Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site

Abstract

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gα_q and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gα_q, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gα_q response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site.

Introduction

Phospholipase C (PLC)² isozymes integrate signaling inputs downstream of diverse receptors to catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) in the inner leaflet of the plasma membrane. This reaction generates two products, inositol 1,4,5-trisphosphate and diacylglycerol, important second messengers and second messenger precursors that regulate multiple cellular processes (1). The mammalian genome encodes six families of PLCs (β, δ, γ, ϵ, η, and ζ) that share a conserved core architecture composed of a pleckstrin homology (PH) domain, four EF hands, a split TIM barrel, and a C2 domain. The TIM barrel contains the active site, and its two halves are separated by the so-called X-Y linker that is thought to occlude the active site and whose motion is central to the regulation of activity. In the PLC-β isozymes, the linker is highly cationic, flanked by a highly anionic region, and disordered. Sondek and co-workers (2) speculated that PLC recruitment to the plasma membrane by protein activators causes electrostatic repulsion of the linker by negatively charged phospholipids that moves it away from the active site to relieve autoinhibition. Proteolysis or genetic removal of the linker elevates basal enzyme activity across many PLC isozymes (3, 4), supporting the generality of this mechanism. However, mutant PLC-βs that lack this linker are activated further by their physiological ligands (2), suggesting the existence of additional regulatory mechanisms that are yet to be discovered.

The four mammalian PLC-β isoforms are stimulated to different extents by Gα subunits of the G_q family, Gβγ subunits, and members of the small Rho GTPase family. All PLC-β isoforms are stimulated by GTP-bound Gα_q, but Gβγ activates only PLC-β1, PLC-β2, and PLC-β3 (5). PLC-β2 is the most responsive to Gβγ stimulation and is also the only isoform that is significantly sensitive to the small G protein Rac1 (6). PLC-β3 responds less than 2-fold to Rac1 (7) but is stimulated more than 100-fold by both Gα_q and Gβγ (8). In previous work, we showed that PLC-β3 and, to a lesser extent, PLC-β2 respond synergistically to Gα_q and Gβγ (8). Such synergism indicates that the binding sites for these activators are independent and do not overlap but is consistent with a two-state allosteric model that requires only one mechanism of activation (8). The PLC-β isoforms are also GTPase-activating proteins for Gα_q (9), accelerating the hydrolysis of Gα-bound GTP to modulate the amplitude, duration, and response kinetics of signaling from G_q-coupled receptors (10).

Although the physiology of PLC-β-regulated signaling pathways is well studied, we have only begun to understand the structural basis of PLC-β activation. Crystal structures of PLC-β bound to Gα_q (11) or Rac1 (12) established the independence of the binding sites for these activators. However, the overall conformation of PLC-β in these structures is remarkably similar to the conformation in the absence of bound activator. This similarity suggests the absence of any large-scale conformational changes during activation, except perhaps at the autoinhibitory X-Y linker, which is not visible and presumably disordered in all available structures. Recent studies identified a helix-turn-helix extension of the C2 domain that is bound to the TIM barrel in the unliganded structure but is refolded and bound to Gα_q upon complex formation (13), supporting the notion that PLC-β activation requires more than simple recruitment to the membrane. Mutational analysis suggested that this helix-turn-helix extension may stabilize the X-Y linker in its inhibitory conformation. Unlike the other PLC families, PLC-β isoforms possess an ∼400-amino acid helical bundle C-terminal to this extension and are separated from it by a protease-sensitive and probably disordered linker. A PLC-β truncation mutant that lacks this helical domain binds Gα_q with about the same affinity as wild-type PLC but displays markedly reduced sensitivity to stimulation by Gα_q (14). Multiple single amino acid mutations in the helical domain also inhibit stimulation by Gα_q (15). However, the helical bundle is dispensable for stimulation by Gβγ or Rac1 (16).

In the absence of a co-crystal structure of the Gβγ·PLC-β complex, the mechanism of PLC-β regulation by Gβγ is the least well understood. Gβγ stimulates the phospholipase activity of PLC-β but inhibits its GTPase-activating protein activity (17). There is no consensus on where Gβγ binds or how it coordinates the dual regulation of PLC-β activities. Two binding sites for Gβγ on PLC-β have been proposed. The N-terminal PH domain is required for stimulation by Gβγ. Removal of the PH domain abolishes Gβγ stimulation without blocking either Gβγ inhibition of G_q GTPase-activating protein activity or stimulation of PLC activity by Gα_q (18). PLC-δ, an isoform not stimulated by Gβγ, gains sensitivity to Gβγ when its PH domain is replaced by the PLC-β PH domain and the first EF hand (19). Gβγ binds the PH domains of many proteins, including some with low nanomolar affinity (20), suggesting that Gβγ may bind to a conserved surface in PH domains. The second proposed site is a conserved helix in the C-terminal half of the TIM barrel. Smrcka and co-workers (21) showed that a peptide corresponding to this region inhibits Gβγ stimulation of PLC-β and that the peptide can be chemically cross-linked to Gβγ. Point mutations in this region also diminished the response to Gβγ (21). These two sites, the PH domain and this region of the TIM barrel surface, are not adjacent in the tertiary structure of PLC-β, and it is hard to envision how both could be involved in Gβγ binding unless there is substantial domain movement.

In this study, we developed a FRET-based assay for the binding of Gβγ to PLC-β3 and to its isolated PH domain, establishing the PH domain as the minimal Gβγ binding site in PLC-β. We also identify a Gβγ binding surface on the PH domain that is consistent with other known Gβγ-PH domain interfaces. This Gβγ binding surface is buried in the available crystal structures of PLC-β, and movement of the PH domain is therefore required for Gβγ-PLC-β binding and activation. We confirm this prediction by showing that anchoring the PH domain by cross-linking it to the EF hand domain blocks PLC-β activation by Gβγ. Based on these data, we propose a new model for PLC-β interaction with Gβγ in which the PH domain is in equilibrium between distinct positions and Gβγ binds and stabilizes a conformation needed for enzyme activation.

Experimental Procedures

Proteins

cDNA encoding PLC-β3 (human) with an N-terminal His₆ tag was cloned into pQE60 for expression in Escherichia coli. Cysteines at positions 193, 516, 614, 892, 1005, 1176, and 1207 were predicted to be solvent-exposed and mutated to serine (1176 to valine) by multiple rounds of QuikChange mutagenesis. The activity and regulation of this modified PLC-β3 purified from E. coli was virtually indistinguishable from wild-type PLC-β3 expressed in Sf9 cells, and we refer to this mutant as wild-type throughout. Point mutations to introduce cysteine residues for cross-linking or fluorescence labeling were made in this background by QuikChange mutagenesis. To quantitate the fraction of intramolecular cross-linking in the Cys⁶⁰,Cys¹⁶⁴ construct, we introduced a TEV protease recognition sequence after Gly⁹⁴ by overlap PCR (22) (⁹⁴GsenlyfqgaaagP⁹⁵; the inserted sequence is in lowercase, and the numbers at the ends indicate native PLC-β3 residue numbers). We refer to this construct as PLC-TEV. All mutants were verified by sequencing over the entire PLC-β3 open reading frame. Wild-type PLC-β3 and variants were purified from transformed BL21DE3/pRep4 cells grown in T7 medium (2% Tryptone, 1% yeast extract, 0.2% glycerol, 0.5% NaCl, 50 mm potassium P_i, pH 7.2) and induced with 60–100 μm isopropyl 1-thio-β-d-galactopyranoside for 9 h at 25 °C. Frozen cell pellets were lysed with 0.5 mg/ml lysozyme in buffer A (20 mm NaHepes (pH 7.5), 5 mm 2-mercaptoethanol, 5 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.2 mm PMSF) plus 500 mm NaCl. After sonication, the suspension was centrifuged for 30 min at 100,000 × g, and the supernatant was mixed with Ni²⁺-nitrilotriacetic acid resin (Qiagen) for 2 h. The resin was washed sequentially with buffer A plus 500 mm NaCl and 5 mm imidazole and buffer A plus 100 mm NaCl and 5 mm imidazole until A₂₈₀ reached baseline. PLC was eluted with buffer A plus 100 mm NaCl and 120 mm imidazole. The eluate was diluted in buffer B (20 mm NaMES (pH 6.0), 0.1 mm EDTA, 1 mm DTT, 0.2 mm PMSF, and 10% glycerol) and applied to a 1-ml SourceS column that was equilibrated with buffer B. After washing the column with 200 mm NaCl in buffer B, protein was eluted by a gradient of 200–500 mm NaCl in buffer B. Peak fractions were pooled, exchanged into buffer C (20 mm NaHepes, 100 mm NaCl (pH 7.5), 0.1 mm DTT, and 10% glycerol) and flash-frozen to −80 °C for storage. N-terminally His₆-tagged PLC-β2 (mouse) was purified from baculovirus-infected insect cells as described earlier (8).

The PH domain (residues 2–147) of human PLC-β3 was amplified by PCR and cloned into a pET28 vector with an N-terminal MBP tag and a TEV protease recognition sequence (a gift from Neal Alto, University of Texas Southwestern Medical Center) (23). The resulting fusion protein had the sequence His₆-MBP-His₆-ENLYFQSG followed by the PLC-β3 PH domain. TEV protease cleaves after the Gln residue in ENLYFQSG. Point mutations were made in this background by QuikChange mutagenesis. Proteins were expressed at 16 °C in BL21DE3 grown in T7 medium for 16–20 h after induction with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside. Cells were lysed in buffer A plus 500 mm NaCl. After sonication, the lysate was centrifuged for 30 min at 100,000 × g. The supernatant was bound to nickel-nitrilotriacetic acid resin (Qiagen) for 2 h. The resin was washed sequentially with buffer A plus 500 mm NaCl and buffer A plus 100 mm NaCl. Bound protein was eluted with buffer A plus 100 mm NaCl and 150 mm imidazole. To remove the MBP tag, proteins were incubated with 1% (w/w) TEV protease for ∼16 h at 4 °C, followed by removal of the cleaved His₆-MBP by passage over amylose resin (New England Biolabs). Proteins were further purified by size exclusion chromatography on a Superdex 75 10/30 column in 20 mm HEPES (pH 7.5), 100 mm NaCl, and 1 mm DTT. Eluted protein was concentrated to ∼3–5 mg/ml, flash-frozen in the presence of 10% glycerol in liquid nitrogen, and stored at −80 °C. All PH domains were verified to be folded by thermal denaturation using SYPRO Orange dye (24).

Gα_q was purified by modifying existing methods (25, 26). Gα_q and His₈-Gγ₂ were expressed from a single baculovirus using the pFastbac-Dual shuttle vector (Invitrogen). The Gα_q-Gγ₂ baculovirus was used to infect Sf9 cells along with baculoviruses encoding Gβ₁ and GST-Ric8A (a gift from Gregory Tall, University of Rochester Medical Center). Because Gα_q isolated from a Gα_q-Ric8A complex had a 50-fold higher EC₅₀ value for PLC-β activation (8, 25), we only used the Gα_q that was bound to and eluted from Gβ₂His₈-Gγ₂. The eluted protein contained a contaminant with phospholipase activity that was removed using a SP-Sepharose column as described above for PLC-β3. The Gγ₂-CFP construct was a fusion of cerulean fluorescent protein (27) to the Ala² residue of human Gγ₂. Gβ₁g₂ and Gb₁g₂-CFP were purified after co-expression with His₆-Gα_i in Sf9 cells as described earlier (26). The Gβ₁γ₂ isoform was used in all experiments. Gα_i and phosducin were purified from E. coli as described previously (28, 29). All protein concentrations were determined by Amido Black binding (30).

Gα_q was activated before the assay by incubation at 16 °C for 14 h with 1 mm GTPγS in 50 mm NaHepes (pH 7.5), 100 mm NaCl, 2 mm MgCl₂, 1 mm DTT, 200 mm (NH₄)₂SO₄, 0.1 mg/ml BSA, and 0.5% CHAPS (8). The fraction of Gα_q bound to GTPγS was determined by [³⁵S]GTPγS binding.

The PH domain and PLC-β3 constructs with a single reactive Cys⁹⁷ were labeled with the thiol-reactive probe Alexa Fluor 594-C₅-maleimide. Prior to labeling, proteins were buffer exchanged into 20 mm Hepes (pH 7.5), 100 mm NaCl to remove dithiothreitol. The protein was reacted with a 10-fold molar excess of the robe on ice for 1–2 h, and the reaction was quenched with β-mercaptoethanol. The dye was removed by adsorption to an appropriate ion exchange resin (Q-Sepharose for the PH domain and SP-Sepharose for PLC-β3). The resin was washed with a buffer supplemented with 200 mm NaCl before elution of protein with a buffer supplemented with 450 mm NaCl. Peak fractions were pooled, concentrated, frozen in liquid N₂, and stored at −80 °C. The labeling efficiency was determined according to the absorption of Alexa Fluor 594 at 590 nm (ϵ = 96,000 cm⁻¹M⁻¹) and the protein concentration.

PLC Assay

PLC activity was measured by monitoring the hydrolysis of [³H]PIP₂ on the surface of large unilamellar vesicles (phosphatidylethanolamine:phosphatidylserine:PIP₂, 20:4:1 molar ratio, 0.25 mm total phospholipid) at 37 °C exactly as described earlier (8). Assays were initiated by the addition of PLC. For experiments that included the isolated PH domain, assays were conducted at 25 °C to prevent precipitation of the PH domain. Data are expressed as moles of inositol 1,4,5-trisphosphate produced per minute per mole PLC.

Fluorescence Measurements

Equilibrium fluorescence measurements were performed on a Fluorolog3-212 spectrophotometer (Horiba Jobin Yvon) using a 3-mm cuvette with both excitation and emission slits set at 5 mm. Gβγ-CFP was diluted in 200 μl of 25 mm Hepes (pH 7.5), 50 mm NaCl, 0.1 mg/ml BSA, 2 mm MgCl₂, and 0.2% cholate. Cholate was omitted when phospholipid vesicles were used. Phospholipid vesicles were prepared using the same procedure as for the PLC assay, except that [³H]PIP₂ was not included. The final concentration of lipids in the FRET binding assay was the same as in the PLC assay. Ca²⁺ and EGTA were omitted from all buffers for FRET measurements. After the addition of acceptor, the mixture was incubated on ice for 30 min. After equilibration to 25 °C for 5 min, fluorescence was recorded between 450 and 650 nm with excitation at 432 nm. FRET was measured as the decrease of Gβγ-CFP fluorescence at 475 nm over the sum of both components at the same concentrations.

Binding and competition data were fit as indicated using the Marquardt-Levenberg algorithm in SigmaPlot. For competitive inhibition of binding or activation, IC₅₀ values from fits to a single-site competition equation were converted to K_i, the equilibrium K_d for the competing ligand, using the formula K_i = IC₅₀ / (1 + [Y] / K_d,_Y) where Y is ligand whose concentration is held constant (usually PH-97Alx or PLC-97Alx), and K_d,_Y is its dissociation constant for Gβγ under the same conditions. Competition by Gα_i-GDP for PH-97Alx binding to Gβγ-CFP (Fig. 2C) was fit to a single-site model as for other competitors to yield the IC₅₀ value. However, the high affinity of Gα_i-GDP for Gβγ-CFP precludes simple conversion of IC₅₀ to K_i because the K_i is well below the total concentration of Gβγ-CFP. We therefore determined K_i by iteratively simulating the experimental data using the total concentrations of each protein and the previously determined K_d of 550 nm for Gβγ-CFP binding to PH-97Alx.

FIGURE 2.

The PH domain specifically inhibits Gβγ stimulation of PLC-β. The effect of the isolated PH domain on the stimulation of PLC-β3 by 10 nm Gβγ (●), 0.5 nm Gα_q-GTPγS (▵) or PLC-β3 basal activity (■). PLC-β3 activity for each G protein in the absence of PH domain is set to 100%. Data are represented as the mean + range from duplicate samples in a single experiment. The solid line shows a fit of the inhibition of Gβγ-stimulated activity to a single-site competition model with an IC₅₀ of 17.8 + 2 μm. Two other experiments gave similar results, with IC₅₀ values of 20 + 1 μm and 12.6 + 1.6 μm for PH domain inhibition of Gβγ-stimulated PLC-β activity.

Intramolecular Cross-linking and Quantification

PLC-β3 variants with Cys⁶⁰ and Cys¹⁶⁴ were cross-linked with bismaleimidoethane (BMOE, Thermo Fisher Scientific). Prior to cross-linking, the protein was buffer exchanged into 20 mm Hepes (pH 7.5), 100 mm NaCl to remove dithiothreitol. Typically, a 10- to 15-fold molar excess of BMOE was added to 10 μm PLC-β3, followed by 30 min of incubation at 0 °C. The reaction was quenched with excess DTT before use.

For quantification of the fraction of PLC cross-linked, the PLC-TEV construct was reacted with BMOE as described. Following quenching, an aliquot was incubated with a 10-fold molar excess of TEV protease at 34 °C for 14 h. The protein was then denatured with sample buffer, run on 6% SDS-PAGE, transferred to a nitrocellulose membrane, and detected with an antibody toward the C terminus of PLC-β3 (B521) (5). Immunoblots were quantitated using ImageJ to measure the intensities of cross-linked and residual non-cross-linked PLC-β3. The fraction of PLC-β3 cross-linked was calculated as the intensity of cross-linked PLC-β3 divided by the sum of both intensities.

Results

Gβγ Binds the PH Domain of PLC-β3

Deletion of the PH domain in PLC-β abolishes regulation by Gβγ (19), suggesting that the PH domain is required for Gβγ interaction with the intact enzyme. To determine whether the PLC-β PH domain is a Gβγ binding site, we developed a FRET-based binding assay. We used CFP fused to Gβγ as the donor and covalently labeled the isolated PLC-β3 PH domain that has a single reactive cysteine (PH-A97C) with an Alexa Fluor 594 acceptor (PH-97Alx). As shown in Fig. 1, PH-97Alx quenches the fluorescence of Gβγ-CFP, accompanied by simultaneous enhancement of Alexa Fluor 594 acceptor fluorescence. We detected no quenching of CFP fluorescence when we added unlabeled PH domain or free dye to Gβγ-CFP or when the labeled PH domain was unfolded by heat denaturation prior to addition. By this assay, Gβγ binds the PH domain with a K_d of 550 nm (Fig. 1B) in detergent solution.

FIGURE 1.

Gβγ binds the PLC-β PH domain. A, FRET between the two fluorophores in Gβγ-CFP and PH-97Alx is demonstrated by donor quenching and acceptor enhancement. 30 nm Gβγ-CFP was incubated in the presence of the indicated concentrations of PH-97Alx in buffer with cholate, and fluorescence was measured as described under “Experimental Procedures.” Data show a decrease of Gβγ-CFP fluorescence at 477 nm and enhancement of PH-Alexa Fluor 594 fluorescence at 617 nm. Fluorescence from direct excitation of Alexa Fluor 594 is subtracted from each spectrum. B, equilibrium binding of Gβγ to the PLC-β3 PH domain. The fluorescence of 30 nm Gβγ-CFP was measured as described in A at increasing concentrations of PH-97Alx. Data show the percentage of CFP fluorescence that is quenched and are the average and range from two independent measurements. The solid line shows a fit to a single-site binding equation with K_d = 550 + 60 nm (standard error of fit) with a maximum of 54% donor quenching. C, inhibition of Gβγ-PH binding by Gα_i-GDP and phosducin. 30 nm Gβγ-CFP was incubated with 700 nm PH-97Alx in the presence of the indicated concentrations of Gα_i-GDP (●) or phosducin (○), and CFP fluorescence was measured as described under “Experimental Procedures.” Data show the percentage of CFP fluorescence that is quenched and are the mean ± S.D. of measurements from two separate experiments, each performed in duplicate. The solid lines are fits to one-site competition models with IC₅₀ = 38 + 7 nm for Gα_i and 69 + 32 nm for phosducin (+ S.E. of fits). D, competition between PH-97Alx and unlabeled PH domain for Gβγ binding. Equilibrium binding of 30 nm Gβγ-CFP to 700 nm PH-97Alx in the presence of PH-A97C was measured by FRET as described under “Experimental Procedures,” and the percentage of CFP fluorescence quenched was calculated. Data are the mean + range of data measured in duplicate in two experiments. The solid line is a fit to a single-site competition model with IC₅₀ = 21 + 5 μm (+ S.E. of fit).

Termination of Gβγ signaling in cells occurs through sequestration by Gα_i-GDP after GTP hydrolysis, and Gα_i-GDP blocks Gβγ stimulation of PLC-β3 (8). As predicted, Gα_i-GDP competes with PH-97Alx for Gβγ binding, as evidenced by the recovery of CFP donor fluorescence (Fig. 1C). The IC₅₀ for Gα_i-GDP was ∼40 nm, and correction for the affinity with which Gβγ binds PH-97Alx (Fig. 1B) (see “Experimental Procedures”) indicates that the K_d for Gα_i-GDP-Gβγ binding is ∼3 nm (range, 2–5 nm based on standard error of fit shown in Fig. 1B), consistent with the subunits existing as a constitutive heterotrimer when Gα_i is in the GDP-bound inactive state. Phosducin, another regulator of Gβγ signaling that shares the same interface on Gβγ as Gα_i-GDP (31), also competitively displaced PH-97Alx from the Gβγ-PH complex with an IC₅₀ of ∼70 nm. From these data, the K_i for Gβγ-phosducin binding is 30 nm, in good agreement with previous reports (29). These observations suggest that the assay reports on specific Gβγ-PH domain binding. PH-A97C also displaced PH-97Alx to bind Gβγ, although with a much higher K_i (Fig. 1D) (see below).

To determine whether PH domain binding is necessary for Gβγ interaction with intact PLC-β, we asked whether the PH domain can inhibit Gβγ-stimulated PLC-β3 activity. As shown in Fig. 2, the addition of isolated PH domain inhibited Gβγ stimulation of PLC-β3 with an IC₅₀ of 18 μm and inhibition of 65% at the highest concentration tested. The PH domain did not alter the basal or Gα_q-GTPγS-stimulated activity of PLC-β3 at any concentration, indicating that the inhibition was an effect on Gβγ stimulation and not on the catalytic activity of PLC-β itself. These data indicate that the isolated PH domain competes with the PH domain in intact PLC-β to specifically block the Gβγ-PLC-β3 interaction. Thus, we propose that the PH domain defines the minimal Gβγ binding region in PLC-β.

The data in Figs. 1–3 measure the affinity of Gβγ binding to the isolated PLC-β PH domain using several different experimental approaches: FRET-based binding measurement, the effect on stimulation of PLC-β catalytic activity, and competition in both of these assays. The tightest interaction is that of the direct binding of Gβγ-CFP to PH-97Alx, with K_d ∼0.5 μm, and the weakest interactions are for competition by unlabeled PH domain for binding or activation in the presence of phospholipid vesicles. Disagreement among these values appears to arise both from the use of phospholipids rather than cholate in the various assay buffers and from the ability of Alexa Fluor labeling to increase affinity of binding. FRET-based binding and competition assays conducted in medium containing phospholipid vesicles report a lower-affinity interaction than when measured in cholate buffer, in agreement with the low-potency inhibition of PLC-β stimulation in vesicles. More significant is the observation that Alexa Fluor labeling increases affinity by 15- to 20-fold (Fig. 3B). Consistent with these observations, we observed only 10% inhibition of Gβγ-CFP-PH-97Alx binding by ∼80 μm PH-A97C in phospholipid-containing buffer. Although we do not understand the mechanism of these effects in any detail, the consistency of results from two assays, with full-length PLC and isolated PH domain, and under different conditions indicates that the variations do not undercut the interpretability of the binding interactions.

FIGURE 3.

Effect of vesicles and labeling on Gβγ interactions. A, equilibrium binding of Gβγ to the PH domain in the presence of phospholipid vesicles was measured by FRET. Data show the percent of CFP fluorescence that is quenched. Data are the average and range of measurements from two separate experiments, with measurements done once in each experiment. The solid line shows the fit to a single-site binding model with a K_d of 7.6 μm and maximal quenching of 57%. Note the difference in concentration of PH-97Alx from Fig. 1B. B, competition by PH-A97C (○) or PH-97Alx (●) for stimulation of PLC-β3 by 10 nm Gβγ. Error bars show the range for duplicate samples in one of two separate experiments. The solid lines show fits to single-site competition models. The IC₅₀ values in the two experiments were 0.7 and 1.4 μm for PH-97Alx and 10.5 and 16.3 μm for PH-A97C.

Rac1 and Gβγ Bind Distinct Surfaces on PLC-β

PLC-β2 and PLC-β3 are directly activated by the small GTPase Rac1. GTPγS-loaded Rac1 binds intact PLC-β with a K_d of ∼25 μm, similar to that for the isolated PH domain (32). Structural studies later confirmed the PH domain as the Rac1 binding site (12). We therefore asked whether Rac1 competes with Gβγ for stimulation of PLC-β activity using PLC-β2, the most Rac1-sensitive isoform. As observed previously (7), activation by Rac1 and Gβγ was essentially additive even at near-saturating concentrations of either ligand (Fig. 4). Further, earlier studies showed that point mutations within the PH domain diminish Rac1-stimulated activity without altering Gβγ-stimulated activity (12). These results indicate that both Rac1 and Gβγ interact with the PLC-β PH domain through distinct binding sites that do not overlap.

FIGURE 4.

Rac1 and Gβγ bind distinct regions on PLC-β. A, stimulation of PLC-β2 by Gβγ was measured in the presence (●) or absence (○) of 15 nm Rac1. Error bars show the range from duplicate measurements in a single experiment. The solid lines show fits to saturation functions with EC₅₀ values of 13 and 17 nm, respectively. A separate experiment with a different batch of proteins gave similar results, with EC₅₀ values of 9.5 and 12 nm. B, Rac1-stimulated PLC-β2 activity was assayed in the presence (●) and absence (○) of 15 nm Gβγ. Data show average and range from two independent measurements done in duplicate. The solid lines are fits to saturation functions with EC₅₀ values of 6.7 and 5.5 nm.

Prediction of the PH Domain-Gβγ Binding Interface by Structural Homology

Because Gβγ binds PH domains in many proteins, it is likely that it recognizes a common PH domain surface. The GRK2 PH domain-Gβγ interface has been extensively characterized biochemically (33) and structurally (34). The top face of the Gβ propeller makes extensive contact with strands β3 and β4 of the GRK2 PH domain core and with an unstructured, basic C-terminal extension past the PH domain, burying ∼2200 Ų of surface area (34). The PH domain in PLC-β adopts a fold similar to the GRK2 PH domain (root mean squared distance 3.8 Å for equivalent Cα atoms) (Fig. 5A), although it does not possess an analogous extension and is immediately followed by the EF hand domain. However, the β strands of the PH domain structurally align with the equivalent GRK2 regions even though the Gβγ contact residues are not strictly conserved (Fig. 5B). Furthermore, we noted that PLC-β1, PLC-β2, and PLC-β3 display a stronger conservation of residues in this region in comparison with PLC-β4, the only isoform that is not detectably stimulated by Gβγ (5).

FIGURE 5.

Structural homology and sequence conservation predict the Gβγ binding face of the PH domain. A, structural alignment of the PH domains of GRK2 (green, PDB code 1OMW) and PLC-β3 (gray, PDB code 3OHM). GRK2 residues involved in Gβγ binding are displayed as red sticks. The root mean squared distance over 95 equivalent Cα positions was 3.8Å. B, sequence alignment of the β2–4 regions of PH domains from PLC-β isoforms and GRK2. The alignment was performed in Promals3D. Residues conserved across PLC-β isoforms are shown in blue, PLC-β4 residues that differ from the other isoforms are shown in yellow, and GRK2 residues that interact with Gβγ are shown red. Conserved residues that are buried in the hydrophobic core of the protein are not colored for clarity. C, equilibrium binding of Gβγ to PLC-β3 PH domain mutants was measured by FRET as described under “Experimental Procedures.” The solid lines are fits to single-site binding models with affinities of 550 nm (wild type), 720 nm (T55R), 1700 nm (D62Q), and 6300 nm (F50Q). D, competition by PH domain mutants with intact PLC-β3 for Gβγ binding. Stimulation of PLC-β3 activity by 10 nm Gβγ was compared in the absence (−) or presence of PH domain mutants (10 μm). Data are the average and range from two independent measurements.

To test whether the PH domains of GRK2 and PLC-β3 bind Gβγ in a similar manner, we examined the role of strands β3 and β4 in the PLC-β PH domain by mutational analyses. The hydrophobic character of this region suggested that Gβγ-PH domain binding is driven by non-polar interactions. To increase the likelihood that a single amino acid substitution at this interface would result in significant perturbation, we chose to introduce the large polar amino acid glutamine. We found that replacement of a conserved hydrophobic residue with glutamine (F50Q) was sufficient to completely abolish binding to Gβγ (Fig. 5C). Consistent with our results, a GRK2 mutant harboring a substitution at the same position in the PH domain (GRK2 R587Q) was insensitive to stimulation by Gβγ (33). Because PLC-β4 is not activated by Gβγ, we predicted that replacement of PLC-β3 residues in this region with the corresponding PLC-β4 residues would diminish Gβγ binding affinity. We picked three positions, Tyr⁵³, Thr⁵⁵, and Asp⁶², which were conserved across Gβγ-responsive PLC-β isoforms but not in PLC-β4 (Asp⁶² is replaced by Glu in PLC-β1 and PLC-β2). Mutations at two of these residues, T55R and D62Q, displayed weaker binding to Gβγ with affinities 1.5- and 3-fold weaker than the wild-type protein. These results suggest that residues from strands β3 and β4 in the PH domain are important and specific binding determinants for Gβγ.

Next, we asked what effect these mutations have on the ability of the PH domain to compete with intact PLC-β3 for Gβγ binding. If a mutation weakened PH domain-Gβγ binding, then it would be predicted not to impede Gβγ stimulation of PLC-β3 catalytic activity. Addition of wild-type PH domain inhibited Gβγ stimulation of PLC-β3, as observed earlier. The PH (F50Q) mutant, which does not bind Gβγ, inhibited Gβγ-stimulated activity by less than 10% so that PLC-β3 activity was within error of the control sample (Fig. 5D). The T55R mutation in the PH domain also reduced its ability to inhibit stimulation by Gβγ, although loss of inhibition was only partial. PH (T55R) inhibited Gβγ-stimulated activity by 45% at 30 μm, the highest concentration tested, compared with 65% for the wild-type PH domain. This loss is consistent with the 1.5-fold reduction in its affinity for Gβγ. The PH (D62Q) mutant, which had 3-fold lower affinity for Gβγ, was similarly compromised in its ability to compete with PLC-β3 for Gβγ binding. At 10 μm, inhibition of Gβγ-stimulated PLC-β3 activity by PH(D62Q) was insignificant, whereas maximal inhibition was only 20% at the highest concentration tested.

Collectively, these data support our hypothesis that strands β3 and β4 in the PH domain form the Gβγ binding face. These results also suggest that the presence of polar residues in PLC-β4 in this region interferes with productive Gβγ interaction, rendering it insensitive to activation by Gβγ.

The Predicted Gβγ Binding Interface of PLC-β Is Not Surface-exposed

To understand how Gβγ interacts with PLC-β, we examined the proposed Gβγ binding residues of the PH domain in PLC-β crystal structures. The interface formed by these residues is distinct from the footprint of Rac1 on the PH domain of PLC-β2 (Fig. 6A), consistent with the lack of competition between Rac1 and Gβγ for binding PLC-β (Fig. 4). Although the Rac1 binding surface is completely surface-exposed, the predicted Gβγ binding face of the PH domain forms an intramolecular interface with the EF hands that buries close to 1200 Ų of total accessible surface area. Close inspection of the PLC-β structure reveals that this interface is composed of only a few hydrogen bonds and van der Waals contacts, suggesting that the interaction between the two surfaces is weak (Fig. 6B). The hydrogen bond interactions between the PH domain and EF hand residues involve residues whose side chain donor and acceptor atoms are 3–4 Å apart, significantly weaker in energy than the average hydrogen bond lengths observed in proteins (∼1.5–2 Å) (35). Taken together with the absence of electrostatic interactions or water bridges at this interface, we hypothesized that the PH domain is mobile in solution, existing in equilibrium between the conformation observed in crystal structures and an “open” conformation where the PH domain moves so that the residues required for Gβγ binding become accessible.

FIGURE 6.

The predicted Gβγ binding face of the PH domain is not surface-exposed. A, top panel, schematic of PLC-β. The C-terminal helical bundle is not shown. Bottom panel, crystal structure of PLC-β2 with the PH domain depicted as a surface (PDB code 2ZKM). Residues predicted to be involved in Gβγ binding based on homology with the GRK2-Gβγ structure (PDB code 1OMW) are shown in red. This surface is not solvent-accessible and does not overlap with the region that binds Rac1 (green). B, details of interactions between the PH domain and EF hands in intact PLC-β3. Residues are colored to match the schematic in A. The numbers indicate the distance between donor and acceptor atoms for residues involved in hydrogen bonds.

If the PH domain of PLC-β has to swing away from the EF hand domain to bind Gβγ, then preventing mobility of the PH domain should obstruct Gβγ access to its binding site. We used intramolecular cross-linking to restrict PH domain motion by introducing cysteines in the PH domain and EF hands to give PLC-β3-Cys⁶⁰,Cys¹⁶⁴. These residues lie at the solvent-exposed edge of the PH domain-EF hand interface (Fig. 7A) so that they can be cross-linked using BMOE, an 8-Å cysteine-reactive cross-linker. Irreversible cross-linking of these residues should block the motion of the PH domain and stabilize it in the conformation observed in PLC-β crystal structures. As predicted, cross-linking PLC-β3-Cys⁶⁰,Cys¹⁶⁴ inhibited Gβγ-stimulated PLC-β activity (Fig. 7B) without blocking stimulation by Gα_q-GTPγS (Fig. 7C). Alkylation of PLC-β3-Cys⁶⁰,Cys¹⁶⁴ with a monovalent maleimide or alkylation with BMOE of single-cysteine mutants, PLC-β3-Cys⁶⁰ or PLC-β3-Cys¹⁶⁴, had no effect. These data indicate that intramolecular cross-linking at the PH domain-EF hand interface interferes with Gβγ-PLC-β3 interaction uniquely without global effects on PLC-β structure or catalytic activity. BMOE cross-linking of PLC-β3-Cys⁶⁰,Cys¹⁶⁴ decreased the maximal Gβγ-stimulated activity of cross-linked PLC-β only partially under optimized conditions, but the EC₅₀ for Gβγ was unchanged (∼20–30 nm, Fig. 7D). If cross-linking partially blocked a Gβγ binding site on PLC-β, we would predict an increase in EC₅₀ for Gβγ activation because of decreased affinity rather than a decrease in maximal response.

FIGURE 7.

Cross-linking PLC-β3 reduces maximal stimulation by Gβγ but does not change the EC₅₀ value. A, close-up view of the PH (blue) and EF hand (yellow) domains of PLC-β3 (PDB code 3OHM). Residues where cysteines were introduced for cross-linking are depicted as orange spheres. B, cross-linking the PH domain and EF hands inhibits stimulation by Gβγ. Wild-type PLC-β3, PLC-β3-Cys⁶⁰, PLC-β3-Cys¹⁶⁴, and PLC-β3-Cys⁶⁰,Cys¹⁶⁴ were assayed for activity in the presence of 15 nm Gβγ after treatment with DMSO (black columns), N-ethylmaleimide (NEM, light gray columns), or BMOE (dark gray columns). Data are normalized for each protein to activity of the DMSO-treated sample and represent the mean ± S.D. from five experiments, each performed in duplicate. C, the same as B but assayed in the presence of 0.5 nm Gα_q-GTPγS. D, stimulation of PLC-β3 before (●) and after (○) treatment with cross-linker in the presence of increasing concentrations of Gβγ. Data are the average and range of duplicate measurements in two separate experiments. The solid lines are fits to saturation functions with EC₅₀ values of 22 + 2 nm (treatment with DMSO) and 25 + 3 nm (treatment with BMOE) (+ S.E. of fits).

The data in Fig. 7D suggest that BMOE cross-linking is incomplete, with the cross-linked PLC insensitive to Gβγ and the rest retaining full sensitivity to Gβγ. Initial attempts to quantify cross-linking failed because the electrophoretic mobility of cross-linked PLC-β3 was indistinguishable from that of the control, and mass spectrometry gave poor peptide coverage in the relevant regions. We therefore designed a mutant PLC-β3 to quantify the fraction of cross-linked species. We introduced a TEV protease cleavage site in an exposed loop of the PH domain of PLC-β3-Cys⁶⁰,Cys¹⁶⁴ to give a construct we refer to as PLC-TEV. Proteolytic cleavage of PLC-TEV should generate two separate polypeptides but would produce a single species in the enzyme that had been correctly cross-linked between Cys⁶⁰ and Cys¹⁶⁴ (Fig. 8A). The relative sizes of cross-linked and non-cross-linked species after proteolysis are such that they can be separated by gel electrophoresis. Treatment of PLC-TEV with protease yielded a species that migrated faster on a SDS-PAGE gel because of loss of the ∼10-kDa N-terminal fragment (Fig. 8A). Treatment with the cross-linker prior to proteolysis abrogated this shift as expected, confirming that this strategy can be used to quantitate the fraction of PLC-β cross-linked. To test whether cross-linking proceeds to completion, we incubated PLC-TEV with the cross-linker for varying times and measured the fraction of cross-linked species. Cross-linking occurred rapidly, on the order of minutes, but plateaued with only ∼70% of total PLC-TEV cross-linked after 30 min (Fig. 8B). Changing the pH or temperature or including a mild reducing agent, tris(2-carboxyethyl)phosphine, in the cross-linking buffer did not increase fractional cross-linking. Incubating a fixed concentration of protein with increasing amounts of BMOE gave similar results. We then analyzed the Gβγ response of these species and observed a negative correlation between the fraction of cross-linked PLC-β and its stimulation by Gβγ (Fig. 8C). This result indicates that the residual activity observed in Fig. 7D was from PLC-β that had not been cross-linked. Importantly, stimulation by Gα_q-GTPγS was not dependent on the cross-linked status. Therefore, our observations are consistent with the idea that Gβγ is unable to stimulate PLC-β when the PH domain is immobilized by tethering to the first EF hand.

FIGURE 8.

Restricting PH domain motion in PLC-β3 blocks the Gβγ response. A, quantifying fraction of PLC-β3 cross-linked. Left panel, schematic of the PH and EF hand domains in the PLC-TEV construct to show the TEV protease site and Cys⁶⁰ and Cys¹⁶⁴, the cysteines cross-linked by BMOE. Right panel, separation of cross-linked and non-cross-linked species in BMOE-treated PLC-β. PLC-TEV was treated with DMSO or BMOE, quenched, and incubated with or without TEV protease. Samples were analyzed to estimate the fraction of PLC cross-linked as described under “Experimental Procedures.” B, time course of intramolecular cross-linking of PLC-β3 with BMOE. PLC-TEV was cross-linked with BMOE. Aliquots were quenched with DTT at the indicated times, and the fraction of PLC cross-linked was measured as described under “Experimental Procedures.” The solid line is a fit to a first-order model with a t_½ of 2 min. C, correlation of fractional loss of Gβγ-stimulation and fraction of protein cross-linked. Aliquots of PLC-TEV from the experiment in B were assayed for activity with either 15 nm Gβγ (○) or 0.5 nm Gα_q-GTPγS (●). Activities are plotted on the y axis. Activity measured for DMSO-treated PLC is set to 100% for both Gα_q and Gβγ. The x axis shows the fraction of protein cross-linked determined by the TEV protease assay. The solid line shows a fit of the Gβγ-stimulated activities to a linear regression model with R² (coefficient of determination) = 0.89.

Restraining PH Domain Motion Blocks Gβγ Binding

Our results suggested that cross-linking PLC-β leads to reduced Gβγ sensitivity by prohibiting its binding to the G protein. We developed a FRET-based assay for Gβγ interaction with PLC-β3 to directly test whether cross-linking blocks Gβγ-PLC-β binding. We labeled PLC-β3 at Cys⁹⁷ in the PH domain with Alexa Fluor 594 (PLC-97Alx), as we had done for the isolated PH domain, and asked whether PLC-97Alx is a FRET acceptor for Gβγ-CFP. PLC-97Alx quenched Gβγ-CFP fluorescence as predicted, similar to PH-97Alx. The K_d for Gβγ-PLC-β3 binding was 220 nm (Fig. 9A). Unlabeled PLC-β3 inhibited binding between Gβγ-CFP and PLC-97Alx with an IC₅₀ of 370 nm (Fig. 9B), equivalent to a K_i of 170 nm, which is approximately equal to the directly determined K_d for labeled PLC-β3. These data indicate that the assay reports on specific binding between Gβγ and PLC-β3.

FIGURE 9.

Cross-linked PLC-β does not bind Gβγ. A, binding of Gβγ to PLC-β3 was measured by FRET. 30 nm Gβγ-CFP was incubated with the indicated concentrations of PLC-97Alx, and fluorescence was measured as described under “Experimental Procedures.” Data show the percentage of CFP fluorescence that is quenched by energy transfer to Alexa Fluor 594. Error bars are the range of measurements from duplicate samples in two separate experiments. The solid line is a fit to a single-site binding model with K_d = 220 + 50 nm (standard error of fit) and a maximum 57% quenching of CFP fluorescence. B, competition between PLC-97Alx and PLC-β3 for Gβγ binding. Binding of 30 nm Gβγ to 250 nm PLC-97Alx at equilibrium was measured by FRET in the presence of the indicated concentrations of PLC-β3. Data show the percentage of CFP fluorescence that is quenched and are the average and range of measurements from two independent experiments. The solid line is a fit to a single-site competition equation with IC₅₀ = 370 + 36 nm (+ S.E. of fit). C, effect of cross-linking PLC-β3 on its binding to Gβγ. Equilibrium binding of 30 nm Gβγ to 300 nm PLC-97Alx was measured by FRET in the presence of PLC-TEV treated with DMSO (○) or BMOE (■). The percentage of CFP fluorescence that is quenched by energy transfer to PLC-97Alx is shown. The data points are from one experiment where the BMOE-treated PLC-TEV was 65% cross-linked. For BMOE-treated PLC-TEV (■), values on the x axis are the concentration of non-cross-linked PLC-β3 at each point. Excess BMOE-treated PLC-TEV was added so that the final concentrations of non-cross-linked PLC-β3 are as indicated. Greater quenching in the absence of competitor compared with B is due to the higher concentration of PLC-97Alx acceptor in this experiment. The solid line is a fit of the competition by DMSO-treated PLC-TEV to a single-site model with IC₅₀ = 360 + 50 nm (+ S.E. of fit). Similar results were obtained when this experiment was repeated using a different batch of BMOE-treated PLC-TEV with a slightly lower fraction cross-linked (53%). Inset, competition by BMOE-treated PLC-TEV but with the abscissa showing total concentrations of PLC-TEV for the same dataset as in the main panel.

We then used this assay to ask whether cross-linked PLC-β binds Gβγ. Because BMOE and Alexa Fluor 594 both react with the sulfhydryl group of cysteines, we could not label cross-linked PLC-β with Alexa Fluor 594. Rather, we used competition to test the binding of cross-linked PLC-β3 to Gβγ. We found that PLC-TEV treated with DMSO competes with PLC-97Alx for Gβγ binding with an IC₅₀ of ∼360 nm or K_i of ∼150 nm. (Fig. 9C), which agrees with the K_i for wild-type PLC-β3 (Fig. 9B). BMOE-treated PLC-TEV also competed for binding (Fig. 9C, inset), but PLC-TEV treated with BMOE is not completely cross-linked and competition apparently reflects only the residual non-cross-linked PLC (see above). The TEV proteolysis assay indicated that 35% of BMOE-treated PLC-TEV used in the experiment in Fig. 9C was not cross-linked, and correction for fractional cross-linking indicated that only the non-cross-linked fraction accounts for the observed inhibition (Fig. 9C). Collectively, these results indicate that PLC-β does not bind Gβγ when cross-linked.

Discussion

Stimulation of PLC-β isoforms by Gβγ is well established, but the identity of the binding site for Gβγ has eluded consensus. In this study, we show that the N-terminal PH domain is the minimal Gβγ binding site. Identification of the Gβγ-binding surface led to a new structural model for Gβγ binding that involves substantial movement of the PH domain that may help anchor and orient PLC-β at the bilayer surface for PIP₂ hydrolysis. This work also describes a reliable and quantitative assay for binding of Gb₁g₂ to PLC-β3 that should be applicable to any PLC-β and Gβγ dimer.

The FRET-based binding assays indicate that Gβγ binds similarly to both the isolated PH domain of PLC-β3 and to intact PLC-β. The free PH domain also inhibited PLC-β activation by Gβγ, but not Gα_q (Fig. 2), by competing with the intact enzyme for binding Gβγ. Binding of Gβγ to the PH domain was inhibited by Gα_i-GDP, consistent with the idea that Gα_i sequesters Gβγ in the G_i heterotrimer until G_i is activated by GTP.

We defined the Gβγ binding surface of the PLC-β PH domain starting with analogy to the crystal structure of Gβγ in complex with GRK2. PH domains in multiple proteins are implicated in Gβγ binding (20), and, of these, Gβγ binding to the PH domain of GRK2 is the best studied (34). The crystal structure of Gβγ in complex with GRK2 shows that Gβγ recognizes a hydrophobic face of the GRK2 PH domain, and this surface was readily mapped to homologous residues of the PLC-β PH domain (Fig. 5). The effect of mutating residues on this face of the PH domain confirms the importance of this hydrophobic surface for regulation and binding by Gβγ. Mutation of the conserved Phe⁵⁰ at this surface to a polar residue abolished Gβγ binding and inhibition of Gβγ-stimulated PLC-β3 activity, and milder mutations at other residues diminished binding to a lesser extent. Previous studies showed that point mutations at Ile⁸⁰, Trp⁹⁹, Met¹⁰¹, Leu¹¹⁷, and Trp³³² in the opposing surface of Gβ reduced stimulation of PLC-β (36, 37). Taken together, these data suggest that non-polar rather than electrostatic interactions contribute most of the free energy of binding between Gβγ and the PH domain.

In the GRK2-Gβγ complex, a cluster of Lys/Arg residues in an extension past the GRK2 PH domain also interacts with the Gβγ surface. Strikingly, the first EF hand domain (EF-1) immediately C-terminal to the PH domain in all four PLC-β isoforms is similarly rich in Lys/Arg residues, and Barr et al. (38) showed that a quadruple charge-reversal mutation in EF-1 is less responsive to Gβγ. They also showed that a GST fusion construct of the PH domain plus EF-1 bound Gβγ in pulldown experiments but that the charge reversal mutation reduced binding. In that same study, Gβγ also bound a fusion construct of GST and the PH domain alone, in agreement with our observation that the PH domain is the minimal Gβγ binding region, but just the PH fusion was less efficient in pulldowns than the PH plus EF-1 construct. These data argue that Gβγ minimally binds the PH domain and that interactions of basic residues in EF-1 may enhance affinity.

The Gβγ-interacting, hydrophobic face of the PH domain we identified is surprisingly buried in an intramolecular interface with the EF hands in crystal structures of PLC-β3 (Fig. 6) so that a substantial conformational change would be required to allow Gβγ binding. We used intramolecular cross-linking of the PH domain to the EF hands to confine the PH domain in the conformation observed in PLC-β crystal structures. Consistent with our proposal, immobilizing the PH domain makes PLC-β refractory to stimulation by Gβγ (Figs. 7 and 8). Furthermore, we showed that Gβγ failed to bind PLC-β3 in which the PH domain was immobilized (Fig. 9). The Gα_q response did not change when PLC-β was cross-linked, indicating that the impaired interaction of cross-linked PLC-β was Gβγ-specific. However, cross-linking-mediated immobilization also likely blocked access to the PH domain surface that faces the TIM barrel. We tested various mutants in this region and found no change in PH domain inhibition of Gβγ-stimulated PLC-β activity, suggesting that this surface is not involved in Gβγ binding.

Gβγ-PLC-β Binding Requires Movement of the PH Domain

If our identification of the Gβγ binding interface in PLC-β is correct, then there must be significant movement to expose it because it is mostly occluded in the resting PLC-β structure by juxtaposition to the EF hand domain (Fig. 6). Furthermore, much of the exposed surface of the PH domain is needed for the Rac1 binding site, and we showed that Rac1 and Gβγ can bind PLC-β simultaneously (Fig. 4). To allow regulatory Gβγ binding, we propose that PLC-β exhibits intrinsic flexibility between closed and open conformational states of the PH domain (Fig. 10). The closed state, in which the PH domain faces the EF hands, is favored under crystallization conditions and, presumably, in solution in the absence of activators. PLC-β can, however, transiently sample an open state in which the PH domain moves away from the EF hands to expose the hydrophobic Gβγ binding face and, by a still not understood mechanism, stimulate catalytic activity. The need for such motion is supported by the observation that locking PLC-β3 in the closed state by cross-linking the PH domain to EF-1 blocks both Gβγ binding and the ability of Gβγ to activate (Figs. 8 and 9).

FIGURE 10.

Model for Gβγ binding to PLC-β. In the inactive state, PLC-β (the domains are colored as in Fig. 6A) exists in equilibrium between the conformation observed in crystal structures (Closed) and a less favored open conformation. The Gβγ-binding surface on the PH domain (red) is occluded by the EF hand domain in the closed conformation. In the open conformation, the PH domain swings up and to the right, away from the EF hand domain, to expose the Gβγ (green) binding surface.

Crystallography of PLC-δ suggests that significant motion of the PH domain in PLC is plausible. In crystals of full-length PLC-δ1, the PH domain and EF-1 were both invisible, suggesting that they undergo significant motion even in crystals (39, 40). Truncation of the PH domain alone stabilized PLC-δ so that its structure could be determined. Further, Drin et al. (41) showed that Gβγ-PLC-β binding reduced intramolecular FRET between the PH domain and the TIM barrel, suggesting that movement of the PH domain away from the TIM barrel to an open state is coupled to Gβγ binding (41). The transition from the closed to the open conformation in PLC-β may also involve loss of EF-1 structure. Unfolding EF-1 would achieve twin objectives: higher affinity Gβγ binding from interaction with the EF-1 basic residues (38), as discussed above, and a longer tether to allow whatever PH domain movement is needed for Gβγ to bind in an appropriately activating orientation.

The requirement for PH domain motion in PLC-β activation by Gβγ does not produce a detailed model for the PLC activation process, but it is consistent with a general model for PLC activation proposed by Sondek and co-workers (2). PLC-β contains an anionic autoinhibitory strand that occludes the active site and that must be moved to allow activation (1). Sondek and co-workers proposed (2) that simply holding the PLC molecule tightly against the bilayer surface shoves the autoinhibitory X-Y linker away from the active site by electrostatic and/or steric repulsion by negatively charged lipids (1). Although we do not know precisely how the Gβγ-PH domain complex is oriented with respect to the bilayer, anchoring PLC-β at the membrane surface is an important part of the Gβγ activation mechanism. Gβγ with a non-prenylated Gγ does not bind to membranes and does not activate PLC-β (42). Less direct support for the role of bilayer anchorage comes from studies of a PLC chimera with a PIP₂-binding PH domain (43).

A mobile PH domain can allow binding of PLC-β to Gβγ and consequent activation, but this mechanism by itself does not constrain PLC-β orientation. Such a simple mechanism might, however, explain why Gβγ is a less efficacious activator of PLC-β than Gα_q. Gα_q binds PLC-β by contacting regions in the EF hand domain, the C2 domain, and a helical extension immediately C-terminal to the C2 domain (11) and may thereby help orient PLC-β with its active site facing the bilayer to facilitate access to the PIP₂ substrate. Gα_q binding has also been proposed to relieve autoinhibition by moving the helical extension away from the autoinhibitory strand to allow it to move away from the active site (13). There are no data to suggest that Gβγ performs either function and may therefore be a weaker activator.

Mobility of the PH domain in binding Gβγ may also reconcile PH-Gβγ binding with the suggestion by Smrcka and co-workers (21) that Gβγ has a second binding site on PLC-β on the TIM barrel domain. This site is essentially on the side of PLC-β opposite from that of the PH domain in crystal structures. If the PH domain exhibits significant motion, it is possible that Gβγ binds simultaneously to the PH and TIM barrel domains, although this proposal awaits rigorous investigation

Our work suggests the presence of a previously unappreciated PLC-β conformation critical to Gβγ binding and regulation of phospholipase activity. PLC-β is also synergistically activated by Gα_q and Gβγ (8), and the G_q GTPase-activating protein activity of PLC-β is inhibited by Gβγ (17). How the physical and functional interactions of Gβγ with PLC-β and Gα_q direct the dynamics of complex assembly/disassembly in this three-protein system is uncertain. Studies focused on visualizing these interactions will shed light on the mechanisms coordinating Gβγ regulation of PLC-β activities.

Author Contributions

G. K. conceived the project, designed and conducted the experiments, analyzed most of the data, and prepared the manuscript. E. M. R. contributed to the data analysis and wrote the paper with G. K.