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J. Biol. Chem., Vol. 276, Issue 45, 41797-41802, November 9, 2001

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Role of the  Subunit Prenyl Moiety in G Protein Complex Interaction with Phospholipase C^*

Vanessa C. Fogg, Inaki Azpiazu, Maurine E. Linder^§, Alan Smrcka^¶, Suzanne Scarlata, and N. Gautam^**

From the Departments of  Anesthesiology, ^** Genetics, and ^§ Cell Biology & Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, the  Departments of Physiology & Biophysics and Molecular Genetics & Microbiology, State University of New York, Stony Brook, New York 11794, and the ^¶ Department of Pharmacology & Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received for publication, August 10, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The G protein complex regulates a wide range of effectors, including the phospholipase C isozymes (PLCs). Prenyl modification of the  subunit is necessary for this activity. Evidence presented here supports a direct interaction between the G protein  subunit prenyl group and PLC isozymes. A geranylgeranylated peptide corresponding to the C-terminal region of the  subunit type, 2, strongly inhibits stimulation of PLC2 and PLC3 activity by the complex. This effect is specific because the same peptide has no effect on stimulation of PLC by an  subunit type, q. Prenylation of the  peptide is required for its inhibitory effect. When interaction of prenylated  subunit peptide to fluorophore-tagged PLC2 was examined by fluorescence spectroscopy, prenylated but not unprenylated peptide increased PLC2 fluorescence emission energy, indicating direct binding of the prenyl moiety to PLC. In addition, fluorescence resonance energy transfer was detected between fluorophore tagged PLC and wild type complex but not an unprenylated mutant complex. We conclude that a major function of the  subunit prenyl group is to facilitate direct protein-protein interaction between the complex and an effector, phospholipase C.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Prenylation with isoprenoid groups is a common post-translational modification of proteins. Isoprenoids are a diverse family of lipid compounds made up of a repeating five-carbon structure called the isoprene unit. Protein prenylation generally consists of the attachment of either of two isoprenoids: the 20-carbon geranylgeranyl group or the 15-carbon farnesyl group (1). Heterotrimeric G proteins () are a class of prenylated proteins that mediate the majority of neurohormonal signaling pathways in mammals. Upon G protein activation by a cell surface receptor, both the  subunit and the complex of a G protein can regulate downstream effectors (2). The  subunit of G is modified with either a geranylgeranyl group or a farnesyl group (3). The isoprenoid is added to a conserved cysteine residue at position 4 from the C-terminal end of the protein, in a consensus motif for prenylation called the CAAX box. The last amino acid in the motif (X) determines whether the cysteine is geranylgeranylated or farnesylated. Most  subunits are geranylgeranylated, but 1, c, and 11 are farnesylated (3).

The role of prenylation of the  subunit in G protein function is still unclear. As with several other proteins, it has been established that the prenyl group on the G protein complex plays a role in anchoring the protein to lipid membranes; complexes mutated at the cysteine residue in the CAAX box of the  subunit no longer associate with the plasma membrane and are located in the cytosol (4, 5). It is thought that the hydrophobic prenyl moiety associates with lipid membranes through lipid-lipid interactions, thus acting as a membrane anchor for proteins. In addition to lipid-lipid interactions, there have been suggestions from studies on small GTP-binding proteins that the prenyl moiety may also interact with proteins and stabilize protein-protein interactions (1).

It is not yet known whether the prenyl moiety of the G protein  subunit is involved in direct interactions with proteins, although prenylation has been shown to be necessary for interaction with receptors and effectors in a number of systems. The  subunit prenyl group has been shown to be a requirement for receptor activation of a G protein (6). Prenylated peptides specific to the C-terminal region of the  subunits have been shown to interact with receptors (7, 8). Prenylation of these peptides was a requirement for this activity. Similar to this requirement in the case of receptors, prenylation of G has been shown to be a requirement for effector interaction also. G containing a mutant unprenylated  subunit does not activate PLC2¹ either in vivo or in vitro (9, 10). Unprenylated complex also does not stimulate adenylyl cyclase type II or inhibit adenylyl cyclase type I (11). However, in all of these studies, the receptors or effectors were either integral membrane proteins (receptor and adenylyl cyclases) or membrane-associated proteins that required lipids as substrates (PLC isozymes). It has thus been unclear whether the prenyl group requirement facilitated complex-membrane interaction or complex interaction with receptor/effector protein.

Previous results have implied that the  subunit prenyl moiety interacts directly with proteins. For instance,  subunit peptides modified with isoprenoids of varying chain lengths differed in their ability to stabilize activated rhodopsin (12). However, there was no direct correlation between overall hydrophobicity with the efficacy of the modified peptides in the receptor stabilization assays. Farnesylated peptides were more active than geranylated (C-10) or geranylgeranylated peptides. These results indicated that the function of the prenyl moiety was more likely stabilization of protein interactions rather than membrane binding. The altered activity of mutant G with prenyl moieties switched from geranylgeranyl to farnesyl or vice versa also implied that the prenyl moiety may be involved in protein interaction (13). Overall, the question of whether the prenyl group plays this important role of stabilizing G protein-receptor or G protein-effector interactions in addition to stabilizing contact with the membrane has remained unresolved. The recent solution of a crystal structure of prenylated Cdc42, a Rho family member, bound to RhoGDI provides the first direct evidence for specific contact between the prenyl moiety and an interacting protein. In this crystal structure, the geranylgeranyl group of Cdc42 occupies a hydrophobic pocket in RhoGDI (14).

To examine whether the  subunit prenyl group is directly involved in interaction with a G protein effector, we synthesized prenylated peptides specific to  subunits. These peptides were then tested for their ability to compete with G in activation assays of PLC2 and PLC3. PLC enzymes are a family of proteins that hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP₂), releasing the second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (15). They are regulated by both G protein  subunits (of the G_q class) and the complex (16). The results from the peptide inhibition assays suggest that the prenylated  peptides compete with the complex for a site on PLC. A fluorescence-based binding assay confirmed direct interaction of the prenylated  peptide with PLC. To examine the role of the  prenyl moiety within the context of the whole complex, we then compared a mutant unprenylated complex with prenylated complex in FRET based assays, which measured direct binding to PLC isozymes in a highly quantitative fashion. The results from these experiments provide strong evidence that the  subunit prenyl group directly facilitates interaction of G with PLC.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

PIP₂ and phosphatidylethanolamine were obtained from Avanti Polar Lipids. [³H] PIP₂ was from PerkinElmer Life Sciences. Nickel-nitrilotriacetic acid resin was from Qiagen. All other reagents were from Sigma.

Construction of Recombinant Baculoviruses

Details of the construction of baculoviruses expressing His-PLC2, His-PLC3, G protein His-i2, q, 1 subunit, 4 subunit, 2 subunit, His-2 subunit, and mutant (C68S) unprenylated 2 subunit have been published (11, 17-21).

Purification of G protein Subunits and PLC Isozymes

The final preparations of all proteins were over 90% pure. Purification of subunits was performed essentially as described before (21).

Purification of 4His2 (Used in the PLC Activity Assays)-- Sf9 cells simultaneously infected with 4 and His-2 baculoviruses were lysed by nitrogen cavitation, and the membranes were extracted with 1% cholate. The detergent extract was applied to a column of nickel resin nickel-nitrilotriacetic acid, washed with buffer A (20 mM Hepes, pH 8.0, 1 mM MgCl₂, 10 mM -mercaptoethanol) containing 300 mM NaCl, 0.5% polyoxyethylene 10 lauryl ether (C₁₂E₁₀) (Sigma) and 10 mM imidazole. complex was eluted with buffer A containing 50 mM NaCl, 1% cholate, and 250 mM imidazole. Peak fractions were concentrated to a final concentration of 1-2 mg/ml.

Purification of Wild Type 12 (Used in the PLC2 Binding Assay)-- Sf9 cells were co-infected with baculoviruses expressing His-i2, 1, and 2. The cells were lysed and proteins peripherally associated with membranes isolated as described before (21). Briefly, extracted proteins were bound to nickel-nitrilotriacetic acid resin. Bound complex is eluted using aluminum fluoride. Peak fractions were dialyzed and concentrated into 20 mM Hepes, pH 8.0, 1 mM EDTA, 3 mM MgCl₂, 3 mM dithiothreitol, and 0.7% CHAPS.

Purification of Mutant 12 C68S (Used in the PLC3 Binding Assay)-- Purification of mutant unprenylated 12 was performed using a modification of the procedure described in Ref. 22. Sf9 cells co-infected with baculoviruses expressing 1 and 2C68S were lysed, and the soluble fraction was subjected to sequential chromatography over Q-Sepharose and hydroxylapatite.

Purification of q-- q was purified as described before (17). Gq was expressed in Sf9 cells using a recombinant baculovirus. To ensure stability of the G subunit, Sf9 cells were also co-infected with baculoviruses expressing  and  subunits. q was purified from membrane extracts by sequential chromatographic steps as described before.

Purification of PLC2 and PLC3-- PLC2 and PLC3 were purified as described previously (19, 20). Briefly, Sf9 cells were infected with baculoviruses expressing histidine-tagged PLC2 or histidine-tagged PLC3. PLC proteins were purified from cell extracts by nickel chromatography.

Peptide Synthesis and Chemical Prenylation

Peptides were synthesized, chemically geranylgeranylated or farnesylated, and purified as described (23). The geranylgeranyl-bromide was obtained from American Radiolabeled Chemicals (St. Louis, MO). Farnesyl was obtained from Aldrich. Briefly, peptide (2 µmol) and prenyl bromide (4 µmol) were mixed in a solution of butanol:methanol:water (1:1:1) previously purged under nitrogen atmosphere. Butyl-hydroxytoluene was provided as an antioxidant. The reaction was started with 0.5 M sodium carbonate. The reaction was allowed to proceed at room temperature under nitrogen atmosphere, in the dark with continuous agitation for 18 and 24 h. The reaction was stopped with acetic acid, and the samples were frozen at 85 °C.

Prenylated peptides are purified by reverse chromatography on a PepRPC fast protein liquid chromatography column HR 10/16 (Amersham Pharmacia Biotech) using a linear (0-100%) gradient of acetonitrile in water containing 0.1% trifluoroacetic acid. The prenylated compounds elute at a position of the gradient corresponding to approximately 50-60% acetonitrile content. Farnesylated peptides elute earlier than their geranylgeranylated counterparts. Peptides were usually converted to prenyl peptide with a 30-50% yield, which after purification and other operations resulted in a yield of 15-25% neat prenylated peptide. Prenyl peptides were stored in butanol:methanol:water (1:1:1, volume) or dimethylsulfoxide at 85 °C. The molecular masses of the prenylated peptide was checked by mass spectrometry. The concentration of the peptide was determined by amino acid analysis. The integrity of the modified peptides in stocks was checked regularly by chromatography in a PepRPC column by fast protein liquid chromatography.

PLC Assays

stimulation of phospholipase C was performed as described previously (19).

Peptide Inhibition of Complex Stimulated PLC2 Activity-- Prenylated peptides were stored in a 1:1:1 solution of butanol:methanol:water at 80 °C. Appropriate amounts were added to tubes and vacuum dried to remove the organic solvents. Peptides were then solubilized by sonication in presonicated lipid substrate (50 µM PIP₂, 200 µM phosphatidylethanolamine, and [³H]PIP₂; ~8000 cpm/assay). 120 pM PLC2 and 100 nM final concentration of 42 were then added to the lipid vesicle/peptide mix. The reactions were started by addition of CaCl₂ and incubated for 15 min at 30 °C. The reactions were stopped by addition of 10% trichloroacetic acid, and bovine serum albumin was added to precipitate proteins and lipids. Inositol 1,4,5-trisphosphate remained in the supernatant, and [³H]inositol 1,4,5-trisphosphate release was quantitated by scintillation counting. Peptide inhibition of complex-stimulated PLC3 activity was performed as above with 600 pM PLC3 and 10 nM final concentration 42 per assay.

q Activation of PLC3-- q activation of PLC3 was performed essentially as described (17) except that q was preactivated by incubation with 10 mM NaF and 30 µM AlCl₃. Peptide effect on q stimulation of PLC3 was tested as above; 5 µM 2-gg peptide was used for these assays.

Measurement of Peptide-PLC and Complex-PLC Associations Using Fluorescence Spectroscopy

Association between PLC2 and G12 or peptide was quantified by fluorescence (24, 25). All studies were done in the presence of extruded lipids (diameter, 100 nm) composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphoserine] (POPS) or a 2:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphocholine] and POPS. The inclusion of lipid was solely to promote solubility of G and of the prenylated peptide. G complexes (wild type or mutant) were reconstituted into membranes by adding concentrated lipid solution to detergent solubilized complex and then removing the detergent by dialysis. Fluorescence studies were carried out by labeling PLC and G with an amine-reactive coumarin or DABCYL (Molecular Probes, Eugene, OR) by adding a 3-4-fold excess of probe to the proteins at pH 8.0, incubating for 30 min, and removing unreacted probe by dialysis (25). PLC and activity were independently assayed using labeled and unlabeled proteins to ensure that labeling did not affect activity.

Titrations were carried out by placing 120 µl of sample in a 3-mm-path length cell and adding small (0.5-2 µl) amounts of the titrating solution. The peptide was dried down with nitrogen and resuspended in the reaction mix or added directly in solution depending on the concentration. Spectra were taken on an ISS spectrofluorometer using an excitation wavelength of 340 for coumarin fluorescence and scanning from 380 to 500, and protein association was analyzed as described below.

Membrane binding of unprenylated G was measured by the 35% decrease in intrinsic fluorescence, exciting at 280 nm, and scanning from 290-400, because lipid bilayers composed of POPS were added to a 120 nM protein solution (24). Association was highly dependent on membrane surface charge and ionic conditions. The partition coefficient of the complex for POPS bilayers at 160 M KCl was found to be 50 + 4 µM (n = 2).

Data Analysis

Binding of the peptide to coumarin-PLC was analyzed by the shift in the emission energy as unlabeled peptide was added to the coumarin-PLC solution. In the FRET assays, association of coumarin-G with DABCYL-PLC was measured by following the decrease in donor emission (coumarin-G) during the addition of a nonfluorescent energy transfer acceptor (DABCYL-PLC).

Association between PLC and the peptide or G was analyzed by a simple bimolecular association. Here, we report only the apparent dissociation constant and do not take into account that the proteins maybe interacting on a quasi-two dimensional membrane surface. Using the PLC₂-G association as an example, for bulk phase equilibria the association between PLC₂ and G is as follows.

(Eq. 1)

The total amount of G in solution is [G]₀ = [G] + [G·PLC], and, similarly, the total amount of PLC is [PLC]₀ = [PLC] + [G·PLC].

The degree of association () of PLC with G is calculated from the normalized ratio of the change in fluorescence properties at each point along the titration curve over the total change for the association. Thus, K_d can be calculated from  by the following equation.

(Eq. 2)

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We examined the ability of prenylated peptides specific to the C terminus of the 2 subunit type to inhibit activation of PLC2 and PLC3. If a PLC isozyme makes direct contact with the prenyl group of the  subunit during interaction with the complex, then prenylated peptides from the  subunit should block such interaction. The sequence of the  subunit peptides and their prenyl modifications are shown in Fig. 1. The 2 peptide corresponds to the C-terminal 12 amino acids of the mature, fully processed 2 subunit. This peptide was either left unmodified or chemically prenylated at the last cysteine with either a geranylgeranyl group (2-gg) or a farnesyl group (2-far). The geranylgeranyl group is the isoprenoid present on native 2 subunits (26). We used the 42 complex in these assays. 42 stimulates both PLC2 and PLC3 with the same efficacy as 12 under identical conditions (21). Basal activities of these PLC isozymes were 10-20% of G-stimulated activity under the conditions used. When tested, the 2-gg peptide completely inhibits G stimulation of both PLC2 and PLC3, reducing PLC activity to basal levels. (Fig. 2). The 2-gg peptide did not have any significant effect on basal activity of PLC2 (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Peptides tested for inhibition of complex stimulated PLC activity. Peptides were modified chemically such that the cysteine at the C terminus formed a thioether linkage with the isoprenoid moieties (described under "Experimental Procedures"). C15, farnesyl; C20, geranylgeranyl.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Geranylgeranylated 2 peptide inhibits stimulation of PLC isozymes. A, effect of increasing concentrations of 2-gg peptide on stimulation of PLC2. 100 nM 42 complex and 120 pM PLC2 were incubated with lipid substrate and varying concentrations of 2-gg peptide as described under "Experimental Procedures." The reactions were started by addition of CaCl2 and incubated at 30 °C for 15 min. The results shown are the means of duplicate samples and are expressed as percentages of -stimulated PLC2 activity in the absence of peptide. This plot is representative of four independent experiments. B, effect of increasing concentrations of 2-gg peptide on stimulation of PLC3. The reactions were performed as in A, but with 10 nM 42 complex and 600 pM PLC3. The results shown are the means of duplicate samples and are expressed as percentages of -stimulated PLC3 activity in the absence of peptide. This plot is representative of three independent experiments.

2-gg peptide inhibition of stimulation could be due to the hydrophobic prenyl group causing a general disturbance of lipid vesicles in the assay, preventing the PLC isozymes from accessing their lipid substrate or from a direct action upon PLC isozymes. To resolve this issue, we examined the effect of the 2-gg peptide on Gq stimulation of phospholipase C3. The G protein  subunit type, Gq, is known to strongly stimulate PLC3 and is thought to do so by a mechanism different from that of G stimulation (27). q-binding and -binding regions on PLC2 have been mapped and are distinct from one another (28, 29). The 2-gg peptide does not significantly affect q stimulation of PLC3, even at a concentration of peptide that completely inhibits stimulation of PLC3 (5 µM) (Fig. 3). This indicates that the effect of the peptide is not the result of either direct inhibition of PLC activity or nonspecific disruption of enzyme access to substrate.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effect of  2-gg peptide on q stimulation of PLC. Gq was preactivated and then incubated with 600 pM PLC3 in the presence of lipid substrate and 5 µM 2-gg peptide as described under "Experimental Procedures." The reactions were started by the addition of CaCl2 and incubated at 30 °C for 15 min. The bars represent the means ± S.E. of two independent experiments, each experiment performed in duplicate.

We next examined the relative contributions of both the geranylgeranyl group and the amino acid portion of the 2-gg peptide in mediating this inhibitory effect. Unprenylated 2 peptide had very little effect on complex stimulation of either PLC2 or PLC3, even at a concentration of 20 µM (Fig. 4A). At this concentration 2-gg inhibits G-stimulated PLC activity almost completely (Fig. 2). Thus, the prenyl moiety is essential for 2-gg inhibition of G action on PLC. To determine whether or not the prenyl moiety alone is sufficient for inhibition of stimulation of PLC, we initially worked with a prenylated cysteine compound. However, problems with solubility of the prenylcysteine prevented further study with this compound. Therefore, to determine the importance of the amino acid sequence in 2-gg inhibition of G activity, we synthesized a geranylgeranylated 2 peptide in which the amino acid sequence of the last 12 residues of the 2 subunit was randomized (2scr-gg). This scrambled peptide inhibited G stimulation of PLC2 and PLC3 as well as the wild type 2-gg peptide (Fig. 4B). Thus, the amino acid sequence of the 2-gg peptide is not important for its inhibitory effect on G stimulation of PLC isozymes. This result is consistent with previous data suggesting that it is the prenyl moiety and not the amino acid sequence of  subunits that is the prime determinant on  subunits for G interaction with PLC (13).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Effect of unprenylated or scrambled 2 peptide on stimulation of PLC isozymes. A, effect of 2 unprenylated peptide on stimulation of PLC2 activity and PLC3 activity. B, comparison of scrambled and wild type 2-gg peptides on stimulation of PLC2 activity and PLC3 activity. The assays were performed as in Fig. 2 and reported similarly. The plots are representative of two independent experiments, each performed in duplicate.

Inhibition of stimulation of PLC isozymes by geranylgeranylated peptides suggests that PLC isozymes may directly contact the prenyl moiety on the G protein  subunit during activation by the complex. A fluorescence-based binding method was used to determine direct interaction of the 2-gg peptide with PLC2. PLC2 was labeled with the fluorescent probe coumarin, whose emission energy and intensity were found to be highly sensitive to the addition of prenylated y2-gg but not its unprenylated counterpart. Specifically, the addition of unlabeled 2-gg peptide produced a 2-fold increase in the emission intensity and a significant shift (330 cm¹) of emission energy from coumarin-labeled PLC (Fig. 5). These shifts allowed us to monitor the association between the peptide and PLC2 without the need to attach a fluorescent label on the peptide that could affect its interaction with PLC. The addition of unprenylated 2 peptide resulted in no change in emission energy (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Association of 2-gg peptide to fluorophore-PLC 2 in the presence of lipid bilayers. The change in emission energy was induced by the addition of various concentrations of 2-gg peptide to two fixed concentrations of coumarin-tagged PLC2 (details under "Experimental Procedures"). Changes in emission energy were normalized to the experimentally determined total change of 330 cm1. The data points represent the means ± S.E. from three independent experiments. The data for the two curves were fit to a bimolecular association curve to give the reported values of Kd. Control studies using the unprenylated peptide (n = 2) indicated that it does not alter the fluorescence emission energy of intensity of coumarin tagged PLC2 (data not shown).

To verify that the changes in fluorescence reflected a true interaction of peptide with protein, the association of 2-gg with PLC2 was measured at two concentrations of PLC2. Upon an increase of PLC2 concentration from 5 to 30 nM, the titration curve shifted in the expected direction to the right (Fig. 5). Curves were fit to a bimolecular association constant, giving values of K_d (1.8 ± 0.9 and 5.7 ± 3.1 µM) that were not significantly different from each other.

To directly assess the importance of the isoprenoid group in interaction with PLC isozymes, we performed a direct binding assay with PLC3 and prenylated or unprenylated complexes. Wild type 2 protein was co-expressed with 1 protein, and the complex was purified as described under "Experimental Procedures." Unprenylated complex was synthesized by mutating the 2 subunit C-terminal cysteine at position 68 to serine. This mutation prevents normal prenylation of the  subunit without affecting its ability to bind to the  subunit (6). Mutant unprenylated 2 protein was co-expressed with 1 protein using the baculovirus insect cell expression system, and the resulting complex was purified as described under "Experimental Procedures." Unprenylated 12 was completely inactive in PLC3 assays, even at concentrations at which wild type 42 stimulation of PLC3 saturates (Fig. 6) (as mentioned before 42 has similar potency compared with 12 in activating PLC3). We then examined whether prenylation is necessary for direct binding of 12 to PLC.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Prenyl modification of the  subunit is required for activation of PLC by the complex. Mutant 2C68S does not activate PLC3. PLC3 assays were performed as described in the presence of increasing amounts of mutant unprenylated 12C68S. A sample containing 50 nM 42 was included as a positive control for the PLC3 assay. Wild type 12 activates PLC3 with the same efficacy as 42. At 50 nM concentration of either 42 or wild type 12, stimulation of PLC3 activity saturates (21). The points shown are the means of duplicate samples. The graph is representative of two independent experiments, each performed in duplicate.

Direct binding between PLC and G was assayed using FRET assays. Wild type and unprenylated 12 complex were labeled with the fluorescent donor probe, coumarin. PLC2 was labeled with the acceptor probe, DABCYL. We have previously measured the affinities between PLC2 and G on membrane surfaces by fluorescence resonance energy transfer and found the membrane concentration to be a critical determinant in the magnitude of the their interaction energies (24). Although prenylation of the complex has been shown to increase the affinity of the complex for cell membranes (5), we found that is possible to conduct binding studies under conditions of lipid concentrations (200 µM POPS) (24) where both prenylated and unprenylated complexes are both completely bound to lipid vesicles. Preliminary studies showed that binding of unprenylated complex to negatively charged lipid bilayers composed of POPS was very strong (K_p = ~50 µM) and in the same range as previously determined for prenylated G (24). In Fig. 7, a comparison of the binding of DABCYL-PLC2 to coumarin-labeled wild type or unprenylated G as measured by FRET is shown. These results show clearly that the affinity of prenylated for PLC is significantly higher compared with the unprenylated complex.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Association of fluorophore tagged complex and PLC detected by measuring FRET. Shown is the association of increasing concentrations of DABCYL-PLC2 to 5 nM coumarin-tagged wild type G12 or coumarin-tagged unprenylated G12C68S in the presence of 200 µM POPS as determined by FRET. The data points represent the means ± S.E. from six independent experiments for each combination. The fraction associated was calculated from the 20% change in fluorescence intensity because of energy transfer between these probes (described under "Experimental Procedures").

The results from these FRET based assays indicate that the  prenyl moiety is essential for direct interaction between the complex and PLC isozymes. The results also suggest that there exists a site on PLC isozymes that binds the G protein  subunit geranylgeranyl moiety. To test whether such a site specifically recognizes the geranylgeranyl group, we substituted the geranylgeranyl moiety in the 2 peptide with a farnesyl group (2-far). 2-far is consistently less effective than 2-gg in inhibiting activation of PLC3 (Fig. 8), indicating that if a site on the PLC enzymes binds the prenyl moiety, it can discriminate between geranylgeranyl and farnesyl isoprenoids.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Farnesylated 2 peptide is less effective compared with geranylgeranylated 2 peptide at inhibiting stimulation of PLC isozymes. stimulation of PLC3 was performed in the presence of increasing amounts of 2-gg or 2-far peptides. The experimental procedures were identical to those used in Fig. 2. The results are expressed as percentages of -stimulated PLC3 activity in the absence of peptide. The points shown are the means of duplicate samples from three independent experiments (bars represent ± S.E.).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

What is the nature of the prenyl group involvement in interaction between and PLC isozymes? As confirmed here (Fig. 6), it is known that prenylation of the complex is required for functional regulation of PLC (9, 10). Because PLC acts on lipid substrates, it is possible that the requirement for prenylation is due the targeting of the complex to membranes. However, it is also possible that the  subunit prenyl moiety interacts directly with PLC and thus stabilizes protein-protein interactions between the complex and PLC. The results presented here favor this latter possibility.

Inhibition of stimulation of PLC activity by prenylated peptides suggests that the prenylated peptides compete with for a native prenyl-binding site on PLC isozymes. The effect of these prenylated peptides is specific; the same peptides have no effect on either basal or, more importantly, q-stimulated PLC3 activity. The differing efficacies of geranylgeranylated and farnesylated peptides on PLC activity is consistent with previous experiments using whole proteins (13) and suggest that such a prenyl-binding site can discriminate between different types of isoprenoids. Furthermore, the ~3-fold difference between the efficacy of the farnesylated and geranylgeranylated peptides (IC₅₀ values of ~ 5 and ~1.8 µM) is strikingly similar to the 3-fold difference in the K_d of farnesyl and geranylgeranyl for rhoGDI (4.8 and 1.6 µM as determined in a fluorescence based assay) (14). Although the potential site on PLC can discriminate between different isoprenoids, our results demonstrate that it does not selectively bind a particular isoprenoid to the exclusion of a related molecule. Lipid-protein interactions are predominantly hydrophobic (14, 30). It is likely that the chemistry of these interactions will not allow for the kind of specificity seen in protein-protein interactions.

Experiments that examined the effect of  subunit-specific peptides on fluorescence emission from fluorophore-tagged PLC provide direct evidence for interaction between the geranylgeranyl moiety and the PLC molecule. In this assay, only the 2-gg peptide induced a significant increase in emission energy from PLC2. Unprenylated 2 peptide had no effect on fluorescence emission from PLC. Because increases in emission energy are directly related to complex formation between the molecules in the assay (24), the K_d for 2-gg binding to PLC2 could be determined (1.8 ± 0.9 to 5.7 ± 3.1 µM). The differential binding of prenylated and unprenylated 2 peptide in this assay reflects similar differences in the assays that measured inhibition of G-stimulated PLC activity by the peptides. Because the unprenylated 2-gg peptide has no effect on fluorescence emission from coumarin-tagged PLC2, these results indicate that interaction of a site on PLC with the prenyl moiety is at the basis of 2-gg peptide binding to PLC.

The results from experiments using the whole complex, wild type and mutant, provide further evidence for the interaction of the prenyl moiety with PLC. A strong FRET signal is detected from DABCYL-tagged PLC2 in the presence of coumarin-tagged prenylated complex. The FRET response to increasing concentrations of PLC indicate highly effective complex formation complex formation; K_D for complex formation between prenylated and PLC was 0.9 nM. Complex formation between unprenylated G and PLC was significantly weakened (estimated K_d = 239 nM). The affinity of prenylated G is thus about 200-fold stronger for PLC compared with unprenylated mutant G. Unprenylated G complex consistently interacted with weaker affinity for PLC even under assay conditions where all of the unprenylated complex was localized along with PLC to lipid surfaces (Fig. 7). Thus, differential interaction of prenylated and unprenylated complexes with PLC is not a consequence of their differential interaction with membranes. Together these results from fluorescence spectroscopy directly support a role for the prenyl modification in stabilizing complex-PLC interaction.

In summary, the results presented here demonstrate that the  subunit prenyl moiety directly facilitates interaction of the G protein complex with an effector, PLC. We predict that PLC has a site that specifically binds the prenyl group. The interaction of the G protein  subunit prenyl moiety with PLC may serve as a general model for isoprenoid-protein interactions.

	ACKNOWLEDGEMENT

We thank Dr. Y. Hou (Gautam Laboratory) for pure 12 complex.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants GM51466 (to M. L.), GM53536 (to A. S.), GM53132 (to S. S.), and GM46963 (to N. G.).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.

To whom correspondence should be addressed: Box 8054, Washington University School of Medicine, St. Louis, MO 63110. Tel.: 314-362-8568; E-mail: gautam@morpheus.wustl.edu.

Published, JBC Papers in Press, September 6, 2001, DOI 10.1074/jbc.M107661200

	ABBREVIATIONS

The abbreviations used are: PLC, phospholipase C; PIP₂, phosphatidylinositol-4,5-biphosphate; Sf9 cells, Spodoptera frugiperda cells; FRET, fluorescence resonance energy transfer; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphoserine].

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES