A Chemical Biology Approach Demonstrates G Protein βγ Subunits Are Sufficient to Mediate Directional Neutrophil Chemotaxis*

Background: G protein βγ (Gβγ) subunits are required for chemokine-dependent directional chemotaxis. Results: A chemical activator of Gβγ signaling activated Gβγ signaling and induced directional chemotaxis of neutrophils. Conclusion: Gβγ signaling is sufficient to induce directional chemotaxis of neutrophils. Significance: Demonstrates that G protein-coupled receptor signals other than Gβγ are not required for directional migration of neutrophils in response to a gradient. Our laboratory has identified a number of small molecules that bind to G protein βγ subunits (Gβγ) by competing for peptide binding to the Gβγ “hot spot.” M119/Gallein were identified as inhibitors of Gβγ subunit signaling. Here we examine the activity of another molecule identified in this screen, 12155, which we show that in contrast to M119/Gallein had no effect on Gβγ-mediated phospholipase C or phosphoinositide 3-kinase (PI3K) γ activation in vitro. Also in direct contrast to M119/Gallein, 12155 caused receptor-independent Ca2+ release, and activated other downstream targets of Gβγ including extracellular signal regulated kinase (ERK), protein kinase B (Akt) in HL60 cells differentiated to neutrophils. We show that 12155 releases Gβγ in vitro from Gαi1β1γ2 heterotrimers by causing its dissociation from GαGDP without inducing nucleotide exchange in the Gα subunit. We used this novel probe to examine the hypothesis that Gβγ release is sufficient to direct chemotaxis of neutrophils in the absence of receptor or G protein α subunit activation. 12155 directed chemotaxis of HL60 cells and primary neutrophils in a transwell migration assay with responses similar to those seen for the natural chemotactic peptide n-formyl-Met-Leu-Phe. These data indicate that release of free Gβγ is sufficient to drive directional chemotaxis in a G protein-coupled receptor signaling-independent manner.

G protein-coupled receptors (GPCR) 2 are transmembrane proteins that regulate a variety of cellular and physiological processes. Upon activation GPCRs bind G protein heterotrimers, consisting of G␣GDP and G␤␥ subunits, and catalyze nucleotide exchange and GTP binding to the G␣ subunit (1). This results in functional dissociation of the GTP-bound G␣ subunits from G␤␥ subunits. The free G protein subunits bind directly to various target proteins initiating activation of downstream signaling pathways. G␤␥ subunits bind most effectors at a common surface that is obscured by G␣GDP in the inactive state known as the "hot spot" (2,3). Although effectors tend to share this binding surface, each interacts with specific subsets of amino acids on G␤␥ (3)(4)(5). These differential binding modes for effectors have been effectively exploited in the discovery of peptides and small molecules that prevent activation of some effectors by G␤␥, without affecting the regulation of others (4,6).
Directional migration of cells toward a chemotactic stimulus is required for tissue formation, wound healing, and immune responses. Chemokine receptors are a large family of GPCRs that respond to gradients of chemokine chemoattractants to mediate directional migration of cells (7). The majority of chemokine receptors are coupled to the G i/o family of G proteins and transduce signals through G␤␥ released from G i heterotrimers (8). Multiple pathways are activated by G␤␥ including phospholipase C␤ (PLC␤) and phosphoinositide 3-kinase ␥ (PI3K␥) (9,10). PI3K␥ activation at the leading edge of immune cells downstream is an important determinant of cell polarity and directional migration through generation of a gradient of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). G␤␥ also directly activates cdc42 by binding to PAK, causing F-actin localization and inhibition of phosphatase and tensin homolog at the leading edge (11).
In addition to G␤␥ signals there are also reports that G␣ 13mediated Rho activation leads to migration of MEFs and cancer cells and that G␣ 13 plays a role in neutrophil migration at the trailing edge of migrating cells (12,13). Along with G protein subunits themselves, regulators of G protein signaling (RGS proteins) were also shown to be mediators of cell migration in B lymphocytes (23). Recently, ␤-arrestin and GRK, which are mediators of the receptor desensitization pathway, have also been shown to play a role in cell migration (14 -17). All these proteins are part of the GPCR signaling system, and may play a part in regulating chemoattractant-directed cell migration.
Although it is clear that G␤␥ is required for directed migration in response to chemotactic peptides, chemotactic GPCR signaling is complex and it is unclear to what extent G␤␥ signaling on its own is sufficient drive directional migration of cells, independently of the GPCR, GPCR modulating proteins, or parallel GPCR-driven signaling pathways.
Here we used a chemical biology approach to address these questions. The small molecule 12155 (NSC12155) was previously identified in a screen of the National Cancer Institute diversity library for G␤␥-binding molecules (4). Here we show that 12155 binds to G␤␥, but does not inhibit signaling from G␤␥, rather it displaces G␣GDP thereby activating G␤␥-mediated signal transduction without activating GPCRs, G␣ subunits, or associated pathways. Using this approach we demonstrate that G␤␥ signaling in neutrophils is sufficient to mediate directional chemotaxis.
Isolation of Mouse Neutrophils-Neutrophils were obtained from the bone marrow of adult C57 Bl6 mice. All procedures were carried on ice using ice-cold buffers. Bone marrow was flushed with PBS, pH 7.4, and red blood cells (RBCs) were lysed using ACK lysis buffer. The white blood cells were separated from the lysed RBCs by centrifugation at 1400 ϫ g for 3 min. The cells were counted and 10 8 cells/ml were used for further isolation. Pure neutrophils were isolated using the neutrophil negative selection kit (StemCell).
Purification of G␣ i -GFP-GFP-G␣ i1 was expressed and purified from High Five cells by modification of a previously described method (19). High Five cells at 1.5 ϫ 10 6 cells/ml were infected with viruses encoding G␤ 1 , His 6 G␥ 2 , and G␣ i1 -GFP, and grown for 60 h with continuous shaking at 27°C. GFP was inserted between amino acids 122 and 123 of G␣ i1 . The cells were harvested and membrane extracts were loaded on an nickel-nitrilotriacetic acid column. After washing, G␣ i -GFP was eluted using AlF 4 and MgCl 2 . The eluted fractions were analyzed by SDS-PAGE, Coomassie Blue staining, Western blotting, and fluorimetric detection of the GFP signal, and fractions containing 80% pure GFP-G␣ i1 were pooled, snap frozen in liquid N 2 , and stored at Ϫ80°C.
SIGK Competition Assay-Compounds were preincubated with 20 nM bG␤ 1 ␥ 2 in a 384-well plate. F88 phage displaying the peptide SIGKAFKILGYPDYD was added. After 15 min, anti-M13 antibody was added and incubated 1 h. The complexes were then bound to streptavidin-coated AlphaScreen donor beads (PerkinElmer Life Sciences) and Protein A AlphaScreen acceptor beads and AlphaScreen signal was read after 1.5 h on a Wallac Envision Multilabel Reader (PerkinElmer Life Sciences).
Alternatively (Table 1) an ELISA-based assay was performed as previously described (4,20). Briefly bG␤ 1 ␥ 2 was immobilized in a streptavidin-coated 96-well plate followed by incubation with compound and F88 phage displaying the peptide SIGKAF-KILGYPDYD for 1 h at room temperature. After washing bound phage was detected with a horseradish peroxidase-conjugated anti-M13 antibody followed by color development with ABTS.
Surface Plasmon Resonance (SPR)-SPR was performed as described in Ref. 21, with modifications. bG␤␥ was immobilized on the surface of streptavidin-coated sensorchips (GE Healthcare) by injecting 500 nM bG␤␥ in 50 mM HEPES, pH 7.6, 1 mM EDTA, 50 mM NaCl, 100 mM KCl, 0.1% polyoxyethylene 10 lauryl ether (C 12 E 10 ), and 1 mM dithiothreitol. Unbound streptavidin was blocked with 1 M biotin and buffer was changed to include 0.1% DMSO. Small molecule(s) were then injected and binding was observed for 1 min followed by dissociation for 5 min, at a flow-rate of 50 l/min. After dissociation, the G␤␥ surface was regenerated using 610 mM MgCl 2 , 205 mM urea, and 610 mM guanidine HCl, followed by the second injection.
For G␣ i binding to G␤␥, 10 mM MgCl 2 and 10 M GDP were added to the above mentioned buffer, about 500 micro-refractive index units G␤␥ was immobilized to the chip and binding of G␣ i was observed at a flow rate of 15 l/min for 5 min, followed by dissociation for 5 min. After the dissociation phase AlF 4 Ϫ was added to the buffer and injected over the surface for complete dissociation of G␣ i from G␤␥. To determine the role of 12155 in inhibition of G␣ i binding to G␤␥, 12155 (10 M) was added to all buffers, the baseline was allowed to stabilize, and the binding of G␣ i was determined as described above. The data were analyzed using BIA evaluation software (Biacore), and K D was determined by globally fitting the binding curves determined at several concentrations of compound with a 1:1 binding model.
HL60 Cell Ca 2ϩ Release (Fluorimeter)-Differentiated HL60 cells were harvested by centrifugation and resuspended in 1 ml of Hanks' balanced salt solution (HBSS) buffer with 1 mM Ca 2ϩ and 10 mM HEPES, pH 7.4. Cells were incubated with 1 M Fura 2-AM (Molecular Probes) for 45 min at 37°C, excess dye was removed by centrifugation and resuspension in HBSS with Ca 2ϩ and 10 mM HEPES, pH 7.4, at 2 ϫ 10 7 cells/ml. Cells were diluted 10-fold into 1.8 ml of HEPES-buffered HBSS without Ca 2ϩ , and release of Ca 2ϩ was monitored with 340 and 380 nm excitation and emission detection at 510 nm in a PTI fluorimeter.
Ca 2ϩ Release Assay (Flexstation)-For experiments in Fig. 3, A and C, the release of Ca 2ϩ was monitored real-time in 96-well plates in a Flex station fluorescence plate reader (Molecular Devices) at 340 and 380 nm excitation and emission detection at 510 nm. Differentiated HL60 cells were suspended in 1 ml of HBSS buffer with 1 mM Ca 2ϩ and 10 mM HEPES, pH 7.4. Cells were incubated with 1 M Fura 2-AM (Molecular Probes) for 45 min at 37°C, excess dye was removed by centrifugation, washed, and re-suspension in HBSS without Ca 2ϩ and 10 mM HEPES, pH 7.4, at 1.5 ϫ 10 6 cells/ml. For the assay 150,000 cells were used per well and 95 l were loaded into each well. For inhibitors cells were pre-treated with M119 or U73122 or U73343, for 15 min prior to the analysis. PTX (100 ng/ml) treatment was for 5 h prior to the experiment. The wells were injected with 5 l of either fMLP (50 nM) or 12155 (10 M), unless stated otherwise.
Ca 2ϩ Calibration-The 340/380 Fura-2 ratio was calibrated as a function of Ca 2ϩ concentration in both the PTI fluorimeter and the Flexstation using a calcium calibration buffer kit (Invitrogen) containing 10 mM EGTA and 1 mM Mg2ϩ. Ca 2ϩ was titrated from 0 to 39 M free Ca 2ϩ with 1 M Fura-2 free acid to generate a standard curve from which Ca 2ϩ concentrations were calculated from the 340/380 ratios obtained from Fura-2loaded cells.
Western Blotting-Differentiated HL60 cells were incubated overnight in RPMI 1640 medium without serum at 37°C. Cells were harvested and treated with DMSO (solvent), 12155 (10 M), M119 (10 M), or fMLP (250 nM) for 5 min. The reaction was stopped by centrifugation of the cells at 13,000 ϫ g for 1 min at 4°C followed by suctioning of the media and lysis in SDS sample buffer. The samples were heated at 95°C for 10 min and loaded on a 12% polyacrylamide gel. The gel was transferred overnight and immunoblotted for ERK p44/42 (Cell Signaling), pERK P-p44/42 (Cell Signaling), or pAKT Ser-473 (Cell Signaling). The secondary antibody was anti-rabbit Li-Cor Ab (Odyssey) and was detected using a Li-Cor imaging system (Odyssey).
Phospholipase C Activity Assay-The assay was done as previously described (22). Briefly, lipids vesicles containing ϳ4000 cpm of [ 3 H-inositol]PIP 2 , 25 M PIP 2 and 100 M phosphatidylethanolamine were mixed with 0.5 ng of PLC␤2. 100 nM G␤␥ was preincubated with DMSO or compounds for 10 min, followed by addition to the PLC and lipid mixture. The reaction was set at 30°C for 30 min and quenched with 5% BSA and 10% TCA. Released soluble [ 3 H]IP 3 was measured by liquid scintillation counting.
Flow Cytometry-The assay was done as described in Refs. 23 and 24, with some modifications. Briefly, 10 nM bG␤ 1 ␥ 2 was immobilized on streptavidin beads (Spherotech) in 20 mM HEPES, pH 8.0, 1 mM DTT, 100 mM KCl, 20 mM NaCl, 0.2 mM free Mg 2ϩ , 0.1% C 12 E 10 , 0.1% bovine serum albumin (BSA), and 10 M GDP, for 1 h at 4°C. For the competition assay 12155 was incubated with the G␤␥-bound beads (10 nM G␤␥) for 30 min at room temperature with shaking prior to addition and incubation with GFP-labeled G␣ i1 GDP (20 nM) in buffer containing 10 M GDP and no GTP for 30 min at room temperature. To determine whether 12155 causes dissociation, a complex of 20 nM GFP-G␣ i1 with 10 nM immobilized bG␤␥ was pre-formed by incubating GFP-G␣ i1 for 1 h and excess unlabeled G␣ i1 (1 M) was added to determine the intrinsic off rate of GFP-G␣ i1 from G␤␥. To determine whether 12155 enhanced the GFP-G␣ i1 off rate, 10 M 12155 or 10 M SIGK for comparison were used to initiate the dissociation reaction. The amount of GFP-G␣ bound to immobilized G␤␥ was assessed using a FACSScan flow cytometer (BD Biosciences) exciting at 488 nm and detecting the emission at 510 nm associated with each bead. In the dissociation assay, after addition of competitor to the preassembled GFP/G␣ i ␤␥ mixture, aliquots were removed at various intervals and analyzed by FACS to determine the amount of remaining bead-bound GFP-G␣ i1 fluorescence.
[ 35 S]GTP␥S Binding Assay-The assay was done as described in Ref. 25.
PI3 Kinase Activity Assay-Activation of PI3K␥ was performed as described (26). 10 ng of purified PI3K␥ was assayed with or without 10 nM G␤ 1 ␥ 2 in the presence or absence of compounds. The assay (60 l final volume) contained sonicated micelles of 600 M bovine liver phosphatidylethanolamine and 300 M bovine liver PI substrated in 40 mM NaHEPES, pH 7.4, 2 mM EGTA, 1 mM DTT, 0.2 mM EDTA, 120 mM NaCl, 5 mM MgCl 2 , 1 mM ␤-glycerophosphate, 50 M sodium orthovanadate, 1 mg/ml of BSA. Reactions were initiated by the addition of 10 M ATP with [␥-32 P]ATP (5 Ci/ assay) and by transfer from 4 to 30°C. Reactions were terminated by addition of 500 l of 2:1 methanol/chloroform solution. A stable two-phase system was generated by addition of 100 l of 2.4 M HCl with 5 mM tetrabutylammonium hydrogen sulfate followed by 440 l of CHCl 3 . Samples were centrifuged, and the lower organic phase containing the lipids was extracted three times with a solution containing 48% methanol, 3% chloroform, 0.5 M HCl, 1 mM EDTA, 10 mM tetrabutylammonium hydrogen sulfate. The lower organic phase was removed and dried under nitrogen, and Cerenkov counting was used to quantitate radioactive 32 P incorporation into the lipid fraction using a counting efficiency of 50%. Previous experiments with thin layer chromatography established that in this assay system with purified protein and lipids, greater than 95% of the label migrates in the PI-3-P position. The experiment was performed twice with quadruplicate determinations in each and the data averaged as shown.
Transwell Migration of HL60 Cells and Neutrophils (Boyden Chamber)-The assay was done as described previously in Ref. 27. Differentiated HL60 cells or primary mouse neutrophils were used as indicated. For PTX treatment, HL60 cells were treated with PTX (100 ng/ml) overnight before the day of the experiment. For wortmannin, the HL60 cells were treated with wortmannin (1 M) for 20 min prior to the experiment. Cells were suspended to 10 ϫ 10 6 cells/ml, in HBSS containing Ca 2ϩ , 10 mM HEPES, pH 7.4, and 0.1% BSA. Chemoattractant 12155 (10 M) or fMLP (250 nM) or vehicle control DMSO were added in the lower chamber and 50 l of cells (10 7 cells/ml) were in the upper chamber. Cells were either pretreated with Gallein or DMSO for 15 min, or 12155 or fMLP were added to the upper chamber just prior to the chemokinesis assay. The assay used a filter with a 3-m pore size (Neuroprobe), and was performed for 1 h at 37°C, 5% CO 2 . Cells that had migrated and adhered to the bottom surface of the filter were stained and 3 fields each were counted using a light microscope. The data are shown as chemotaxis index, which is the ratio of the number of cells migrated in the presence of the chemoattractant versus those migrated in presence of DMSO.
Statistical Analysis-All statistical analyses were performed by one-way analysis of variance using the Tukey post-test or a paired t test in figures with only two columns. The value of the analysis is as follows: *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001 and N.S., not significant.

Binds to G␤␥ and Prevents
Binding of SIGK Peptide to G␤␥-Using a chemical screening approach we previously identified small molecules that bind to G␤␥ subunits and modulate the activity of G␤␥ subunits in cells, in vitro, and in animals (4,(27)(28)(29). The primary screen was based on computationally docking small molecules from the National Cancer Institute diversity library to a peptide/effector/G␣ subunitbinding surface on G␤␥ (4). Binding of selected molecules to G␤␥ was confirmed in a competition assay for SIGK peptide binding to G␤ 1 ␥ 2 (4). The small molecule 12155 ( Fig. 1A) was identified in this screen. 12155 inhibited binding of phage displayed SIGK to bG␤␥ in a concentration-dependent manner in a competition AlphaScreen assay with an IC 50 of 5 M and a Hill slope of Ϫ1.7 (Fig. 1B). Direct binding of 12155 to G␤␥ was confirmed using SPR yielding a dissociation constant of 1.5 M (Fig. 1C).
To understand the requirements for 12155 binding to G␤␥ a series of related molecules were obtained from the National Cancer Institute and tested in a competition ELISA (Table 1). 12155 (1,3-bis(4-amino-2-methylquinolin-6-yl)urea) is composed of two aminomethylquinoline moieties connected by a urea linkage. Aminomethylquinoline alone did not bind, nor did 1,3-di-6-quinolylurea (71881) lacking the amino and methyl substitutions on the linked quinoline moieties. Substitution of the urea linkage with longer or bulkier linkages also reduced binding. These data indicate a specific binding mode for 12155 binding to G␤␥ requiring two covalently connected amino quinoline moieties with a defined spacing between them.
12155 Has No Effect on G␤␥-mediated PLC␤ or PI3K␥ Activation in Vitro-12155 was tested for its ability to inhibit PLC␤ and PI3K in vitro (Fig. 1D). Surprisingly 12155 had no effect on G␤␥-mediated PLC␤2 (Fig. 1D), PLC␤3 (data not shown), or PI3K␥ activation (Fig. 1E) at concentrations up to 100 M. M119 and Gallein are well characterized small molecule inhibitors of G␤␥ that were also identified in this screen, and have previously been shown to inhibit G␤␥-dependent PLC␤ and PI3K␥ activation (4,27). In the same assays 10 M M119 strongly inhibited G␤␥-dependent regulation.
12155 Causes G␤␥-dependent Ca 2ϩ Release-The contrast between the properties of 12155 and M119 with respect to the ability to inhibit activation of downstream effectors by G␤␥ is striking, and is consistent with our previous observations that unique G␤␥-binding modes of different small molecules have distinct effects on G␤␥ signaling properties. To further explore the activity of 12155 we examined its action on G␤␥ signaling pathways in HL60 cells differentiated to neutrophils. In neutrophils, chemoattractant peptide receptors such as the receptor for fMLP, FPR1, regulate multiple cellular processes such as cell migration and inflammatory mediator release through G␤␥dependent signaling pathways. These pathways include PLC␤dependent Ca 2ϩ signaling, PI3K␥-dependent PIP 3 formation and Akt activation, and PAK and Cdc42 regulation.
To examine G␤␥-dependent PLC regulation we monitored fMLP-dependent Ca 2ϩ release in cells loaded with the Ca 2ϩ indicator dye Fura-2 in media with low extracellular Ca 2ϩ to ensure that the primary source of Ca 2ϩ is IP 3 -dependent release from internal stores. fMLP stimulated a rapid and robust Ca 2ϩ increase that decayed to baseline over the course of 3-4 min ( Fig. 2A). Surprisingly addition of 12155 alone stimulated a rapid and robust release of Ca 2ϩ in the absence of fMLP that was comparable in magnitude and character to the fMLP-stimulated response (Fig. 2B). In contrast to 12155, M119 itself did not cause an increase in Ca 2ϩ and inhibited the fMLP-dependent signal (Fig. 2C) in agreement with previously reported results (4). The amplitude of the Ca 2ϩ response increased as a function of increasing concentrations of 12155 (Fig. 2D). An EC 50 could not be accurately estimated because the response did not saturate at the concentrations tested. Nevertheless, robust responses were observed at 10 M.
Based on the fact that 12155 binds to G␤␥ in vitro and causes Ca 2ϩ release in cells we hypothesized that 12155 binds directly to G␤␥ in cells and releases it from G␣ subunits, and because it does not block interactions between G␤␥ and PLC␤ or PI3K␥, it allows signaling from 12155-bound G␤␥ to proceed. If this is true the 12155-dependent Ca 2ϩ release should depend on PLC and G␤␥ subunit signaling. To confirm that 12155-dependent Ca 2ϩ release is dependent on PLC activation, cells were pretreated with the PLC inhibitor U73122 prior to 12155 addition. 10 M U73122, but not U73343 inhibited Ca 2ϩ release by 12155 (Fig. 3A), indicating that 12155-dependent Ca 2ϩ release was PLC-dependent. U73122 had no effect on Ca 2ϩ release on its own (data not shown). Although U73122 has been reported to have off target effects, many cases of bona fide inhibition of PLC signaling by U73122 in cells have been reported, and the results are consistent with what would be expected in these cells. To show that the activity of 12155 was dependent on G␤␥, cells were treated with the G␤␥ inhibitor M119 prior to addition of 12155. M119 pretreatment inhibited 12155-mediated Ca 2ϩ release in a concentration-dependent manner (Fig. 3B). These data support the idea that 12155 causes release of Ca 2ϩ from intracellular stores in a G␤␥-and PLC-dependent manner.
To determine whether the activity of 12155 was independent of the receptor, cells were treated PTX to prevent activation of G proteins by G i/o-coupled GPCRs including FPR1. In PTXtreated cells, 12155 robustly stimulated release of Ca 2ϩ (Fig.  3C), whereas the same treatment completely eliminated fMLPdependent Ca 2ϩ signals, suggesting the activity of 12155 to be independent of G i/o -coupled receptors. PTX did cause some inhibition of the 12155-dependent response. This could suggest some receptor-dependent component of 12155-mediated Ca 2ϩ release, but the inhibition also could be due to adverse effects of PTX on the cells or could influence the sensitivity of G␣ i -G␤␥ interactions to dissociation by 12155. Regardless, the signifi-cant PTX-insensitive component is consistent with a mechanism involving direct action of 12155 at G␤␥ subunits to promote PLC signaling.
To further confirm the action of 12155 on G␤␥ signaling in HL60 cells we examined ERK and Akt pathways known to be regulated by G␤␥ signaling in these cells. Cells were treated with DMSO, 12155, M119, or fMLP. 12155 but not M119 increased the levels of phosphorylated ERK (Fig. 4A) and AKT (Fig. 4B) although not to the same level as fMLP. PTX treatment completely eliminated Akt activation by fMLP but not by 12155 (Fig. 4C). Activation of Akt is consistent with the well estab-lished role of G␤␥ in regulation of PI3K in neutrophils. Together these data support that 12155 activates G␤␥ signaling in a receptor-independent manner in neutrophil-like differentiated HL60 cells.
12155 Causes Dissociation of G␤␥ from G␣-GPCRs stimulate G␤␥ signaling by catalyzing GDP release from, and GTP binding to, the G␣ subunit resulting in functional dissociation of G␣ from G␤␥ subunits. We hypothesized that 12155 activates G␤␥ signaling by binding directly to G␤␥ in G protein heterotrimers and causing dissociation of G␣GDP from G␤␥, or by binding to free G␤␥ and disrupting the G␣-G␤␥ binding equilibrium to favor an increase in free G␤␥ concentration, through a mechanism that does not involve receptor activation or nucleotide exchange. First we used SPR to determine whether 12155 can interfere with G␣ i1 interactions with G␤␥. In these experiments biotinylated G␤ 1 ␥ 2 (bG␤ 1 ␥ 2 ) was immobilized and G␣ i1 was injected in the mobile phase at the indicated concentrations (Fig. 5A). Global fitting of the data from three independent experiments gave a dissociation constant of 50 Ϯ 17 (S.E.) nM. In Fig. 5B are representative traces for G␣ binding to G␤␥ in the presence and absence of 12155 included in the mobile phase. 12155 completely inhibited binding of G␣ to G␤␥ (Fig. 5B) indicating that 12155 can compete for G␣ binding to G␤␥.
Attempts to use SPR to determine whether 12155 could enhance dissociation of G␣ from G␤␥ were not successful due to complications from interpreting the binding curves that include 12155 binding and intrinsic G␣ dissociation. We previously employed an assay (24) to examine association and dissociation of fluorescent G␣ subunits from bG␤␥ subunits bound to beads using flow cytometry (31). This method allows for measurement of protein-protein interactions at concentrations near the K D of interactions in the low nanomolar to high picomolar range and allows for the kinetics of association and dissociation of G protein subunits to be measured (24,31). First an equilibrium competition analysis was performed where 12155 was added to G␤␥ prior to GFP-G␣ i1 and incubated to allow the binding to come to equilibrium prior to measurement of GFP-G␣ i1 binding to G␤␥ by flow cytometry. 12155 inhibited binding of GFP-G␣ i to G␤␥ in a concentration-dependent manner (Fig. 5C) consistent with its apparent affinity for G␤␥ and the SPR data.
This experiment establishes that 12155 binding to G␤␥ is sufficient to compete for G␤␥ binding to G␣GDP. In cells 12155 could activate signaling by preventing rebinding of G␣GDP to G␤␥ after basal G␣ subunit dissociation, or 12155 could bind to G␤␥ in the context of the heterotrimer and promote G␤␥ dissociation from G␣GDP as previously reported for  SIGK (23,31). To assess the latter, 12155 was added to a preformed complex of GFP-G␣ i and G␤␥ and the rate of dissociation of G␣ from G␤␥ was measured. The rate of G␣ dissociation in the presence of 12155 was compared with the rate of intrinsic GFP-G␣ i GDP dissociation measured by addition of a 100-fold excess of unlabeled G␣ i GDP to prevent GFP-G␣ i GDP rebinding to G␤␥, and to the rate of SIGK-induced dissociation (Fig.  5D). As previously reported, SIGK enhanced the rate of G␣ dissociation from G␤␥ (k off ϭ 0.12 min Ϫ1 ) relative to the intrinsic G␣ dissociation rate (k off ϭ 0.05 min Ϫ1 ). Similarly 12155 increased the rate of dissociation of the GFP-G␣GDP from G␤␥ (k off ϭ 0.12 min Ϫ1 ) indicating that 12155 is capable of promoting dissociation of G␤␥ from G␣GDP without causing nucleotide exchange.
To confirm that 12155 does not affect G␣ i activity we measured GTP␥S binding to G␣ subunits in native membranes prepared from CHO cells overexpressing the -opioid receptor. As expected, treatment of -opioid receptor expressing CHO membranes with the -opioid agonist DAMGO increased binding of [ 35 S]GTP␥S indicative of the ability of this receptor to couple to G␣ i activation (Fig. 5E) 12155 Acts as a Chemoattractant for HL60 Neutrophils-Development of an agonist that activates G␤␥, independently of the receptor, and G␣ subunit nucleotide exchange, provides a powerful tool to study the role of free G␤␥ in an acute cellular setting. G␤␥ plays an important role in chemoattractant receptor-dependent cell migration (27,32). Although G␤␥ is important for migration of cells, it is unclear if release of free G␤␥ is sufficient to mediate directional chemotaxis in the absence of activation of G␣ subunits or other GPCR-mediated pathways.
To determine whether G␤␥ release from G␣GDP is sufficient to drive neutrophil chemotaxis we asked whether 12155  could cause directional HL60 cell migration in a transwell migration assay. In this assay chemoattractants are placed in a lower chamber, which is separated from cells in the upper chamber by a membrane with pores that allow migration of cells from the upper chamber to the lower chamber in response to the gradient of chemoattractant established between the upper and lower chambers. Thus this method measures the ability of cells to sense and migrate directionally in response to chemotactic gradients. fMLP stimulated robust migration of HL60 cells into the lower chamber whereas gallein, a G␤␥ inhibitor, does not. Interestingly, 12155 robustly stimulated cell migration to an extent equivalent to fMLP (Fig. 6A).
To examine chemotaxis of primary cells, neutrophils were isolated from mouse bone marrow and tested in the transwell . 12155 induces dissociation of G␣GDP from G␤␥ without causing nucleotide exchange in the G␣ subunit. A, surface plasmon resonance analysis of G␣ i1 GDP binding to immobilized bB␤␥. Shown is one of three independent experiments with independent bG␤␥ immobilizations. The K d of 50 Ϯ 17 nM is the average value from separate global fittings to a single site-binding model for each experiment. B, shown are representative SPR traces with and without 12155 included in the mobile phase. This experiment was repeated in 3 independent experiments at multiple concentrations. A K d could not be calculated because specific binding of G␣ was not observed. C, 12155 inhibits GFP-G␣i 1 binding in a equilibrium completion experiment. 12155 was premixed with bead-bound bG␤ 1 ␥ 2 followed by incubation with GFP-G␣ i1 and bead-associated GFP fluorescence was detected by flow cytometry after 30 min at 488 nm and detecting emission at 530. Data are pooled from two separate experiments performed in triplicate and fit with a standard inhibition equation (GraphPad Prism). D, 12155 enhances the kinetics of dissociation of GFP-G␣ i1 GDP from G␤␥ in a flow cytometry-based assay. GFP-G␣ i was pre-bound to bead-bound bG␤␥, 10 M 12155 along with unlabeled G␣ i (to prevent rebinding of GFP-G␣ i ), DMSO ϩ G␣ i1 , or 10 M SIGK ϩ G␣ i1 was added to initiate measurement of the dissociation rate. The amount of GFP-G␣ i bound to bG␤␥ was assayed in aliquots at the indicated times as in B. Data are pooled from three independent experiments performed in triplicate. Lines are curve fits with a single exponential decay function using GraphPad Prism 6. E, 12155 does not induce nucleotide exchange in the G␣ subunit. Membranes from CHO cells overexpressing the -opioid receptor were treated with 12155 (10 M), DAMGO (300 nM) or both. The amount of [ 35 S]GTP␥S bound to G␣ i was determined by filter binding the proteins and counting the radioactivity bound to the G␣ i subunit. The data represents the mean Ϯ S.E. of triplicate determinations (n ϭ 4). (**, p Ͻ 0.01; ***, p Ͻ 0.001).
assay for their ability to respond to a gradient of 12155 (Fig. 6B). 12155 potently, and effectively, stimulated primary neutrophil chemotaxis with an EC 50 of ϳ5-10 M, although the response did not saturate at 30 M. These data are consistent with its concentration dependence for activation of Ca 2ϩ release, although the concentrations required are significantly higher than for binding the purified protein in vitro. This is not surprising because the compound has to cross cell membranes and interfere with subunit association in cells.
Accumulation of cells in the lower chamber could be the result of stimulation of random migration that lacks directionality (chemokinesis) or directional migration in response to a gradient (chemotaxis). To distinguish between directional and random migration chemoattractant is placed in both the upper and lower chambers at equal concentrations thereby eliminating gradient formation. When 12155 or fMLP were added as a uniform stimulus, there was no significant migration of differentiated HL60 cells to the lower chamber (Fig. 6C). This indicates that a gradient of 12155 is sufficient to act as a chemoattractant and direct chemotaxis of HL60 cells and primary neutrophils.
12155 Causes Receptor-independent G␤␥-mediated PI3Kdependent Cell Migration-To show the migration induced by 12155 to be independent of GPCR activation, HL60 cells were treated with PTX prior to 12155 or fMLP. PTX completely inhibited fMLP-dependent chemotaxis as expected but only partially inhibited 12155-stimulated cell migration (Fig. 7A). To provide evidence that 12155 mediates chemotaxis through G␤␥, cells were treated M119. We have previously shown that M119 inhibits PI3K and G␤␥-mediated cell migration in HL60 cells and neutrophils (27). M119 completely blocked both fMLP-and 12155-mediated cell migration (Fig. 7B).
PI3K␥ is a central molecule in G␤␥-mediated cell migration (9) and 12155 activates G␤␥-mediated Akt phosphorylation (Fig. 4B). Cells were pretreated with wortmannin to inhibit PI3K and chemotaxis was measured in response to gradients of 12155 and fMLP. Wortmannin inhibited 12155-mediated cell migration, supporting the idea that 12155 activates PI3K-dependent cell migration (Fig. 7, C and D). Together these data indicate that a gradient of 12155 serves as a chemotactic stimulus to drive directional cell migration by directly driving dissociation of G␤␥ from G␣GDP indicating that chemotaxis does not require GPCR activation and can be promoted solely by the activation of G␤␥-dependent signaling pathways.

DISCUSSION
Here we show that a small molecule 12155 activates G␤␥ signaling and is a sufficient stimulus to drive directional chemotaxis of neutrophils. The data shows that 12155 activates G␤␥ signaling by binding directly to G␤␥ and causing release of G␣GDP without causing nucleotide exchange. 12155 was found in a competition screen for binding of the SIGK-phage displayed peptide to G␤␥. A cell permeable version of SIGK, myristoyl-SIGK, was shown by our laboratory to stimulate G␤␥-dependent ERK phosphorylation in smooth muscle and other cell types (23). SIGK binds at a surface on G␤␥ that corresponds to the binding site for the G␣ subunit switch II helix and competes for binding of effectors such as phospholipase C␤ and PI3K␥ but not type I adenylyl cyclase or N-type Ca 2ϩ channels. Our data supported a model that explained the ability of cell-permeable SIGK to stimulate ERK activation: SIGK binding to G␤␥ in G␤␥-G␣GDP heterotrimers in cells enhances the release of G␣GDP, leaving G␤␥ bound to SIGK. This released SIGK-bound G␤␥ could then signal to ERK because bound SIGK did not interfere with effector surfaces on G␤␥ required for activation of this pathway. SIGK did not activate PLC␤ or PI3K␥ because with SIGK bound to the released G␤␥ the binding sites for PLC␤ and PI3K␥ were occupied with SIGK. 12155 is one of several molecules that were found in the screen for binding to G␤␥ in a competition assay for SIGK binding. Unlike other molecules identified in this screen such as M119, 12155 did not inhibit G␤␥-dependent regulation of PLC␤ or PI3K␥. Thus, in contrast to SIGK, when 12155-bound G␤␥ is free from G␣GDP it can signal downstream to multiple G␤␥-dependent signaling pathways.
Our data do not clearly discriminate between a cellular mechanism where 12155 binds to heterotrimers and drives G␤␥ dissociation or where 12155 binds to free G␤␥ to prevent rebinding to G␣GDP. 12155 enhances the G␣GDP dissociation rate from G␤␥␣GDP heterotrimers by greater than 2-fold in vitro, indicating that it can bind to heterotrimers. How this rate enhancement in vitro relates to the rate of free G␤␥ release in cells, and whether this is sufficient to drive the observed rapid Ca 2ϩ increase is not clear. Ca 2ϩ responses are highly amplified so a small but rapid increase free G␤␥ could be sufficient to drive a rapid Ca 2ϩ increase. Regardless of the specific mechanism, the data support a model where 12155 treatment of cells leads to an increase in free G␤␥ without activation of G␣.
The chemical biology based approach used here has several advantages, one of the most important of which is the ability to rapidly perturb native systems without requiring overexpression of proteins. Protein overexpression can upset the balance of natural stoichiometries and requires 24 to 48 h during which time the system can adapt to the intended perturbation. In this particular system the use of a chemical activator also allowed for establishment of a gradient with which a directional chemotaxis assay could be established. A possible concern is the potential for off-target effects complicating interpretation of the experiments. 12155 is also known as surfen and is known to bind to, and antagonize, interactions with heparin sulfate (33, 34) as well as bind to and inhibit anthrax lethal factor (35). Our data are highly consistent with the idea that the chemotaxis mediated by 12155 is due to its ability to bind to G␤␥ including the observation that M119 inhibition of G␤␥ signaling inhibits 12155 directed migration, whereas PTX is only weakly effective.
Previous studies have identified G␤␥ to be important in inducing cell migration, with inhibition of its signaling leading to inhibition of migration (27,32). Here we show that G␤␥ release from G␣GDP alone is sufficient to induce chemotaxis in solution. This indicates that the GPCRs, activated G␣ i or G␣ 13 subunits, other GPCR accessory proteins, such as GRK2 and ␤-arrestin, or the regulators of G proteins signaling (RGS proteins) G␣ 13 , are not required to mediate directional chemotaxis of neutrophils in solution. Numerous reports have suggested G␣ subunits along with RGS, GRK2, and ␤-arrestin to play a role in migration (12)(13)(14)(15)(16)(17), but these proteins may serve to modulate G␤␥-mediated chemotaxis. A recent report suggested that there is a role for G␣GDP in directing cell migration that could be involved in 12155mediated signaling and could be the subject for further investigation (36). All of the migration assays in this work were conducted in a transwell migration chamber in the absence of extracellular matrix. This allowed us to assess the role of G␤␥ in setting the compass and driving directional chemotaxis but does not mimic neutrophil migration during an infection that includes adhesion to endothelial cells, rolling, trans-endothelial migration, and interstitial migration where other aspects of GPCR signaling may play important roles.
Other studies have examined the relative roles of G␣ and G␤␥ signaling downstream of the receptor to discern their relative roles in directing chemotaxis. One study used HEK293 cells transfected with CXCR1 and a G␣ q/z chimera that couples to CXCR1 but does not regulate G␣ i effectors such as adenylyl cyclase (8). In these cells IL-8 was able to act as a chemotactic stimulus even though G␣ i was not activated implying that G␣ i signaling is not required for directional chemotaxis of HEK293 cells. Another study used the rapamycin-dependent FKBP-FRB dimerization system to bypass the G protein signaling system in HL60 cells and directly activate PI3K activity to produce PIP 3 . They demonstrate that uniform application of rapamycin causes polarization of the cells and increased migration, although directional movement of cells in a gradient was not tested (30). Both of these systems although providing valuable information are somewhat artificial either using transfected HEK293 or HL60 cells. In our study we show that in primary neutrophils a gradient of compound that releases free G␤␥ from G␣ i GDP can act as a chemoattractant.
In this study we demonstrated the utility of a small molecule G␤␥ activator in a cellular setting, to activate G␤␥-mediated signaling pathways. This compound will be helpful in elucidating more pathways and functions related to G␤␥ or GPCR-dependent signaling. Understanding the details of where 12155 binds and how it modulates release of G␤␥ from G␣ i GDP is a question of interest. We propose the existence of a key residue(s) on G␤␥, on binding to which, 12155 induces dissociation of G␤␥ from G␣. Finding the binding site of 12155 by a mutational analysis of the binding site or through solving the three-dimensional co-crystal structure of 12155 with G␤␥ can further illuminate this hypothesis.