hatched bars) of 100 µM GTPgamma S. Right panels, soluble fractions of insect cells expressing PLCbeta 1 (1.3 µg of protein/sample), PLCbeta 1Delta (0.25 µg of protein/sample), PLCbeta 2 (1.0 µg of protein/sample), PLCbeta 2Delta (1.0 µg of protein/sample), PLCbeta 3 (2.1 µg of protein/sample), or PLCbeta 3Delta (1.0 µg of protein/sample) were incubated either in the absence (empty bars) or in the presence of 300 nM beta 1gamma 1 (hatched bars) for 6 min (upper right panel), 7 min (middle right panel), or 4 min (lower right panel). Phospholipase C activity was measured at 150 nm free Ca2+ in the presence of phospholipid vesicles containing PI-4,5-P2. The results are given as the percentage of the basal activities, which were set to 100%. For any given pair of wild-type and mutant PLCbeta isozyme, the basal activities varied by less than a factor of two (not shown).

Simultaneous Stimulation of PLCbeta 2 by Rac2 and beta gamma Subunits-- The next experiments were designed to examine whether the different specificities of PLCbeta isozyme stimulation by Rho GTPases and beta gamma dimers may reflect independent regulation of PLCbeta 2 by both stimulators. We have previously shown that beta 5gamma 2-His is a more potent stimulator of PLCbeta 2Delta than is beta 1gamma 2-His and that the latter, but not the former beta gamma dimer, activates PLCbeta 3 (33). A comparison of the effects of beta 5gamma 2-His and beta 1gamma 2-His on the activity of wild-type PLCbeta 1, PLCbeta 2, and PLCbeta 3 is shown in Fig. 7. Among the PLCbeta isoforms tested, PLCbeta 3 was the isoform most sensitive to stimulation by beta 1gamma 2-His, followed by PLCbeta 2 and PLCbeta 1, which was hardly activated by this beta gamma preparation. In marked contrast, beta 5gamma 2-His proved to be a potent and efficacious activator of full-length PLCbeta 2, but it did not affect full-length PLCbeta 1 or PLCbeta 3. Because activation of PLCbeta 2 by beta 5gamma 2 occurred at low concentrations (EC50: ~10 nM) and reached saturation within the range of beta gamma dimer concentrations tested, beta 5gamma 2-His was chosen as the beta gamma dimer to compare the effects of Rac2 and beta gamma dimers on PLCbeta 2 activity when added alone or in combination at maximally effective concentrations (Fig. 8). Because stimulation of PLCbeta 2 by beta gamma dimers is sensitive to high concentrations of detergent (32), detergent-free Rac2-LyGDI heterodimers were used as a source of Rac2. In our hands, Rac2-LyGDI heterodimers and monomeric Rac2 stimulated PLCbeta 2 with equal potency and efficacy in the presence of phospholipid vesicles containing the phospholipase C substrate PI-4,5-P2 (31). Fig. 8 shows that addition of GTPgamma S allowed activation of Rac2 from the heterodimer (EC50: ~20 nM) leading to an 3-fold stimulation of PLCbeta 2Delta and that stimulation of the enzyme was observed both in the absence and in the presence of beta 5gamma 2-His (EC50: ~20 nM). The extent of stimulation by GTPgamma S-activated Rac2-LyGDI was additive with that obtained by beta 5gamma 2-His, suggesting independent stimulation of PLCbeta 2 by Rho GTPases and beta gamma subunits. Interestingly, partial inhibition of beta 5gamma 2-His-mediated PLCbeta 2 stimulation by Rac2-LyGDI was measured in the presence of GDP. Because there was no effect of Rac2-LyGDI on basal PLCbeta 2 activity, this result may indicate that inactive Rac2-LyGDI may interact with beta 5gamma 2-His and/or noncompetitively interfere with beta 5gamma 2-His-mediated PLCbeta 2 activation.


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Fig. 7.   Stimulation of PLCbeta isozymes by beta 5gamma 2 and beta 1gamma 2 dimers. Soluble proteins of insect cells expressing PLCbeta 1 (0.3 µg of protein/sample), PLCbeta 2 (0.5 µg of protein/sample), or PLCbeta 3 (4.0 µg of protein/sample) were reconstituted with increasing amounts of purified recombinant beta 1gamma 2-His (filled triangles) and beta 5gamma 2-His (filled squares). PLC activity was measured for 30 min at 150 nM free Ca2+ in the presence of phospholipid vesicles containing PI-4,5-P2.


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Fig. 8.   Effect of G protein beta 5gamma 2 dimers on stimulation of PLCbeta 2Delta by Rac2-LyGDI. Purified PLCbeta 2Delta (0.5 µg/sample) was incubated in the absence (empty and filled triangles) or presence (empty and filled circles) of 30 nM beta 5gamma 2-His and increasing concentrations of purified Rac2-LyGDI with phospholipid vesicles containing PI-4,5-P2. The incubation was performed in the presence of 100 µM GDP (empty symbols) or 100 µM GTPgamma S (filled symbols).

Structural Requirements of PLCbeta Stimulation by Rho GTPases versus beta gamma Dimers-- The independent stimulation of PLCbeta 2 by beta gamma subunits and Rho GTPases prompted us to delineate the structural elements of PLCbeta 2 required for enzyme activation. The site of interaction of PLCbeta 2 with beta gamma was localized by others to the region between the catalytic subdomain Y residues Glu574 and Lys583 (39, 40). However, beta gamma dimers have also been reported to bind to the isolated PH domains of PLCbeta 1 and PLCbeta 2 (41). To identify the sites on PLCbeta 2 relevant for activation by Rac2 and beta gamma , we took advantage of the fact that both stimulators elicited only a slight effect on PLCbeta 1Delta , but markedly activated PLCbeta 2Delta . The effects of the two stimulators were examined on the activity of PLCbeta 1-PLCbeta 2 chimera in which N-terminal portions of PLCbeta 2Delta had been replaced by the corresponding regions of PLCbeta 1Delta (Fig. 9). Two chimera, designated A and B, carrying the putative PH domain and the PH domain together with the four EF-hand motifs of PLCbeta 1Delta , respectively, were analyzed. A third chimera, comprising the PH domain, the EF-hand motifs, the catalytic subdomain X of PLCbeta 1Delta , and the catalytic subdomain Y of PLCbeta 2Delta , was catalytically inactive and hence not used for further analysis. Although chimera A was expressed in baculovirus-infected insect cells at levels considerable lower than were PLCbeta 2Delta and chimera B, the two chimeras and PLCbeta 2Delta were indistinguishable in terms of the dependence of their catalytic activity on Ca2+ (not shown). Fig. 10 shows that substitution of the N-terminal portions of PLCbeta 2Delta for the corresponding regions of PLCbeta 1Delta led to reduction of the degree of beta 1gamma 1-mediated stimulation. Specifically, at the highest concentration of beta 1gamma 1 tested (4 µM), the degrees of stimulation were 129-, 88-, 66-, and 8-fold for PLCbeta 2Delta , chimera A, chimera B, and PLCbeta 1Delta (Fig. 10A). Because the effects of beta 1gamma 1 did not reach saturation within the range of concentrations tested, it is currently unclear whether the reduced stimulation is due to a decrease in the potency or efficacy of beta 1gamma 1. Very interestingly, substitution of the putative PH domain of PLCbeta 2Delta for its counterpart of PLCbeta 1Delta caused an almost complete (>95%) loss of stimulation by GTPgamma S-activated Rac2 (Fig. 10B). In additional experiments (not shown), we found that chimeras A and B were also resistant to stimulation by GTPgamma S-activated Cdc42Hs. Taken together, these results not only show that the structural requirements of PLCbeta 2 stimulation by beta gamma dimers and by the Rho GTPases Rac2 and Cdc42Hs are distinct but also suggest that the putative PH domain of PLCbeta 2 is critically involved in mediating its activation by Rho GTPases.


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Fig. 9.   Linear representation of PLCbeta 2Delta , chimera A, chimera B, and PLCbeta 1Delta . The amino acid sequences were aligned using the ClustalW program contained in the PC/GENE software package (Intelligenetics, Mountain View, CA). Chimeras A and B are composed of the first 142 and 307 residues, respectively, of PLCbeta 1, followed by the 695 and 530 C-terminal residues, respectively, of PLCbeta 2Delta . AB, antibody recognition site.


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Fig. 10.   Stimulation of PLCbeta 2Delta , chimera A, chimera B, and PLCbeta 1Delta by beta gamma dimers and Rac2. A, aliquots of soluble fractions of insect cells expressing PLCbeta 2Delta (filled circles), chimera A (filled triangles), chimera B (filled squares), or PLCbeta 1Delta (filled diamonds) were incubated at increasing concentrations of beta 1gamma 1 with phospholipid vesicles containing PI-4,5-P2. The incubation was performed for 6 min at 150 nM free Ca2+. In B, PLC activity was measured as in A, except that the assay was performed at increasing concentrations of Rac2 in the presence of either 100 µM GDP (open symbols) or 100 µM GTPgamma S (filled symbols). Prior to this experiment, the amounts of the samples containing soluble PLC isoforms had been adjusted to give similar basal activities in the presence of 150 nM free Ca2+ and 2 mM sodium cholate (not shown) (PLCbeta 2Delta , 2 µg of protein/sample; chimera A, 60 µg of protein/sample; chimera B, 3 µg of protein/sample; PLCbeta 1Delta , 0.3 µg of protein/sample). There was no effect of beta 1gamma 1 (1 µM) or Rac2 (1 µM) on phospholipase C activity of soluble fractions (60 µg of protein/sample) of non-infected insect cells or insect cells infected with baculovirus encoding E. coli beta -galactosidase.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Specificity of PLCbeta Stimulation by Rho GTPases-- We have previously shown that the Rho GTPases Rac1 and Cdc42Hs, but not RhoA, stimulate the activity of PLCbeta 2 (21, 31). In this study, we demonstrate that the PLCbeta isozymes PLCbeta 1, PLCbeta 2, and PLCbeta 3 are differentially sensitive to stimulation by Rho GTPases and G protein beta gamma dimers. Activated Rac2 is shown to stimulate PLCbeta isozymes with the rank order of potency and efficacy of PLCbeta 2 > PLCbeta 3 >=  PLCbeta 1. This rank order is clearly different from the order observed for G protein beta gamma dimers, which is PLCbeta 3 > PLCbeta 2 > PLCbeta 1 for most beta gamma dimers, e.g. beta 1gamma 1 or beta 1gamma 2-His (cf. Figs. 2 and 7 and Refs. 9, 42, and 43) and PLCbeta 2 > PLCbeta 1 >>> PLCbeta 3 for beta 5gamma 2-His (cf. Fig. 7). Furthermore, the results reported here show that both Rac1 and Rac2 are more potent activators of PLCbeta 2 than Cdc42Hs. All three PLCbeta -stimulating Rho GTPases, Rac2, Rac1, and Cdc42Hs, preferentially activate PLCbeta 2 (PLCbeta 2 > PLCbeta 3 >=  PLCbeta 1) (not shown). RhoA did not stimulate any of the three PLCbeta isozymes tested under the same conditions (not shown). Notably, the specificity of PLCbeta activation by Rho GTPases described here did not depend on the presence of detergents, which could conceivably differentially affect the activities of the PLCbeta isozymes, because purified detergent-free Rac2-LyGDI heterodimers (31) stimulated the PLCbeta isozymes studied here with the same rank order as did detergent-solubilized monomeric, C-terminally processed Rac2 (not shown). Interestingly, the rank order of PLCbeta stimulation by Rho GTPases also differs from that reported for activation of these enzymes by members of the alpha q subfamily of G protein alpha  subunits: PLCbeta 1 >=  PLCbeta 3 > PLCbeta 2 (3, 4). Together with the observation that the C-terminal region of PLCbeta isozymes is required for the stimulation by alpha q subunits (43, 44), but not for stimulation by Rho GTPases (20 and Fig. 6), this finding suggests that the stimulation of PLCbeta by alpha  subunits of heterotrimeric G proteins differs mechanistically from stimulation of this enzyme by Rho GTPases. Thus, PLCbeta isozymes can be activated, in the nanomolar concentration range, by both subunits of G proteins and by Rho GTPases but with distinct PLCbeta isozyme specificities.

The Role of the C-terminal Regions of PLCbeta s in Their Regulation by Rho GTPases and beta gamma -- Proteolytic cleavage of PLCbeta 3 by calpain at a site upstream of the C2 domain has been suggested to enhance beta gamma -mediated stimulation (36, 37). Our data show, however, that truncation of recombinant PLCbeta 1, PLCbeta 2, and PLCbeta 3 at a site corresponding to the calpain cleavage site in PLCbeta 1 had no effect on their sensitivity to beta gamma stimulation. The finding that the deletion of the C-terminal region of PLCbeta 3 did not enhance beta gamma stimulation suggests that the increased PLCbeta 3 stimulation following calpain cleavage reported by Banno and coworkers (37) may not simply result from the removal of an inhibitory constraint built up by the C-terminal amino acids of PLCbeta 3. Instead, it is more likely that the C-terminal region generated by treatment of PLCbeta 3 with calpain still interacts with the remaining part of the enzyme, to inhibit its activity, whereas it is absent from the deletion mutant studied here. Consistent with earlier results (20), Rac2 stimulation of PLCbeta 2 was enhanced in the absence of the C-terminal region of the enzyme. However, because this effect was not observed in the case of PLCbeta 1 and PLCbeta 3, the PLCbeta 2 specificity described here is clearly not a consequence of C-terminal proteolysis of the enzymes.

Simultaneous Stimulation of PLCbeta Isozymes by Rho GTPases and G Proteins-- The fact that the stimulatory effects of beta 5gamma 2 and GTPgamma S-activated Rac2 on PLCbeta 2 were strictly additive at saturating concentrations of the two activators suggests that there are separate sites on PLCbeta 2 for the interaction with beta gamma and Rac2. Similar observations have been made previously for the activation of PLCbeta 2 and PLCbeta 3 by alpha q and beta gamma (9, 45). In additional experiments, we have found that neither beta gamma -mediated activations nor alpha q-mediated activations of PLCbeta 3 or PLCbeta 1 were influenced by GTPgamma S-activated Rac2. Collectively, these results suggest that PLCbeta isozymes can be isozyme-specifically activated by three different stimulators, G protein alpha  subunits, G protein beta gamma dimers, and Rho GTPases, via independent regulatory sites.

Structural Requirements of PLCbeta Stimulation by Rho GTPases versus beta gamma Dimers-- The generation of chimeric PLCbeta enzymes made up of portions from an isoform stimulated only poorly by beta gamma dimers and Rho GTPases, PLCbeta 1Delta , together with the remaining portions of PLCbeta 2Delta , an isoform markedly sensitive to both activators, allowed to delineate the structural elements of PLCbeta 2 required for the regulation by beta gamma and Rac2. The fact that chimeras A and B were still markedly activated by beta gamma dimers suggests that the catalytic subdomains of PLCbeta 2 are both necessary and sufficient for beta gamma stimulation. This is consistent with a previous report showing that a region within the catalytic Y domain of PLCbeta 2 contains the stimulatory beta gamma interaction site (40). The lower extent of stimulation of chimeras A and B by beta gamma relative to PLCbeta 2Delta is in line with the suggestion that an additional binding site for beta gamma dimers may exist in the putative PH domain of PLCbeta 2 (41), which, albeit not absolutely required for beta gamma stimulation, may increase the affinity of the beta gamma -PLCbeta 2 interaction. In addition, our results suggest that the region corresponding to the putative PH domain of PLCbeta 1, an isozyme barely activated by beta gamma , is capable of substitute for the corresponding region of PLCbeta 2. This is consistent with the recent report describing interaction of an isolated PH domain of PLCbeta 1 with beta gamma dimers (41). Interestingly, construction of a chimera consisting of the putative PH domain of PLCbeta 2 and the catalytic subdomains X and Y of PLCdelta , an enzyme that is not regulated by beta gamma dimers, resulted in a beta gamma -regulated enzyme (46). This suggests that beta gamma may interact with multiple sites in phospholipase C isozymes and that these sites can be provided even by isozymes that are poorly (e.g. PLCbeta 1) or not at all (e.g. PLCdelta 1) sensitive to beta gamma stimulation. An important outcome of our experiments on chimeric PLCbeta 1-PLCbeta 2 enzymes is the observation that the substitution of the putative PH domain of PLCbeta 2 by the corresponding region of PLCbeta 1 abolished Rac2-mediated stimulation of the enzyme. This result not only demonstrates different structural requirements of PLCbeta 2 stimulation by beta gamma and Rac2, but also shows, for the first time, that the putative PH domain of PLCbeta 2 is specifically and critically involved in mediating the regulation of the activity of the PLCbeta isozyme. It is currently unknown whether Rac2 directly binds to the PH domain of PLCbeta 2 or whether Rac2 induces a conformational change of the enzyme involving the PH domain. Interestingly, the PH domain of PLCbeta 2 was found to be required for both membrane targeting and catalytic activity of recombinant PLCbeta 2 in transfected COS-7 cells (44). The functional role of the region corresponding to the putative PH domain in PLCbeta isozymes is poorly understood. The isolated PH domain of PLCbeta 1 has been reported to bind inositol phospholipids (PI-3-P > PI-4,5-P2, PI-3,4,5-P3) (47). A cooperative mechanism involving phosphatidylinositol 3-phosphate and beta gamma subunits has been proposed to regulate plasma membrane localization and activation of PLCbeta 1 through the putative PH domain of this enzyme. A similar scenario involving Rac2 and beta gamma could be depicted in the case of PLCbeta 2. Thus, PLCbeta isozymes seem to act as a point of convergence of transmembrane signaling: PLCbeta 1 integrating signals emanating from inositol phospholipid 3-kinase and G protein alpha q subunits, PLCbeta 3 those from G protein alpha q and beta gamma subunits, and PLCbeta 2 those from G protein beta gamma subunits and Rac/Cdc42Hs.

The finding that the activity of PLCbeta isozymes can be specifically regulated by three different stimulators, G protein alpha  subunits, G protein beta gamma dimers, and the Rho GTPases Rac and Cdc42Hs also enhances the cellular repertoire to coordinate, both spatially and temporally, responses to extracellular signals acting through stimulation of PLCbeta isozymes and thereby to enhance the degree of signal specificity. An intriguing possibility is that the Rho GTPases Rac and Cdc42Hs act as organizers to mediate recruitment of PLCbeta 2 to allow activation by G protein-coupled receptors only at specific sites within the cell and/or only within a specific time frame during or after receptor activation. The mechanisms involved in the recruitment of PLCbeta isozymes to their phospholipid are still not known. It seems clear, however, that the known activators, alpha q and beta gamma , are not involved in this process (48, 49). Interestingly, although PLCbeta 3 is strongly activated by beta gamma dimers in cell-free systems, this isoform is, in marked contrast to PLCbeta 2, not stimulated by chemoattractant receptor activation or exogenous beta gamma dimers in transiently transfected COS-7 cells (17). These results support the notion that each PLCbeta isoform may require distinct additional components either for recruitment or stimulation of the catalytic activity. For example, activated members of the alpha q subfamily of G protein alpha  subunits have been shown to permit PLCbeta stimulation by receptors acting through beta gamma subunits of pertussis toxin-sensitive G proteins (50). The results presented herein suggest that Rac1, Rac2, and Cdc42Hs may contribute to the specificity and/or efficacy of PLCbeta signaling. Interestingly, both PLCbeta 2 and Rac2 are the major representatives of the PLCbeta and Rac GTPase subfamilies, respectively, in myeloid cells (16, 51). Both proteins are activated in response to activation of chemoattractant receptors in a pertussis-toxin sensitive manner (17, 52, 53, 54). The recent finding, that expression of a dominant-interfering form of Cdc42Hs in myeloid-differentiated HL-60 cells drastically reduced the formyl peptide receptor mediation of (a) formation of inositol 1,4,5-trisphosphate, (b) increase in the concentration of intracellular Ca2+, and (c) rapid activation of Rac2 (55), supports our hypothesis that Rac2 and possibly Rac1 and Cdc42Hs are critically involved in receptor-mediated regulation of PLCbeta 2 activity in intact cells. Although the mechanisms by which chemoattractant receptors stimulate Rac/Cdc42 are not defined and the functional relevance of phosphatidylinositol 3-kinase to this process is a subject of debate, the recently identified beta gamma - and PI-3,4,5-P3-dependent Rac exchanger P-Rex 1 appears to fill the gap between chemoattractant receptors and Rac GTPases in leukocytes (56).

In conclusion, our results demonstrate that the specificity of PLCbeta stimulation by the Rho GTPases Rac and Cdc42Hs differs from the specificity observed for alpha q/11 subunits and beta gamma dimers of heterotrimeric G proteins and that PLCbeta 2 represents the PLCbeta isozyme most sensitive to stimulation by the Rho GTPases Rac and Cdc42 among the PLCbeta isozymes investigated in this study. Moreover, there are separate sites on PLCbeta isozymes for the regulation by Rho GTPases, G protein alpha  subunits, and beta gamma subunits allowing independent activation by these stimulators.

    ACKNOWLEDGEMENTS

We are grateful to Norbert Zanker, Renate Straub, and Susanne Gierschik for excellent technical assistance. We thank Drs. Geurts van Kessel and Martien van Asseldonk for the generous gift of the cDNA-encoding PLCbeta 3.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft (Grant SFB 497).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 49-731-5002-3874; Fax: 49-731-5002-3872; E-mail: daria.illenberger@medizin.uni-ulm.de.

Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M208282200

    ABBREVIATIONS

The abbreviations used are: PLC, phospholipase C; LyGDI, a Rho guanine nucleotide dissociation inhibitor originally identified in lymphocytes, but subsequently also found in other cells; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); PH, pleckstrin homology; PI-3-P, phosphatidylinositol 3-phosphate; PI-4, 5-P2, phosphatidylinositol 4,5-bisphosphate; PI-3, 4,5-P3, phosphatidylinositol 3,4,5-trisphosphate.

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RESULTS
DISCUSSION
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