Role of the γ Subunit Prenyl Moiety in G Protein βγ Complex Interaction with Phospholipase Cβ

The G protein βγ complex regulates a wide range of effectors, including the phospholipase Cβ isozymes (PLCβs). Prenyl modification of the γ subunit is necessary for this activity. Evidence presented here supports a direct interaction between the G protein γ subunit prenyl group and PLCβ isozymes. A geranylgeranylated peptide corresponding to the C-terminal region of the γ subunit type, γ2, strongly inhibits stimulation of PLCβ2 and PLCβ3 activity by the βγ complex. This effect is specific because the same peptide has no effect on stimulation of PLCβ by an α subunit type, αq. Prenylation of the γ peptide is required for its inhibitory effect. When interaction of prenylated γ subunit peptide to fluorophore-tagged PLCβ2 was examined by fluorescence spectroscopy, prenylated but not unprenylated peptide increased PLCβ2 fluorescence emission energy, indicating direct binding of the prenyl moiety to PLCβ. In addition, fluorescence resonance energy transfer was detected between fluorophore tagged PLCβ and wild type βγ complex but not an unprenylated mutant βγ complex. We conclude that a major function of the γ subunit prenyl group is to facilitate direct protein-protein interaction between the βγ complex and an effector, phospholipase Cβ.

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 Ϫ4 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 PLC␤2 1 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 PLC␤2 and PLC␤3. PLC␤ enzymes are a family of proteins that hydrolyze phosphatidylinositol 4,5bisphosphate (PIP 2 ), 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
Materials PIP 2 and phosphatidylethanolamine were obtained from Avanti Polar Lipids. [ 3 H] PIP 2 was from PerkinElmer Life Sciences. Nickelnitrilotriacetic acid resin was from Qiagen. All other reagents were from Sigma.

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 Mutant ␤1␥2 C68S (Used in the PLC␤3 Binding Assay)-Purification of mutant unprenylated ␤1␥2 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). G␣q 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.

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 mol) and prenyl bromide (4 mol) were mixed in a solution of butanol:methanol: water (1:1:1) previously purged under nitrogen atmosphere. Butylhydroxytoluene 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 Ϫ85°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 Ϫ85°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.
Peptide Inhibition of ␤␥ Complex Stimulated PLC␤2 Activity-Prenylated peptides were stored in a 1:1:1 solution of butanol:methanol: water at Ϫ80°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 M PIP 2 , 200 M phosphatidylethanolamine, and [ 3 H]PIP 2 ; ϳ8000 cpm/assay). 120 pM PLC␤2 and 100 nM final concentration of ␤4␥2 were then added to the lipid vesicle/peptide mix. The reactions were started by addition of CaCl 2 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 [ 3 H]inositol 1,4,5-trisphosphate release was quantitated by scintillation counting. Peptide inhibition of ␤␥ complex-stimulated PLC␤3 activity was performed as above with 600 pM PLC␤3 and 10 nM final concentration ␤4␥2 per assay.
␣q Activation of PLC␤3-␣q activation of PLC␤3 was performed essentially as described (17) except that ␣q was preactivated by incubation with 10 mM NaF and 30 M AlCl 3 . Peptide effect on ␣q stimulation of PLC␤3 was tested as above; 5 M ␥2-gg peptide was used for these assays.

Measurement of Peptide-PLC␤ and ␤␥ Complex-PLC␤ Associations Using Fluorescence Spectroscopy
Association between PLC␤2 and G␤1␥2 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-snglycero-3-[phosphoserine] (POPS) or a 2:1 mixture of 1-palmitoyl-2oleoyl-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 aminereactive 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 l of sample in a 3-mmpath length cell and adding small (0.5-2 l) 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 ϩ 4 M (n ϭ 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 PLC␤ 2 -G␤␥ association as an example, for bulk phase equilibria the association between PLC␤ 2 and G␤␥ is as follows.
[ 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.

RESULTS
We examined the ability of prenylated peptides specific to the C terminus of the ␥2 subunit type to inhibit ␤␥ activation of PLC␤2 and PLC␤3. 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 ␤4␥2 complex in these assays. ␤4␥2 stimulates both PLC␤2 and PLC␤3 with the same efficacy as ␤1␥2 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 PLC␤2 and PLC␤3, reducing PLC␤ activity to basal levels. (Fig. 2). The ␥2-gg peptide did not have any significant effect on basal activity of PLC␤2 (data not shown).
␥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 G␣q stimulation of phospholipase C␤3. The G protein ␣ subunit type, G␣q, is known to strongly stimulate PLC␤3 and is thought to do so by a mechanism different from that of G␤␥ stimulation (27). ␣q-binding and ␤␥-binding regions on PLC␤2 have been mapped and are distinct from one another (28,29). The ␥2-gg peptide does not significantly affect ␣q stimulation of PLC␤3, even at a concentration of peptide that completely inhibits ␤␥ stimulation of PLC␤3 (5 M) (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.
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 PLC␤2 or PLC␤3, even at a concentration of 20 M (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 PLC␤2 and PLC␤3 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).
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 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.
binding method was used to determine direct interaction of the ␥2-gg peptide with PLC␤2. PLC␤2 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 cm Ϫ1 ) of emission energy from coumarin-labeled PLC␤ (Fig. 5). These shifts allowed us to monitor the association between the peptide and PLC␤2 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).
To verify that the changes in fluorescence reflected a true interaction of peptide with protein, the association of ␥2-gg with PLC␤2 was measured at two concentrations of PLC␤2. Upon an increase of PLC␤2 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 Ϯ 0.9 and 5.7 Ϯ 3.1 M) 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 PLC␤3 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 ␤1␥2 was completely inactive in PLC␤3 assays, even at concentrations at which wild type ␤4␥2 stimulation of PLC␤3 saturates (Fig. 6) (as mentioned before ␤4␥2 has similar potency compared with ␤1␥2 in activating PLC␤3). We then examined whether prenylation is necessary for direct binding of ␤1␥2 to PLC␤.
Direct binding between PLC␤ and G␤␥ was assayed using FRET assays. Wild type and unprenylated ␤1␥2 complex were labeled with the fluorescent donor probe, coumarin. PLC␤2 was labeled with the acceptor probe, DABCYL. We have previously measured the affinities between PLC␤2 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 M 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 ϭ ϳ50 M) and in the same range as previously determined for prenylated G␤␥ (24). In Fig. 7, a comparison of the binding of DABCYL-PLC␤2 to coumarinlabeled 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.
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 PLC␤3 (Fig. 8), indicating that if a site on the PLC␤ enzymes binds the prenyl moiety, it can discriminate between geranylgeranyl and farnesyl isoprenoids. DISCUSSION 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␤. ⌻he 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 PLC␤3 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 ϳ3-fold difference between the efficacy of the farnesylated and geranylgeranylated peptides (IC 50 values of ϳ 5 and ϳ1.8 M) is strikingly similar to the 3-fold difference in the K d of farnesyl and geranylgeranyl for rhoGDI (4.8 and 1.6 M 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 PLC␤2. 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 PLC␤2 could be determined (1.8 Ϯ 0.9 to 5.7 Ϯ 3.1 M). 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 PLC␤2, 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 PLC␤2 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 ϭ 239 nM). The affinity of prenylated G␤␥ is thus about 200-fold stronger for PLC␤ compared with unprenylated mutant G␤␥. Unprenylated G␤␥ com-plex 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.