Reassembly of Phospholipase C-β2from Separated Domains

Phosphatidylinositol-specific phospholipase C-βs (PLC-βs) are the only PLC isoforms that are regulated by G protein subunits. To further understand the regulation of PLC-β2 by G proteins and the functional roles of PLC-β2 structural domains, we tested whether the separately expressed amino and carboxyl halves of PLC-β2could associate to form catalytically active enzymes as two polypeptides, and we explored how the complexes thus formed would be regulated by G protein βγ subunits (Gβγ). We expressed cDNA constructs encoding PLC-β2 fragments of different lengths in COS-7 cells and demonstrated by coimmunoprecipitation that the coexpressed fragments could assemble and functionally reconstitute an active PLC-β2. The pleckstrin homology domain of PLC-β2 was required for its targeting to the membrane and for substrate hydrolysis. Reconstituted enzymes that contained the linker region that joins the two catalytic domains were as active or more active than the wild-type PLC-β2. When the linker region was removed, basal PLC-β2 enzymatic activity was increased further, suggesting that the linker region exerts an inhibitory effect on basal PLC-β2 activity. The reconstituted enzymes, like wild-type PLC-β2, were activated by Gβγ; when the C-terminal region was present in these constructs, they were also activated by Gαq. Gβγ and Gαq activated these PLC-β2 constructs equally in the presence or absence of the linker region. We conclude that the linker region is an inhibitory element in PLC-β2and that Gβγ and Gαq do not stimulate PLC-β2 through easing the inhibition of enzymatic activity by the linker region.

All the ten mammalian PLC isozymes identified to date are modular proteins. As shown in Fig. 1, the PLCs contain a pleckstrin homology (PH) domain, four EF-hand motifs, a catalytic domain (composed of X and Y regions separated by a linker region) and a C2 domain. PLC-␤s have an additional 400-residue C-terminal region, which is required for activation by G␣ q (9,10) and may also contribute to membrane localization (11).
Among the PLC isozymes, only members of the PLC-␤ family (PLC-␤ 1-3 ) are activated by G␤␥. Part of the G␤␥-binding site on PLC-␤ 2 is located in the Y region as shown by cross-linking (12) and copurification (13). G␤␥ can also bind to the isolated PH domains from PLC-␤ 2 (14). Indirect evidence suggests that this interaction may lead to activation of PLC-␤ 2 (15). Despite these progresses, the mechanism whereby G␤␥ activates PLC-␤ 2 is still unclear. It seems unlikely that PLC-␤ 2 activation by G␤␥ involves membrane translocation of PLC-␤ 2 to the plasma membrane, because G␤␥ does not significantly alter the binding affinity of PLC-␤ 2 to phospholipid vesicles (16 -18).
The goal of this study was to further understand the mechanism of substrate hydrolysis of PLC-␤ 2 , its regulation by G protein subunits, and the functional contribution of some of the PLC-␤ 2 domains to enzyme function. Although some PLC domains are homologous to known domains in other proteins, and highly homologous domains can be found among the various PLC isoforms, these domains may have different functions in the different isoforms. For example, PH domains are found in many proteins, but only some of them can bind WD-repeatcontaining proteins (such as G␤␥) (for review see Ref. 19). In addition, the PH domains of various PLC isoforms show very different affinities to phospholipids (14,20,21). It is therefore necessary to test individual proteins to find out which role(s) a particular domain plays in a particular context.
The roles of other specific PLC domains in basal and ligandregulated PLC catalytic activities have not yet been clearly identified. For example, the role of the linker region between X and Y regions of the catalytic domain is less well understood. In the crystal structure of the PLC-␦ 1 molecule, the X and Y regions are tightly associated to form a triose phosphate isomerase barrel-like structure (22). Although the X and Y regions are well conserved among the PLC isozymes, the linker region possesses little similarity among the PLC isozymes. For example, PLC-␥s have a long linker region that contains two SH2 domains, one SH3 domain, and an additional PH domain, whereas the linker regions in PLC-␤ and PLC-␦ are less than 100 residues long and contain no obvious structural domains within them. In the crystal structure of PLC-␦ 1 , the linker region shows a disordered structure (22). The linker region is not essential for PLC catalytic activity. Coexpression of the Nand C-terminal fragments of PLC-␥ 1 lacking the linker region produces a catalytically active complex with an activity substantially higher than the holoenzyme (23). Trypsin digestion of PLC-␦ 1 cleaves the enzyme at the linker region and generates two associated fragments that retain catalytic activity (24). Proteolysis at or near the linker region of a truncated form of PLC-␤ 2 after it had folded into an active enzyme suggested that the linker region served as an inhibitory element (25). In this study, the linker region was cleaved but not removed and the exact site of tryptic or V8 protease cleavage was not determined. Because the authors used a truncated form of PLC-␤ 2 that was not stimulated by G␣ q , it was not possible to determine the effect of proteolysis of the enzyme in or near its linker region on G␣ q -dependent PLC activity.
It is usually straightforward to analyze the contributions of domains at the N or C termini of a protein, because truncated forms of the enzyme can be made, and these truncated proteins are often active. In addition to analyzing the role of the Nterminal PH domain and the C terminus, we were particularly interested in the linker region. Because it is often difficult to study the function of internal domains due to misfolding of proteins with internal deletions, we attempted to reconstitute PLC-␤ 2 from two separate fragments, each containing one of the two catalytic X and Y regions. We tested whether or not the N-and C-terminal halves of PLC-␤ 2 could associate to form catalytically active enzymes when expressed as two separate polypeptides and whether reconstituted PLC-␤ 2 could still be activated by G␤␥ and G␣ q. Using PLC fragments of different lengths, we examined the functional contribution of the PH domain, the linker region and the C-terminal region to basal activity and G␣-and G␤␥-mediated PLC-␤ 2 activation. Answers to these questions will expand our understanding of the mechanism of substrate hydrolysis by PLC-␤ 2 and its regulation by G proteins.

EXPERIMENTAL PROCEDURES
cDNA Constructs-Plasmids containing cDNA sequences encoding various fragments of human PLC-␤ 2 were constructed by polymerase chain reaction. Full-length wild-type PLC-␤ 2 in pMT2 vector (a gift from M. Simon of the California Institute of Technology, Pasadena, CA) was used as template. The primer at the 5Ј-end included a HindIII site, a Kozak sequence (GCCGCC), and a start codon. The primer at the 3Ј-end included an EcoRI site and a stop codon. To add a FLAG or hemagglutinin (HA) epitope tag to a construct, one of the two primers contained the sequence encoding the epitope. The polymerase chain reaction products were digested with HindIII and EcoRI and cloned into an HindIII/ EcoRI-cut pcDNA3 vector. All the sequences were confirmed by DNA sequencing.
Cell Culture and Transfection-COS-7 cells were maintained in complete growth medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 g/ml streptomycin). Cells in 6-well plates (for immunoprecipitation) or 12-well plates (for PLC activity assay) were transfected using Lipo-fectAMINE (Life Technologies). Prior to transfection, cells were transferred to Opti-MEM I medium (Life Technologies) for 1 h. The medium was replaced with 1 ml (6-well plates) or 500 l (12-well plates) of Opti-MEM I containing preformed DNA-LipofectAMINE complexes. The final concentration of DNA in the medium was 1.5 or 2.5 g/ml. The exact amount of each plasmid was as indicated in the legends to individual figures. The ratio (w/w) between DNA and LipofectAMINE was always kept at 1:8. After 5 h, 2 ml (6-well plates) or 500 l (12-well plates) of complete growth medium were added to each well. The me-dium was replaced with complete growth medium the next day.

35
S Metabolic Labeling and Immunoprecipitation-Forty-eight hours after transfection, cells on 6-well plates were starved for 2 h in 2 ml of starvation medium (RPMI 1640 without glutamine, methionine, and cysteine (Sigma) supplemented with 10% dialyzed, heat-inactivated fetal bovine serum and 2 mM L-glutamine). The cells were then metabolically labeled in 1 ml of starvation medium containing 150 Ci of [ 35 S]-Express Protein Labeling Mix (NEN) for 4 h. The cells were rinsed with PBS and lysed in 1 ml of lysis buffer (50 mM HEPES-Na (pH 7.5), 6 mM MgCl 2 , 1 mM EDTA, 75 mM sucrose, 3 mM benzamidine, 1% (v/v) Triton X-100, and 1 mM dithiothreitol) at 4°C for 30 min. The cell lysates were precleared with 30 l of protein G-agarose (Roche Molecular Biochemicals) or 50 l of protein A-Sepharose (Sigma) slurry (50% (v/v) in PBS) for 30 min. After a 10-min centrifugation, the supernatants were mixed with 2 l of M2 anti-FLAG antibody (Sigma) or anti-HA-epitope antibody 12CA5 (Babco) at 4°C overnight. The samples were centrifuged at 15,000 ϫ g for 15 min. The supernatants were then mixed with 30 l of protein G-agarose or 50 l of protein A-Sepharose slurry for 1.5 h. The resins were washed twice at 4°C for 15 min each with 1 ml of lysis buffer containing 150 mM NaCl and once at room temperature for 15 min with 1 ml of PBS. 25 l of 3ϫ sample buffer (187.5 mM Tris-Cl (pH 6.8), 6% SDS, 30% glycerol, 0.003% bromphenol blue) was added to each of the final pellets, and 20 l was loaded onto an SDS-PAGE gel. The gel was Coomassie Blue-stained, destained, treated with EN 3 HANCE (NEN), dried, and used for autoradiography with intensifying screens at Ϫ80°C.
Inositol Phosphate Production in COS-7 Cells-PLC activity was analyzed as production of inositol phosphates (26,27). Twenty-four hours after transfection, the medium was replaced with 1 ml of inositolfree DMEM supplemented with 5% fetal bovine serum. Two hours later, the medium was again replaced with the same medium containing 2 Ci of myo-[ 3 H]inositol. After 15 min, 10 l of 1 M LiCl was added to each well (the final LiCl concentration was 10 mM). No difference in the uptake/incorporation of myo-[ 3 H]inositol was found in cells incubated with LiCl-containing medium for 1 h and 24 h. Forty-eight hours after transfection, the cells were washed with 1 ml of PBS and extracted twice for 30 min each with 500 l of 20 mM formic acid. The extracts were combined and neutralized to pH 7.5 with a solution containing 7.5 mM HEPES and 150 mM KOH. The neutralized extracts were loaded onto 0.5 ml AG1-X8 (Bio-Rad) anion exchange columns. Prior to use, the columns were washed with 2 ml of 1 M NaOH and 2 ml of 1 M formic acid and equilibrated with H 2 O to neutrality. After the extracts were loaded onto the columns, the columns were washed with 5 ml of H 2 O and 5 ml of 5 mM Borax and 60 mM sodium formate. The inositol phosphates were eluted with 3 ml of 0.9 M ammonium formate and 0.1 M formic acid. The eluates were counted in a scintillation counter.
Subcellular Fractionation-COS-7 cells cultured in 6-well plates were transfected and metabolically labeled with [ 35 S]-Express Protein Labeling Mix as described above. The cells were washed with PBS and detached by incubation in 500 l of Trypsin-EDTA solution (1ϫ) (Sigma) at 37°C for 1 min. After mixing with 2 ml of DMEM/8% fetal bovine serum, the cells were collected by centrifugation at 500 ϫ g at 4°C for 5 min. After being washed with 3 ml of a buffer identical to the lysis buffer used in immunoprecipitation but containing no Triton X-100, the cells were resuspended in 600 l of the same buffer and went through freeze-and-thaw in ethanol/dry ice three times (3 min per period). The broken cells were then passed trough a 23-gauge (or smaller) needle ten times to shear DNA and centrifuged at 100,000 ϫ g in a Beckman SW55 rotor at 4°C for 30 min. The supernatant was the soluble fraction. The pellet was resuspended in 600 l of lysis buffer containing 1% Triton X-100 and incubated at 4°C for 30 min. The supernatant, after a 5-min centrifugation at 15,000 ϫ g was the particulate fraction. Both the soluble and the particulate fractions were later used in immunoprecipitation.
Western Blot Analysis-Forty-eight hours after transfection, cells in 6-well plates were washed with 2 ml of PBS and harvested in 1 ml of lysis buffer. The cells were lysed at 4°C for 30 min. After a 10-min centrifugation at 15,000 ϫ g, an aliquot (10 l) of the supernatant was loaded on an SDS-PAGE mini-gel, and the resolved proteins were wet-electroblotted to a nitrocellulose membrane and probed with specific primary and peroxidase-conjugated secondary antibodies using a chemiluminescence kit according to the manufacturer's instructions (NEN).

RESULTS
Design of PLC-␤ 2 Plasmids-We constructed several mammalian expression plasmids encoding various fragments of PLC-␤ 2 , as shown in Fig. 1. To further understand the mechanism of substrate hydrolysis of PLC-␤ 2 and its regulation by G protein subunits, we used these constructs to determine whether the amino and carboxyl halves of PLC-␤ 2 could associate when expressed as two polypeptides and, if they could, how the complexes thus formed would be regulated by G␤␥ and G␣ q . These constructs were designed to allow us to test the role of the PH domain, to compare the activity and G protein regulation of enzyme with the linker region either attached to the C-terminal fragment or completely removed, and to compare the activity of reconstituted enzyme with and without the Cterminal domain required for activation by G␣ q . To all these fragments (except construct A), a FLAG or an HA epitope tag-encoding sequence was attached at one end (see Fig. 1). Construct A had both a FLAG tag at the N terminus and an HA tag at the C terminus to compare the results of immunoprecipitation through the FLAG and HA tags.
Assembly of PLC-␤ 2 Fragments Expressed in COS-7 Cells-Antibodies directed against the epitope tags were used to (co)immunoprecipitate metabolically labeled PLC-␤ 2 fragments that had been expressed in COS-7 cells. The representative autoradiograph in Fig. 2A shows that most of the fragments were robustly expressed. Only A and E fragments were expressed at significantly lower levels (about one-tenth to onefifth) when compared with their corresponding PH domaincontaining fragments (AЈ fragment and wild-type, respectively).
PLC-␤ 2 has an endogenous proteolytic site that cleaves off the C-terminal region necessary for G␣ q activation (10). The wild-type enzyme expressed in COS-7 cells was partially cleaved at this site giving rise to a fragment of about the same size as AЈ fragment. The AЈ fragment was further cleaved to a polypeptide of approximately the same size as the BЈ fragment. Similarly, A and E fragments had shorter polypeptides of the same size as the B fragment, suggesting that there is another proteolytic site near the C terminus of the X region. The remaining proteolytic or background bands were not identified but represented only a small fraction of the total protein.
Lanes 8 -11 of Fig. 2A show that the BЈ fragment could bind C, CЈ, D, and DЈ fragments (CЈ and DЈ fragments lacked the linker region, whereas C and D fragments included this region). The expression level of the BЈ fragment was higher when the C-terminal fragments were coexpressed, suggesting that they stabilized the BЈ fragment. Immunoprecipitation through the FLAG tag on the BЈ fragment was able to coimmunoprecipitate the C, CЈ, D, and DЈ fragments, indicating that the BЈ fragment was able to form complexes with each. The BЈ fragment coimmunoprecipitated approximately equal amounts of C and CЈ fragments. The numbers of methionines in these fragments were: 15 in BЈ, 17 in C, 16 in CЈ, 27 in D, and 26 in DЈ. The D and DЈ fragments were cleaved at or near the site described by Park et al. (10) to generate C and CЈ fragments, which also bound to the BЈ fragment. About 90% of D fragment and 75% of DЈ fragment were cleaved. The implication of this cleavage for interpretation of activity measurements will be described below. The B fragment, which lacked the PH domain, could also coimmunoprecipitate a C-terminal fragment (C, CЈ, D, and DЈ fragments), but the capacity was lower when compared with the BЈ fragment (Fig. 2B). Therefore, removal of the PH domain reduced but did not block assembly. Immunoprecipitation and coimmunoprecipitation could also be performed through the HA epitope tag on A, C, CЈ, D, and DЈ fragments, but the efficiency was lower. For this reason, we performed the coimmunoprecipitation in all our other experiments through the FLAG epitope tag.
We also examined whether the PLC-␤ 2 fragments could form complexes after they had been synthesized. When the BЈ fragment and C, CЈ, D, or DЈ fragments were expressed in COS-7 cells separately and later mixed after cell lysis, none of the C, CЈ, D, nor DЈ fragments were coimmunoprecipitated by the BЈ FIG. 1. Schematic representation of a human PLC-␤ 2 molecule and its constructs with epitope tags. PH, pleckstrin homology domain; EF, EF-hands; X and Y, X and Y regions of the catalytic domain; L, X-Y linker region; C2, C2 domain. The long black bar at the Cterminal end is the C-terminal region. See text for more details.

FIG. 2. Expression of PLC-␤ 2 fragments in COS-7 cells.
COS-7 cells in 6-well plates were cotransfected with plasmids encoding various PLC-␤ 2 fragments. 625 ng/ml of each DNA was used. Vector DNA (pcDNA3) was added as needed to make a total DNA concentration of 1.5 g/ml. The cells were metabolically labeled with [ 35 S]methionine/ cysteine. The PLC-␤ 2 fragments were immunoprecipitated with the anti-FLAG tag antibody as described under "Experimental Procedures" and resolved on two 10% SDS-PAGE gels. Positions of expressed PLC-␤ 2 fragments are indicated by arrows. When more than one band of a protein is present, only the band of the largest size was indicated. The first lane on the left in both gels is a control (transfected with vector only). fragment (data not shown). Therefore, the PLC-␤ 2 fragments need to be coexpressed in cells to form complexes with one another. In addition, we failed to coimmunoprecipitate G␤ 1 ␥ 2 through a FLAG tag on the PLC-␤ 2 fragments (including the wild-type PLC-␤ 2 ) or to coimmunoprecipitate the PLC-␤ 2 fragments with G␤ 1 ␥ 2 through an HA tag on G␤ 1 or G␥ 2 , suggesting that the interaction was weak.
Roles of the PH Domain for the Enzymatic Activity and Subcellular Distribution of PLC-␤ 2 Fragments-We next tested the catalytic activity of the PLC-␤ 2 fragments measured as production of inositol phosphates. Full-length, wild-type PLC-␤ 2 was used as control. As shown previously in this laboratory (26,27), inositol phosphate production increased when COS-7 cells were transfected with PLC-␤ 2 itself (Fig. 3A). Coexpression of G␤ 1 ␥ 2 caused a pronounced rise in PLC activity. PLC-␤ 2 truncated at the C terminus (AЈ fragment) had basal and G␤ 1 ␥ 2 -stimulated activity equal to the full-length enzyme. Even though the wildtype enzyme was substantially cleaved, if the activity of the wild-type enzyme was much higher than AЈ fragment, the mixture should still show higher activity than the AЈ fragment. These results were consistent with previous reports (9, 10, 28). However, when the PH domain was removed from the fulllength or truncated enzyme (E fragment and A fragment, respectively), both were inactive. Fig. 3A also shows that individual fragments containing only one of the two catalytic regions (B, BЈ, C, CЈ, D, and DЈ) had no PLC activity whether or not the PH domain was present. Fig. 3B illustrates that basal and G␤ 1 ␥ 2 -stimulated PLC-␤ 2 activity could be reconstituted from two fragments each containing one of the catalytic domains only if the PH domain was present. The characteristics of the reconstituted activity will be discussed below. These results indicate that the PH domain was required for PLC-␤ 2 to hydrolyze its substrate in COS-7 cells.
Because the fragments and reconstituted complexes lacking the PH domain (i.e. E, A, and complexes formed with the B fragment) were expressed at levels significantly lower than those containing the PH domain (the wild-type enzyme, AЈ, and complexes formed with the BЈ fragment) (Fig. 2), it was possible that their lower catalytic activity was simply a result of lower expression. To test this possibility, we compared the expression and PLC activity of B ϩ CЈ at a higher DNA dose (625 ng of DNA/ml at transfection) with those of BЈ ϩ CЈ transfected with one-tenth of this DNA dose (62.5 ng/ml). We chose this pair, because, in contrast to the wild-type enzyme and the AЈ fragment, at the lower DNA dose BЈ ϩ CЈ were expressed well and had a basal activity substantially higher than the blank. Despite a higher expression level of the B fragment (due to higher DNA dosage) compared with the BЈ fragment and a similar amount of coimmunoprecipitated CЈ, B ϩ CЈ showed no enzymatic activity. Similar results were also observed for other fragments (data not shown). Although this experiment did not completely exclude the possibility that the absence of PLC activity of E and A was in part due to low expression, our results indicated that removal of the PH domain abolished the function of PLC-␤ 2 in COS-7 cells.
Removal of the PH domain also altered the subcellular distribution of the expressed proteins (Fig. 4). When the PH domain was present (wild-type, AЈ, and BЈ fragments), ϳ30% of the enzyme was found in the particulate fraction. Constructs lacking the PH domain (E, A, and B fragments) were found almost exclusively in the soluble fraction. Therefore, the inaccessibility to a membrane-associated substrate may account for the observed loss of PLC-␤ 2 activity in constructs lacking the PH domain. However, using these experimental approaches in transfected cells, we could not distinguish an intrinsic loss of catalytic activity in truncated fragments from effects secondary to alterations in subcellular localization.
Effects of G␣ i on G␤ 1 ␥ 2 Activation of Wild Type and Reconstituted PLC-␤ 2 -G␤␥ needs to dissociate from G␣ to interact with its effectors. Therefore, excess G␣ i should block the G␤␥ activation of PLC-␤ 2 by scavenging free G␤␥ to form heterotrimers (26,27). This is an important control, because it shows that G␤␥ is activating PLC-␤ 2 with characteristics expected for a heterotrimeric G protein. Fig. 5 shows that, although G␣ i1 itself did not exhibit any significant effect in any group, it

FIG. 3. Activity of PLC-␤ 2 fragments and reconstituted PLC-␤ 2 .
A, activity of single PLC-␤ 2 fragments. COS-7 cells in each duplicate wells in 12-well plates were transfected with 625 ng/ml of each DNA. In all cases, vector DNA was added to give a total DNA concentration of 2.5 g/ml. Black bars, no G␤ 1 ␥ 2 ; shaded bars, in the presence of G␤ 1 ␥ 2 . WT, wild-type. A representative experiment analyzed in duplicate is shown. The error bars indicate the ranges of duplicate determinations. Each construct was tested at least three times. B, activity of two cotransfected PLC-␤ 2 fragments. Experimental conditions were identical to those in A of this figure except that the concentration of each PLC-␤ 2 DNA was 125 ng/ml. Wild-type PLC-␤ 2 and the AЈ fragment were used as positive controls. The PLC activity dropped to a level equal to or slightly lower than the basal activity. Cotransfection of the COS-7 cells with plasmids encoding G␣ i1 and/or G␤␥ protein subunits had no effect on the amount of immunoprecipitated PLC-␤ 2 fragments (data not shown).
Effects of the Linker Region on Basal and G␤ 1 ␥ 2 -stimulated Activity of Reconstituted PLC-␤ 2 -The above experiments showed that the N-terminal fragment containing the PH domain (BЈ fragment) could reconstitute active enzyme when coexpressed with the C-terminal fragments. The reconstituted complexes had higher basal PLC activities than the wild-type PLC-␤ 2 and, like the wild-type PLC-␤ 2 and AЈ fragment, were activated by G␤ 1 ␥ 2 (Figs. 3B and 5). Among the four complexes, those lacking the linker region (BЈ ϩ CЈ and BЈ ϩ DЈ) had higher basal and G␤ 1 ␥ 2 -stimulated activity than those with the linker region connected to a C-terminal fragment (BЈ ϩ C and BЈ ϩ D).
Although the reconstituted complexes had G␤ 1 ␥ 2 -stimulated activities that were equal to or greater than that of the wildtype enzyme, this was entirely due to increased basal activity. There was no significant change in the increment due to G␤ 1 ␥ 2 between the wild-type enzyme and any mutant/complex but BЈ ϩ C, whose increase was higher than that of any other group. This indicates that, although cleavage or removal of the linker region leads to increased basal activity, it does not affect the activation of PLC-␤ 2 by G␤ 1 ␥ 2 .
Regulation of PLC-␤ 2 Activity by G␣ q -PLC-␤s are the only PLC isozymes that are activated by G␣ q (9, 29 -31). This activation involves the long C-terminal region characteristic of the PLC-␤ isozymes. We had shown above (Figs. 3 and 5) that the recombined PLC-␤ 2 complexes could be activated by G␤ 1 ␥ 2 and tested next whether they could also be activated by G␣ q (Fig. 6). We used the wild-type PLC-␤ 2 and AЈ fragments, which lacks the C-terminal region, as positive and negative controls, respectively. Among the four complexes, no significant activation by G␣ q was observed in the combinations lacking the C-terminal region (BЈ ϩ C or BЈ ϩ CЈ). In contrast, BЈ ϩ D and BЈ ϩ DЈ were activated by G␣ q . As with G␤␥ activation, the increment in PLC-␤ 2 activity due to G␣ q was very similar in wild-type PLC-␤ 2 , BЈ ϩ D and BЈ ϩ DЈ. Again, removal of the linker region (as in BЈ ϩ DЈ and BЈ ϩ CЈ) increased the basal activity, but the basal activity and the G␣ q -induced increase in activity were additive. These results documented that the reconstituted PLC-␤ 2 complexes containing the C-terminal region were still subject to regulation by G␣ q . We also tested the effect of G␣ q on fragments and reconstituted enzymes without the PH domain (E, B ϩ CЈ, and B ϩ DЈ). These fragments or reconstituted proteins showed no increase in PLC activity, providing further evidence that removal of the PH domain abolished the function of PLC-␤ 2 (Fig. 6B).

DISCUSSION
Our study is the first to reconstitute active enzymes with PLC-␤ fragments. In this study, we have characterized several constructs encoding various fragments of human PLC-␤ 2 . We reassembled PLC-␤ 2 from enzyme fragments each containing one of the two catalytic regions (Fig. 2) and found that the PH domain was required for both enzymatic activity (Figs. 3) and membrane targeting of PLC-␤ 2 (Fig. 4). These reassembled enzymes were still subject to regulation by G protein subunits (Figs. 5 and 6). We identified the X-Y linker region as an inhibitory element in the intact enzyme. However, changes at the linker region did not affect the regulation of PLC-␤ 2 by G protein subunits (Figs. 3, 5, and 6).
The Roles of the PH Domain-We found that, although the targeting of PLC-␤ 2 fragments lacking the PH domain to the membrane may be impaired (Fig. 4), these fragments were still assembled (Fig. 2). Moreover, the PH domain was essential for PLC-␤ 2 to hydrolyze its substrates in COS-7 cells (Fig. 3). We could not distinguish whether removal of the PH domain in PLC-␤ 2 prevented the enzyme from getting access to its substrates in the plasma membrane, caused a loss in enzymatic activity, or both. A PLC-␦ 1 construct in which the PH domain was replaced by glutathione S-transferase has full enzymatic activity (32), suggesting that the major role of the PH domain in PLC-␦ 1 is to ensure membrane localization. The PH domain of PLC-␦ 1 binds to the PIP 2 polar headgroup with an affinity FIG. 5. Regulation of PLC-␤ 2 fragments by G␤ 1 ␥ 2 and G␣ i1 . COS-7 cells in duplicate wells in 12-well plates were transfected with 125 ng/ml of each PLC-␤ 2 DNA, but the concentrations of G␤ 1 , G␥ 2 , and G␣ i1 DNA were kept at 625 ng/ml each. In all cases, vector DNA was added to give a total DNA concentration of 2.5 g/ml. A representative experiment out of four independent experiments analyzed in duplicate is shown. The error bars indicate the ranges of duplicate determinations.
FIG. 6. Activation of PLC-␤ 2 fragments by G␣ q . A, activation of PLC-␤ 2 fragments containing the PH domain. COS-7 cells in each duplicate wells in 12-well plates were transfected with 125 ng/ml of each PLC-␤ 2 DNA. The concentration of G␣ q DNA was 625 ng/ml. In all cases, vector DNA was added to give a total DNA concentration of 1.5 g/ml. The counts of the blank group (vector DNA only) with or without G␣ q were subtracted from those of the other groups under the same conditions. The error bars indicate standard deviations of three independent experiments. Black bars, no G␣ q ; shaded bars, in the presence of G␣ q . B, absence of activation of PLC-␤ 2 fragments lacking the PH domain. The error bars indicate the ranges of duplicate determinations in the same experiment, which was repeated twice with similar results. and specificity comparable to the native enzyme (20) and is proposed as the anchor localizing the enzyme to the plasma membrane in the "tether-and-fix" model based on the crystal structure of PLC-␦ 1 (22). The PH domain of PLC-␥ 1 binds to PIP 3 strongly and specifically and targets the enzyme to the membrane in response to growth factor stimulation (21). In contrast, PLC-␤ 1 , PLC-␤ 2 and their PH domains bind to phospholipid membrane surfaces with lower affinities, and the binding is PIP 2 concentration-independent (14). We observed that PLC-␤ 2 fragments lacking the PH domain were not found in the particulate fraction, whereas constructs containing the PH domain were partially targeted to the particulate fraction (Fig. 4). Therefore, this domain is also involved in membrane targeting of PLC-␤ 2 . Besides binding to some inositol phosphates, PH domains identified in some proteins bind to proteins containing WD-repeats (33). An example is the strong interaction between ␤-adrenergic receptor kinase and G␤␥ (34). The isolated PH domain of PLC-␤ 2 binds to G␤␥ with an affinity comparable to that of the full-length PLC-␤ 2 (14), but the significance of this interaction for the enzyme's regulation by G␤␥ needs to be further tested.
The Roles of the Linker Region-The X and Y regions form the catalytic domain of PLC. In the present study, we demonstrated by coimmunoprecipitation experiments that, when COS-7 cells were cotransfected with two plasmids each containing the DNA sequence encoding one of the two catalytic regions, the two in vivo coexpressed fragments associated tightly with each other (Fig. 2). In contrast, fragments that were separately expressed and then combined could not bind to each other, suggesting that the association occurs during translation.
The reassembled enzymes possessed catalytic activity similar to or higher than that of the wild-type PLC-␤ 2 (Figs. 3B and 5). The most dramatic elevation of the basal catalytic activity was found in the two combinations lacking the linker region, BЈ ϩ CЈ and BЈ ϩ DЈ. These results suggest that the linker region, when present in the intact enzyme, inhibits basal PLC-␤ 2 activity. When the linker region was attached to the C-terminal fragment containing the Y domain, but (in contrast to wild-type PLC-␤ 2 ) was not linked to the X domain, thereby allowing for more flexibility, the basal activity almost doubled (Figs. 3B and 5, compare the wild-type PLC-␤ 2 with BЈ ϩ D). However, the linker region may still interfere with PIP 2 hydrolysis, because complete removal of the linker region resulted in even greater increase in the basal activity.
The long C-terminal region was not essential for the basal or G␤␥-stimulated activity of PLC-␤ 2 (Fig. 3A). However, the highest basal activity of all was given by BЈ ϩ DЈ that lacked the linker region but retained the long C-terminal domain. As was shown in Fig. 2, the C-terminal domain did not lead to the formation of more reassembled PLC-␤ 2 . Therefore, we conclude that the presence of the C-terminal domain allows those complexes that do reassemble to acquire a more active conformation.
We found that even when the linker region was cleaved or completely removed, PLC-␤ 2 was activated by G␤ 1 ␥ 2 and this activation was completely blocked by G␣ i1 (Fig. 5). These PLC-␤ 2 fragments were also subject to activation by G␣ q . Consistent with previous findings (9,10), activation by G␣ q was contingent upon the presence of the C-terminal region (Fig. 6). In addition, all PLC-␤ 2 fragments showed similar increment in PLC-␤ 2 activity upon activation by G␤ 1 ␥ 2 or G␣ q . Therefore, it is highly unlikely that the G protein ␣ q and ␤␥ subunits regulate PLC-␤ 2 by direct effects on the linker region.
Conclusions-Our experiments show that the PH domain is required for the basal as well as the G␣ q -and G␤␥-stimulated PLC-␤ 2 activity in a heterogeneous cell expression system. Like PLC-␥ 1 , functional PLC-␤ 2 can be reconstituted from two coexpressed enzyme fragments, each containing one of the two catalytic regions. The linker region is an inhibitory element in PLC-␤ 2 , but cleavage or removal of the linker region does not affect the G protein-mediated regulation of PLC-␤ 2 . Therefore, G␤␥ and G␣ q appear to activate PLC-␤ 2 by mechanisms other than easing the inhibition of PLC-␤ 2 activity by the linker region, thereby providing evidence for a regulatory pathway for PLC-␤ 2 -involving mechanisms distinct from other PLC isoforms.