Differential Inhibitor of Gβγ Signaling to AKT and ERK Derived from Phosducin-like Protein

Abstract

Differential inhibitors of Gβγ-effector regions are required to dissect the biological contribution of specific Gβγ-initiated signaling pathways. Here, we characterize PhLP-M1-G149, a Gβγ-interacting construct derived from phosducin-like protein 1 (PhLP) as a differential inhibitor of Gβγ, which, in endothelial cells, prevented sphingosine 1-phosphate-induced phosphorylation of AKT, glycogen synthase kinase 3β, cell migration, and tubulogenesis, while having no effect on ERK phosphorylation or hepatocyte growth factor-dependent responses. This construct attenuated the recruitment of phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane and the signaling to AKT in response to Gβγ overexpression. In coimmunoprecipitation experiments, PhLP-M1-G149 interfered with the interaction between PI3Kγ and Gβγ. Other PhLP-derived constructs interacted with Gβγ but were not effective inhibitors of Gβγ signaling to AKT or ERK. Our results indicate that PhLP-M1-G149 is a suitable tool to differentially modulate the Gβγ-initiated pathway linking this heterodimer to AKT, endothelial cell migration, and in vitro angiogenesis. It can be also useful to further characterize the molecular determinants of the Gβγ-PI3Kγ interaction.

Heterotrimeric G protein signaling depends on the actions of GTP-loaded Gα and free Gβγ, the two functional components of the heterotrimer, leading to the generation of second messengers and cell specific functional events (1, 2). Differential inhibitors of Gβγ are required to dissect the biological impact of different Gβγ-dependent effectors. Gβγ actions can be blocked by competition with peptides derived from its effectors. For example, the effect of Gβγ on adenylyl cyclase II, G protein-activated inward rectifier K⁺ channel, G protein-coupled receptor kinase 2, and phospholipase Cβ3, is attenuated by a peptide from adenylyl cyclase II (3). In addition, RACK1 (receptor for activated C kinase 1) selectively inhibits the effect the chemokine receptor CXCR2 on the activation of phospholipase Cβ2 and adenylyl cyclase II in HEK293 cells, without affecting other functions of Gβγ (4). Recently, Smrcka and colleagues characterized the effect of small molecule inhibitors of Gβγ, suggesting their potential application in therapeutic strategies targeting particular Gβγ-dependent pathways (5). Emerging possibilities to target this heterodimer in pathological situations such as inflammation and angiogenesis are based on the role of Gβγ in cell survival and chemotaxis. To the best of our knowledge, no molecular tool is yet available to differentially inhibit Gβγ signaling to AKT.³

Gβγ is a key transducer of sphingosine 1-phosphate (S1P)-elicited angiogenic signals promoting endothelial cell migration, proliferation, and survival (6–12). Multiple Gβγ-dependent effectors are potentially involved in the molecular events required for endothelial cell migration. These include lipid kinases such as PI3Kγ and PI3Kβ (13), and a novel family of Rac guanine nucleotide exchange factors, represented by P-REX1, which is activated by Gβγ and phosphatidylinositol 3,4,5-trisphosphate (14–16). Gβγ signaling is frequently attributed to pertussis toxin-sensitive G_i coupled receptors, and it has been consistently revealed by the antagonistic effect of the carboxyl-terminal region of G protein-coupled receptor kinase 2, which sequesters Gβγ thereby inhibiting all its intracellular actions (17). In addition, mutational analysis of Gβ revealed that different residues, all of them mapping to the interface of contact between Gβγ and Gα, are important for the activation of distinct Gβγ effector molecules (18).

Phosducin was originally identified as a phosphoprotein restricted to the retina and pineal gland forming a complex with Gβγ (19, 20). It was considered a protein kinase A-sensitive regulator of G protein-mediated signaling (21, 22). Further studies identified a family of phosducin-like proteins (PhLPs) (23, 24). Phosducin and Gα share affinity for the same region of Gβγ, as revealed by the structural analysis of Gβγ in complex with Gα or phosducin and by in vitro binding experiments (25). This area of interaction includes some of the residues considered necessary for the activation of Gβγ-dependent effectors (18, 26). It was initially postulated that phosducin and related proteins, by interfering with the availability of free Gβγ, exert an inhibitory role on Gβγ signaling. However, recent genetic evidence raised an apparently conflicting situation; the knockout of PhLP in fungi resulted in a phenotype equivalent to the absence of Gβγ, contrary to its expected role as an inhibitor (27). Novel experimental evidence indicated that PhLP has a positive effect on Gβγ signaling due to its participation in the assembly of the heterodimer, helping to stabilize free Gβ subunits leaving the ribosome after synthesis (28–31).

Despite the positive role of full-length PhLP in the assembly of Gβγ heterodimers, it is still possible that different fragments of this protein, which could retain their interaction with distinct regions of Gβγ, might function as inhibitors of Gβγ signaling. Accordingly, we characterized here the effect of different PhLP-derived constructs on the signaling pathways elicited by S1P or HGF in endothelial cells. In addition, we explored the mechanism by which PhLP-M1-G149 interferes with Gβγ preventing the activation of AKT.

EXPERIMENTAL PROCEDURES

Bioinformatic Tools

The structure of Gβγ/phosducin (25, 32, 33) was analyzed with the CN3D program, available from the NCBI web site (www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). This program was used to identify the amino acids at the Gβγ/phosducin-contact interface within 3–3.5 Å.

Phosducin-like Protein-derived Constructs

Based on the structure of the complex formed by Gβγ and Phd (25, 32, 33), and considering the homology between Phd and PhLP, five PhLP-derived constructs were obtained by PCR using as template the cDNA of human PhLP (24) (NCBI accession AF076463, kindly provided by Cheryl Craft, University of Southern California, Los Angeles). According to the structural analysis, these constructs would interact with different amino acids at the effector region of Gβγ. The corresponding cDNAs were cloned into the mammalian expression vectors pCEFL-EGFP, pCEFL-GST, or pCEFL-EGFP-CAAX. EGFP or GST was expressed at the amino terminus of the constructs, whereas in the case of the EGFP-CAAX, the CAAX box was at the carboxyl terminus. All the constructs were cloned with BamHI and EcoRI restriction sites (5′ and 3′, respectively) that were included in the corresponding primers. The sequences of the oligonucleotides are as follows: PhLP (M1-E283) sense, ataGGATCCatgcccgagagcctggacagc and antisense, ataGAATTCtcattcaacatcttcttcttc; (PhLP-M1-G149) sense, ataGGATCCatgcccgagagcctggacagccc and antisense, ataGAATTCcccatatctaggcccaaaactc; (PhLP-Q94-E283) sense, ataGGATCCcagagtaggaatggcaaagattc and antisense, ataGAATTCtcattcaacatcttcttcttct; (PhLP-Q94-G149) sense, ataGGATCCcagagtaggaatggcaaagattc and antisense, ataGAATTCcccatatctaggcccaaaactcagc; (PhLP-M1-K102) sense, ataGGATCCcagagtaggaatggcaaagattc and antisense, ataGAATTCtcattcaacatcttcttcttct; and (PhLP-R147-E283) sense, ataGGATCC-agatatgggtttgtgtatgagc and antisense, ataGAATTCtcattcaacatcttcttcttct. For the EGFP-CAAX constructs, the stop codon in the antisense oligonucleotides was omitted.

Cell Lines and Transfections

Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEMç Sigma) supplemented with 10% fetal bovine serum (Terra Cell International). For transient transfections, tissue culture plates were treated for 10 min with phosphate-buffered saline (PBS) containing 5 μg/ml poly-d-lysine before seeding the cells to prevent them from detaching from the plates during the transfection procedure and thereafter. Cells (2.2 × 10⁶) were seeded the day before transfection in 100-mm dishes in DMEM supplemented with 10% fetal bovine serum. HEK 293T cells were routinely transfected with Lipofectamine Plus reagent (Invitrogen). Cells were incubated for 5 h in serum-free DMEM with DNA plasmids premixed with Lipofectamine-Plus reagent according to the manufacturer's instructions and were then incubated overnight at 37 °C in DMEM containing 10% fetal bovine serum. The total amount of DNA in all transfections was 4 μg/plate. When required, empty pCEFL vector was used to maintain a constant amount of DNA.

Interactions between Gβγ and PhLP Constructs

The interaction between Gβγ and PhLP-derived constructs was assessed in HEK293T as indicated in the figure legends. Gβγ pulldown experiments were done using both subunits fused to His₆ amino-terminal tags. Briefly, cells were washed with ice-cold PBS and lysed at 4 °C in TBS-T buffer containing 50 mm Tris, pH 7.5, 0.15 m NaCl, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin, and insoluble material was removed by centrifugation at 14,000 rpm for 10 min at 4 °C. His₆-tagged Gβγ was isolated by incubating with 40 μl of Talon beads (Metal Affinity Resin, Clontech) for 2 h at 4 °C. Beads were washed three times with lysis buffer and boiled for 5 min in 1× Laemmli sample buffer. Expression of proteins and the putative interactions between them were revealed by Western blot using anti-His₆ monoclonal antibodies (Sigma) or GFP monoclonal antibodies Covance). Immunoblots were visualized by enhanced chemiluminescence detection (Pierce). Similar experiments were done to investigate the effect of PhLP-Q94-G149 construct in the interaction between PhLP-M1-G149 and Gβγ. Briefly, HEK 293T cells in 10-cm dishes were transfected with 1.0 μg of PhLP-M1-G149, 0.3 μg each His₆-Gβ₁γ₂ and increasing concentration of the plasmid coding for PhLP-Q94-G149 construct (0.3, 1, and 3 μg). Pulldown assays were performed 2 days after transfection.

For experiments assessing the interaction between Gβ₁ mutants and PhLP constructs the following Gβ mutants were used: Gβ₁(L55A) (residue at the amino-terminal interface (18)), Gβ₁(K57A), Gβ₁(S98A), Gβ₁(T143A), and Gβ₁(W332A) (all of them at the switch interface) (18) were obtained by using the site-directed mutagenesis system (QuikChange, Stratagene). Their interaction with PhLP, PhLP-M1-G149, or PhLP-Q94-E283 was assessed in HEK293T cells cotransfected with 1 μg of the different pCEFL-HA-Gβ₁ mutants (as indicated in Fig. 2) or pCEFL-HA Gβ₁, and 1 μg of pCEFL-Gγ2, 2 μg of pCEFL-GST-PhLP, 3 μg of pCEFL-GST-PhLP-M1-G149, or 3 μg of pCEFL-GST-PhLP-Q94-E283. Pulldown experiments were done 2 days after transfection. The interaction between different Gβγ heterodimers and PhLP constructs was assessed in HEK293T cells. Briefly, FLAG-tagged Gβ subunits (in pCDNA3, obtained from the Guthrie cDNA Resource Center) were cotransfected with pCEFL-Gγ₂ (1 μg each), and 2 μg of pCEFL-GST-PhLP, or 3 μg of pCEFL-GST-PhLP-M1-G149, or 3 μg of pCEFL-GST-PhLP-Q94-E283, pCEFL empty plasmid was used to adjust the amount of transfected DNA. Two days after transfection, PhLP constructs were isolated by pulldown assay using glutathione-Sepharose beads, and the presence of associated Gβ was revealed by Western blot. Cells were lysed with TNTE buffer (50 mm Tris-HCl buffer, pH 7.5, 150 mm NaCl, 5 mm EDTA containing 1% Triton X-100) plus of the previously indicated protease inhibitors, cell lysates were incubated with 40 μl of glutathione-Sepharose 4B beads (Amersham Biosciences) rocking for 1 h at 4 °C. Sepharose beads were collected by centrifugation and washed three times with TNTE buffer. Bound proteins were resolved by SDS-PAGE and detected by Western blot using anti-HA (Covance), anti-FLAG (Sigma), or anti-GST (Santa Cruz Biotechnology, Santa Cruz, CA) monoclonal antibodies. Whole cell lysates (25 μg of protein per lane) were used for Western blots, whereas ∼2 mg of protein were used for pulldown assays. Data showed are representative of at least three independent experiments.

FIGURE 2.

Analysis of the interaction between Gβγ mutants and constructs derived from PhLP. Upper panel: Gβ₁ with the indicated point mutations at the interface known to interact with Gα or effectors (18) was cotransfected with Gγ₂ and PhLP constructs in HEK293T cells. Interaction between Gβγ (wild-type Gβ or the indicated mutant) and PhLP constructs, fused to GST was detected by pulldown assays. The presence of Gβ or the indicated mutants interacting with PhLP or the constructs PhLP-M1-G149 or PhLP-Q94-E283 was detected by Western blot using HA antibodies that recognized this tag fused at the amino-terminal of Gβ. As a negative control, a construct corresponding to GST and wild-type Gβγ was used. The expression of transfected GST-tagged PhLP is shown in the pulldown assays, and the expression of Gβ and the indicated mutants is shown in total cell lysates (TCL). Bottom panel: the importance of Gβ₁Ser98 and Gβ₁W332, analyzed with NCBI's CN3D program, is highlighted in the structure of the complex formed by phosducin and Gβγ (25); as shown, these amino acids establish two interacting spots with the central region of phosducin. According to the pulldown experiments shown in the upper panel, the first interacting contact, depending on Gβ₁Ser98, is critical for the interaction with full-length PhLP and is also important for PhL-M1-G149.

The influence of PhLP constructs on the expression of different Gβ subunits was assessed in HEK293T cells cotransfected with FLAG-tagged Gβ subunits, Gγ₂, and PhLP, PhLP-M1-G149, or PhLP-Q94-E283. Two days after transfection, cells were harvested, 10 μg of total cell lysates was used for SDS-PAGE, and the expression of the different Gβ subunits was detected by Western blot using anti-FLAG antibodies.

Influence of PhLP Constructs on the Interaction between Gβγ and PI3Kγ

The interaction between Gβγ and PI3Kγ was assessed by coimmunoprecipitation experiments in HEK 293T cells transfected with plasmids expressing 3X-FLAG Gβ₁, Gγ₂, PI3Kγ (EE-tagged p110 and EE-tagged p101), kindly provided by L. R. Stephens (34) and PhLP constructs as indicated in Fig. 9. Briefly, 2 days after transfection, cells, grown in 10-cm dishes, were placed on ice, rinsed once with phosphate-buffered saline, and lysed in 1 ml of ice-cold TNTE buffer containing protease inhibitors; insoluble material was removed by centrifugation. After clearing, 3 μl of EE antibody (Covance) was added to the supernatant and rotated overnight at 4 °C. Next day, 25 μl of protein A-Sepharose was added and rotated again for 2 h at 4 °C. Immunoprecipitates were washed three times with 1 ml of lysis buffer and resuspended in 1× protein sample buffer. Lysates containing ∼50 μg of total cellular protein or immunoprecipitates were analyzed by Western blotting after SDS-polyacrylamide gel electrophoresis and visualized by enhanced chemiluminescence detection (Millipore).

FIGURE 9.

Molecular mechanism of PhLP-M1-G149-mediated inhibition. A, the effect of PhLP-M1-G149, PhLP, or PhLP-Q94-E283 on the interaction between EE-tagged PI3Kγ and FLAG-Gβγ was assessed by immunoprecipitation in transfected HEK293T cells (upper panel). The expression of the transfected proteins was verified in total cell lysates (TCL, lower panel). The graph on the right panel shows the average of five independent experiments normalized to the result obtained in EGFP-transfected cells, vertical lines represent the ± S.E. (*, p < 0.05 respect to control). B, the effect of PhLP, PhLP-M1-G149, or PhLP-Q94-E283 on the recruitment of PI3Kγ to the membrane in response to Gβγ overexpression was assessed by immunofluorescence in endothelial cells. Confocal images show the localization of PI3Kγ detected with antibodies against the EE tag fused to p101 and p110 subunits (middle and bottom panels). The expression of PhLP constructs, fused to EGFP and a carboxyl terminus CAAX box, is shown in the upper panel. EGFP-CAAX was used as control.

The influence of PhLP constructs on the recruitment of PI3Kγ to the membrane in response to Gβγ overexpression was determined by immunofluorescence in PAE cells. Cells seeded on fibronectin (20 μg/ml)-coated coverslips were transfected with 100 ng each, of His₆-Gβ₁γ₂, EE-PI3Kγ (p101 and p110), and 200 ng of PhLP constructs or 100 ng of EGFP-CAAX as indicated in Fig. 9. Serum-starved cells were processed for immunofluorescence, 2 days after transfection, using previously described conditions (35). Briefly, cells were washed with PBS, fixed with fresh 4% paraformaldehyde in PBS, pH 7.4, for 20 min, washed five times with PBS and permeabilized with 100% methanol for 6 min at −20 °C. and incubated with 1% bovine serum albumin for 30 min at 37 °C. PI3Kγ was detected with EE monoclonal antibody (Covance) used at 1/500 in 0.5% bovine serum albumin for 1 h at 37 °C, washed, and followed by incubation with Cy5 secondary antibody (Jackson) used at 1/200 in 1% bovine serum albumin for 40 min at room temperature. Cells were mounted with Vectashield (Vector Laboratories), and images were acquired with a DMIRE2 confocal laser-scanning microscope (Leica Microsystems, Deerfield, IL) by the use of a 63×, numerical aperture 1.4 oil immersion objective and a zoom of 2.

Stably Transfected Endothelial Cells

All EGFP-tagged constructs derived from PhLP were transfected into porcine aortic endothelial (PAE) cells using Polyfect reagent (Qiagen); control cells were transfected with pCEFL-EGFP. Clones were selected 48 h after transfection with 2.0 mg/ml G418 disulfate salt (Geneticin 418, Sigma) in DMEM supplemented with penicillin/streptomycin (BIOSOURCE, International) and 10% fetal bovine serum. Expression of the transfected constructs was determined by fluorescence-activated cell sorting, immunoblotting, and fluorescence microscopy.

ERK, AKT, and GSK3β Assays

Stably transfected endothelial cells were cultured in DMEM, including 10% serum and G418 antibiotic until they reached confluence. In brief, cells were serum-starved for 8 h and either stimulated with 1 μm S1P, 10 ng/ml HGF, or vehicle for the indicated period. Cells were washed and lysed with 1× boiling Laemmli sample buffer. Total cell lysates were used for Western blots as indicated previously. Phosphorylation of AKT, ERK1/2, or GSK3β and expression of these proteins were detected using the following rabbit antibodies: phospho-AKT-1/2/3 (Ser-473) antibody (Santa Cruz Biotechnology); phospho-p44/42 mitogen-activated protein kinase (MAPK, Thr-202/Tyr-204) antibody (Cell Signaling) and phospho-GSK3β (Ser-9) antibody (Cell Signaling). Protein expression was analyzed using: mouse monoclonal anti-protein kinase B (PKB/AKT1, Sigma), rabbit polyclonal anti-MAPK (Santa Cruz Biotechnology), and rabbit polyclonal anti-glycogen synthase kinase-3 (GSK3, Cell Signaling).

The effect of Gβγ overexpression on AKT and ERK phosphorylation was assessed in COS7 cells. Briefly, cells were cultured in 6-well plates in DMEM with 10% fetal bovine serum until they reached a 70% of confluence. Cells were then transfected with 0.5 μg of plasmids coding for different Gβ subunits (1–5), Gγ₂, and PhLP constructs as indicated in the figure legends. Next day, cells were serum-starved, and the day after they were lysed in 200 μl of ice-cold TBS-T buffer containing the previously indicated protease inhibitors and phosphatase inhibitors (20 mm β-glycerophosphate and 1 mm sodium vanadate), insoluble material was removed by centrifugation. Phosphorylation of AKT or ERK1/2 was detected as described above.

Effect of PI3K Inhibitors in Endothelial Cells

The effect of PI3Kγ and PI3Kβ inhibitors (36) on the activation of AKT in PAE cells was assessed in serum-starved confluent cultures grown in DMEM in 6-well plates. In brief, cells were serum-starved the night before the experiment. Next day, media was replaced before incubation with the inhibitors, either PIK93 for PI3Kγ (IC₅₀, 0.016 μm), TGX115 (ZK 28), for PI3Kβ (IC₅₀, 0.13 μm), or vehicle, which were incubated in 1 ml of serum-free media before stimulation as indicated in Fig. 5. The maximum concentration used in our experiments was 0.1 μm. Stimulated or control cells were washed and lysed with boiling 1× Laemmli sample buffer. Total cell lysates were used for Western blots to detect the phosphorylation of AKT using anti-phospho-AKT (Ser-473) antibody (Cell Signaling, 9271). The experiments using PI3K inhibitors were done by MLGH in Tamas Balla's laboratory at NICHD, NIH using inhibitors kindly provided by Zacchary Knight and Kevan Shokat (University of California, San Francisco).

FIGURE 5.

Effect of PTX and PhLP-derived constructs on S1P or HGF-induced activation of AKT and ERK in endothelial cells. A, upper panels: endothelial cells were stimulated with 1 μm S1P or 10 ng/ml HGF for the indicated time. After stimulation, phosphorylated AKT and ERK (pAKT and pERK) were detected by Western blotting using phospho-specific antibodies. As a reference, the expression of AKT and ERK was detected in parallel using antibodies that recognize the kinases regardless of their phosphorylation state (bottom panels). B, effect of pertussis toxin (PTX, 100 ng/ml) on S1P or HGF signaling to AKT and ERK in EGFP-transfected endothelial cells. C, effect of PI3Kγ and PI3Kβ inhibitors on the activation of AKT elicited by S1P or HGF. D and E, effect of PhLP-derived constructs on S1P or HGF signaling to AKT and ERK in endothelial cells. The activation of AKT and ERK (pAKT and pERK) was examined with phospho-specific antibodies, and the expression of AKT and ERK was detected in parallel. EGFP-transfected cells were used as control. Bars represent the mean densitometric values of three independent experiments, as the one shown below the graph, normalized to the maximal agonist dependent effect (100%) obtained in HGF-stimulated cells; in the case of PhLP-M1-G149-transfected cells, the results correspond to six independent experiments; vertical lines represent the ± S.E. (*, p < 0.05 respect to control). F, effect of pertussis toxin (PTX) on S1P and HGF signaling to AKT and ERK in PhLP-M1-G149-transfected endothelial cells.

Cell Migration Assays

Migration assays of stably transfected endothelial cells were performed in Boyden chambers. Filters with 8-μm pores were coated overnight with 10 μg/ml fibronectin. The bottom wells of the chamber were filled with 150 μl of media containing (or lacking) chemoattractant. Stably transfected endothelial cells were harvested by trypsinization, washed by centrifugation, and resuspended 1 × 10⁵ cells in 100 μl of fresh DMEM. After the cells were inoculated into the upper chamber and incubated for 6 h at 37 °C. The migrated cells on the bottom of the chamber were fixed with methanol and stained with 10 mg/ml crystal violet. The cells that remained on the top side of the filter (those that did not migrate) were removed with a cotton swab. The level of migration was determined by densitometric quantitation of the stained filters, using ImageJ software.

In Vitro Angiogenesis Assay

Stably transfected endothelial cells were serum-starved 15 h before the experiment, detached by gently trypsinization, washed, counted, and resuspended in DMEM. Previously, Matrigel basement membrane matrix (BD Biosciences, 354230) diluted 1:2 in cold serum-free DMEM was plated into 96-well tissue culture plates and incubated for 1 h at 37 °C before seeding the cells. Then, 10 × 10³ cells were added on top the Matrigel in the absence or presence of different stimuli (1 μm S1P or 0.5 ng/ml HGF). After 9 h of incubation, the experiment was stopped, and the results were assessed morphologically using light microscopy. The length of the tubes was measured using Image J software.

Statistical Analysis

Test and control samples in the functional assays were compared for statistical significance by using Student's t test, a p < 0.05 was considered significant.

RESULTS

Structural Analysis of Gβγ-Phosducin Complex and Interaction between Gβγ and Constructs Derived from PhLP

In this study, we tested the possibility that constructs derived from PhLP, a Gβγ-interacting protein, differentially inhibit Gβγ signaling to AKT and ERK. To identify the sites of interaction between different regions of phosducin and Gβγ, we analyzed the crystal structure of phosducin-Gβγ shown in Fig. 1A (25, 32, 33). According to the analysis of three different structures of Gβγ-phosducin complexes: 1B9Y (33), 2TRC (25), and 1AOR (32), and assuming that phosducin and PhLP interact with the same residues in Gβγ, the amino acids shown in white color in Fig. 1B, bottom right, are those in Gβγ interacting with PhLP amino-terminal region (PhLP-M1-K102): Lys-57, Asn-230, Thr-274, and Asp-290; those shown in white color in the left panel, interact with PhLP central region (PhLP-Q94-G149): Gln-75, Ser-98, and Trp-332 (Trp-332 within a distance of 3.5 Å), and those shown in white color in the upper right panel, interact with PhLP carboxyl-terminal region (PhLP-R147-E283, within a distance of 3.0 Å (25, 32)): Gln-44, Arg-46, Thr-47, Arg-304, and His-311. Previous studies with Gβ point mutants revealed that some of these residues are required for the activation of different effectors (13). In particular, the residues of Gβγ involved in the interaction with phosphoinositide 3-kinase (PI3K), a known effector of Gβγ leading to AKT activation and other downstream effectors, remain to be determined. To begin addressing this issue, we engineered five PhLP-derived constructs that, according to the structure, are most likely to maintain their conformation and ability to interact with Gβγ (Fig. 1, B (middle panel) and C). These constructs covered the amino- (PhLP-M1-K102), central- (PhL-Q94-G149), and carboxyl- (PhLP-R147-E283) regions of PhLP (Fig. 1, B (middle panel, indicated in different intensities of gray) and C). All the constructs were expressed as amino-terminal EGFP-tagged (GFP) fusion proteins. Their expression was confirmed in HEK-293T cells (Fig. 1C, left panel).

FIGURE 1.

Structural analysis of Gβγ-phosducin complex and interaction between Gβγ and constructs derived from PhLP. A, structure of the heterotrimer formed by Gβ (light gray), Gγ (dark gray), and phosducin (white) (25). B, phosducin-interacting amino acids (within 3–3.5 Å) highlighted in white in Gβγ as detected with the NCBI's CN3D program. Those interacting with the different regions of phosducin are shown as white residues in structures of Gβγ next to the corresponding interacting region of Phd. C, based on the structure of phosducin, constructs corresponding to the different regions of PhLP were generated. The amino-terminal domain (M1-K102) corresponds to the dark gray region, the central region (Q94-G149) corresponds to the light gray region and the carboxyl-terminal domain (R147-E283) corresponds to the black part of the structure; right panel. The interaction between PhLP-derived constructs and Gβγ was assessed in HEK-293T transiently transfected with His₆-tagged Gβ₁γ₂ and EGFP-tagged PhLP constructs. His₆-Gβ₁γ₂ was isolated with a cobalt-based metal affinity resin and interacting PhLP derived constructs were detected by Western blotting using anti-GFP antibodies. As a control, a construct corresponding to the carboxyl-terminal region of GRK-2 fused to GFP was used. The expression of transfected EGFP-tagged PhLP or the indicated constructs is shown in total lysates and those interacting with His₆-Gβ₁γ₂ are shown in the pulldown assay (PD: His). His₆-Gβ₁γ₂ is shown in the bottom panel.

Analysis of the Interaction between Gβγ and Constructs Derived from PhLP

The interaction between Gβγ- and PhLP-derived constructs was assessed in HEK-293T. His-tagged Gβγ was isolated under non-denaturing conditions using a cobalt-based metal affinity resin. EGFP-tagged PhLP constructs present in the Gβγ pulldown assays were detected by Western blot. All the constructs that included the central region of PhLP (Q94-G149) showed a strong interaction with Gβγ, comparable to the interaction detected between Gβγ and the carboxyl-terminal region of GRK2 used as control. In contrast, the constructs lacking this region (PhLP amino-terminal region, PhLP-M1-K102) and (PhLP carboxyl-terminal region, PhLP-R147-E283) showed a reduced ability to interact with Gβγ (Fig. 1C). The expression of His₆-tagged Gβγ was demonstrated by Western blot with anti-His antibody (Fig. 1C, lower panel). These results showed that the different regions of PhLP, expressed as EGFP-tagged derived peptides, interact with Gβγ, suggesting that they might modulate specific Gβγ-downstream pathways and therefore could be used to dissect Gβγ signaling events.

To assess the contribution of key residues at the interface of Gβ₁ that are known to interact with Gα or effectors (18) to the interaction between Gβγ and PhLP constructs, we assessed the interaction of different Gβ₁ mutants, expressed with Gγ₂, in PhLP pulldown experiments. As shown in Fig. 2, wild-type Gβγ interacted with PhLP and the constructs PhLP-M1-G149 or PhLP-Q94-E283 in pulldown experiments using PhLP constructs fused to GST. Interestingly, Gβ₁Ser98Ala was unable to interact with full-length PhLP, indicating that the interacting interface formed by GβSer98 with the central region of PhLP is critical for the interaction. In Phd, the amino acids that interact with Gβ₁Ser98 are Phd-E85, Phd-L90, and Phd-R94, establishing a region of electrostatic interactions between Gβγ and Phd (25, 32, 33). The importance of Gβ₁Ser98 and Gβ₁W332 was analyzed with the CN3D program (NCBI), and the predicted interface is highlighted in the structure of the complex formed by Phosducin and Gβγ (25) shown in Fig. 2, lower panel.

The experiments with Gβ point mutants and those comparing the interaction between Gβγ and different PhLP constructs suggested the importance of the central region of PhLP on the interaction with Gβγ. Thus, we tested the ability of the construct corresponding to this region, PhLP-Q94-G149, to compete with the construct PhLP-M1-G149. This includes the central region and extends toward the amino terminus. As shown in Fig. 3, top panel, the central region of PhLP competed with PhLP-M1-G149 for the interaction with Gβγ, confirming the importance of the PhLP fragment Q94-G149 in the interaction with Gβγ (Fig. 3).

FIGURE 3.

Importance of the central region of PhLP in the interaction with Gβγ. Pulldown experiments as those described in Fig. 1C were done to test the effect of increasing concentration of PhLP-Q94-G149 in the interaction between PhLP-M1-G149 and Gβγ. As shown in the upper panel, the central region of PhLP competes in the interaction of PhLP-M1-G149 with Gβγ, pointing out to the critical role of the central region of PhLP in the interaction with Gβγ. The expression of the transfected proteins was verified by Western blot in total cell lysates (TCL, lower panel). As a negative control, EGFP was used in the pulldown assays along with PhLP-M1-G149 (tagged with EGFP) and Gβγ.

The interaction between different Gβγ heterodimers and PhLP constructs was analyzed by pulldown experiments using PhLP constructs fused to GST. As shown in Fig. 4, Gβγ heterodimers containing either Gβ₁, Gβ₂, Gβ₃, or Gβ₄, all interacted with PhLP, PhLP-M1-G149, or PhLP-Q94-E283. Interestingly, PhLP-Q94-E283 also interacted with FLAG-tagged Gβ₅γ₂. The interaction was specific as shown in the pulldown assays in which GST and FLAG-Gβ₁γ₂ were used as negative controls.

FIGURE 4.

Analysis of the interaction between different Gβγ heterodimers and constructs derived from PhLP. The indicated FLAG-tagged Gβ subunits were cotransfected with Gγ₂ and the indicated PhLP construct in HEK293T cells. The interaction with PhLP or the indicated constructs, fused to GST was detected by pulldown assays. The presence of Gβ interacting with PhLP or the constructs PhLP-M1-G149 or PhLP-Q94-E283 was detected by Western blot using FLAG antibodies that recognized this tag fused at the amino-terminal of Gβ (upper panel). Cells transfected with GST and FLAG-Gβ₁γ₂ were used as negative controls (C). The expression of transfected GST-tagged PhLP and the indicated constructs is shown in the pulldown assays (lower panel), and the expression of transfected FLAG-Gβ is shown in total cell lysates (TCL, middle panel).

Effect of PTX and PhLP-derived Constructs on S1P and HGF Signaling to AKT and ERK in Endothelial Cells

We next tested whether PhLP constructs can differentially interfere with Gβγ signaling in endothelial cells stimulated with S1P or HGF. Both agonists are known to induce activation of PI3K-AKT, ERK, and chemotactic migration in endothelial cells (10, 12, 37). The action of S1P activating these pathways is known to be mediated through G_i-coupled receptors (10, 12, 38). Here, using antibodies against the phosphorylated forms of AKT and ERK, we demonstrated that 1 μm S1P and 10 ng/ml HGF induced a rapid phosphorylation of AKT and ERK in PAE cells (Fig. 5A). Pertussis toxin (PTX) differentially inhibited the effect of S1P, but not of HGF, on AKT and ERK phosphorylation (Fig. 5B). These data indicated that S1P regulates signaling to AKT and ERK through a G_i heterotrimeric protein in PAE cells. In addition, S1P and HGF signal to AKT via the intervention of different PI3K isoforms as demonstrated by a differential effect of specific inhibitors. As shown in Fig. 5C, S1P signaling to AKT depends on the activity of PI3Kγ, and the effect of HGF is independent of this isoform. The potential effect of the different PhLP constructs as modulators of Gβγ signaling to AKT and ERK was determined in stably transfected PAE cells stimulated with S1P or HGF. PhLP-M1-G149, the construct corresponding to the amino plus the central regions of PhLP, prevented the activation of AKT in cells stimulated with S1P while having no effect on the activation of ERK (Fig. 5, D and E). Other PhLP-derived constructs did not interfere with the ability of S1P to promote the phosphorylation of AKT and ERK (Fig. 5, D and E). We noticed a slight increase in the basal phosphorylation of ERK in cells expressing PhLP-M1-G149 and PhLP-Q94-E283, but these constructs did not affect the agonist-dependent activation of ERK. On the other hand, cells expressing the smaller constructs, corresponding to the amino-, central-, and carboxyl-terminal regions of PhLP did not show differences, with respect to the control in the activation of AKT and ERK (supplemental Fig. S1). The effect of S1P on AKT and ERK in cells expressing PhLP-M1-G149 was still sensitive to inhibition by PTX (Fig. 5F), indicating that the coupling properties of S1P receptors in these cells were not modified by expression of the PhLP-M1-G149 construct. Neither PhLP-derived constructs nor PTX inhibited the effect of HGF (Fig. 5, D–F).

Effect of PhLP-M1-G149 on GSK3β Phosphorylation in Endothelial Cells

Glycogen synthase kinase 3β (GSK3β) is a target of the PI3K signaling pathway, being phosphorylated by AKT and p70S6 kinase (39, 40). To assess the role of G_i in the phosphorylation of GSK3β in endothelial cells responding to S1P or HGF, PTX-treated cells were stimulated with these agonists as indicated in Fig. 6A. Both, S1P and HGF promoted the phosphorylation of GSK3β, an effect that was sensitive to PTX in S1P-stimulated cells (Fig. 6A, upper panel). To further assess whether PhLP-M1-G149 prevents the phosphorylation of AKT substrates, we evaluated the phosphorylation of GSK3β in cells expressing PhLP-M1-G149 and stimulated with S1P or HGF. The results, shown in Fig. 6 (B and C), indicated that PhLP-M1-G149 prevented the functionality of AKT (assessed by its ability to phosphorylate GSK3β) in cells responding to S1P. Full-length PhLP or PhLP-Q94-E283 did not interfere with the phosphorylation of GSK3β in cells responding to S1P or HGF (Fig. 6, B and C).

FIGURE 6.

Effect of PTX, PhLP-M1-G149, PhLP, or PhLP-Q94-E283 on the phosphorylation of GSK3β induced by S1P or HGF in endothelial cells. A, effect of pertussis toxin (PTX, 100 ng/ml) on S1P- or HGF-induced phosphorylation of GSK3β (pGSK3β, an AKT substrate) in EGFP-transfected endothelial cells. EGFP-expressing PAE cells were stimulated for 5 min either with 1 μm S1P or 10 ng/ml HGF, phosphorylation of GSK3β was detected in total cell lysates using antibodies recognizing the kinase phosphorylated in Ser-9. As a reference, the expression of GSK3β was detected in parallel using antibodies that recognize the kinase regardless of its phosphorylation state (bottom panel). B, effect of PhLP, PhLP-M1-G149, or PhLP-Q94-E283 on S1P- or HGF-induced phosphorylation of GSK3β in stably transfected endothelial cells; PAE cells expressing EGFP were used as control. After stimulation, total cell lysates were analyzed by Western blotting using phospho-specific antibodies against GSK3β or antibodies recognizing the kinase regardless of its phosphorylation state. C, bars represent the mean densitometric values of phospho-GSK3β from three independent experiments, as the one shown in B, normalized to the maximal effect (100%) obtained in HGF-stimulated cells; vertical lines represent the ± S.E. (*, p < 0.05 respect to control).

Effect of PhLP-M1-G149 on Endothelial Cell Migration

To examine the effect of PhLP or PhLP-M1-G149 on endothelial cell migration, we assessed the chemotactic response to S1P or HGF of endothelial cells expressing these constructs or control cells expressing EGFP. Interestingly, cells expressing PhLP-M1-G149 showed a notably reduced migration in response to S1P, while the effect of HGF was not affected (Fig. 7, A and B). Full-length PhLP did not interfere with the migration of endothelial cells in response to S1P or HGF, in fact, an increase in the effect of S1P was detected (Fig. 7, A and B).

FIGURE 7.

Effect of PhLP-M1-G149 or PhLP on S1P- or HGF-induced migration of endothelial cells. Stably transfected endothelial cells expressing EGFP, PhLP (full-length M1-E283), or PhLP-M1-G149 were subjected to migration assays in Boyden chambers containing either vehicle, 1 μm S1P or 10 ng/ml HGF in the bottom wells; cells that migrated through fibronectin-coated polycarbonate filters (with 8-μm pores) were fixed and stained. A, a representative filter showing cells that migrated under the different conditions. B, bars represent the mean densitometric values of stained filters from six independent migration assays, as the one shown in A, normalized to the migration obtained in HGF-stimulated cells; vertical lines represent the ± S.E. (**, p < 0.05 respect to control).

Effect of PhLP-M1-G149 on in Vitro Angiogenesis

Considering the angiogenic properties of S1P, we tested if the expression of PhLP-M1-G149 affects this response. We assessed the effect of S1P and HGF on the in vitro angiogenic response of PhLP-M1-G149-transfected endothelial cells. Our results indicated that PhLP-M1-G149 was able to interfere with S1P-induced angiogenesis, whereas the effect of HGF was not affected. In parallel, cells transfected with EGFP or EGFP-tagged PhLP showed normal angiogenic responses to S1P or HGF (Fig. 8, A and B).

FIGURE 8.

Effect of PhLP-M1-G149 or PhLP on S1P or HGF-induced in vitro angiogenesis. Stably transfected endothelial cells expressing EGFP, PhLP-M1-G149, or PhLP (full-length M1-E283) were subjected to in vitro angiogenesis assay in 96-well tissue culture plates containing Matrigel. These cells were stimulated with vehicle, 1 μm S1P, or 0.5 ng/ml HGF in serum-free DMEM. A, a representative area showing cells that form tube-like structures under 1 μm S1P and 0.5 ng/ml HGF. B, bars represent the mean capillary tube length from three independent assays, as the one shown in A, normalized to the tube formation obtained in HGF-stimulated cells; vertical lines represent the ± S.E.

Molecular Mechanism of PhLP-M1-G149-mediated Inhibition

To assess the molecular mechanism by which PhLP-M1-G149 affects Gβγ signaling to AKT, we tested the hypothesis that this construct interferes with the ability of Gβγ to interact with PI3Kγ and the ability of this heterodimer to recruit PI3Kγ to the plasma membrane. As shown in Fig. 9A, FLAG-tagged Gβ₁γ₂ interacted with PI3Kγ in coimmunoprecipitation experiments. This interaction was significantly reduced in cells expressing PhLP-M1-G149, but not in cells expressing full-length PhLP or PhLP-Q94-E283. In addition, the association of PI3Kγ to the plasma membrane in response to the overexpression of Gβγ was attenuated by PhLP-M1-G149, expressed in endothelial cells as a GFP-tagged protein with a CAAX box at the carboxyl terminus to restrict its expression to the membrane. Full-length PhLP or the carboxyl-terminal construct did not interfere with the recruitment of PI3Kγ to the membrane (Fig. 9B). In experiments using higher amounts of transfected Gβγ, PhLP-M1-G149 lost its ability to compete with PI3Kγ (not shown), further indicating that it recognizes the same interacting interface in Gβγ.

Intriguingly, our results showed that full-length PhLP did not interfere with Gβγ signaling. Because it has been recently reported that PhLP contributes to Gβ expression (31), we tested whether PhLP, PhLP-M1-G149, or PhLP-Q94-E283 could affect the expression of different Gβγ heterodimers. As shown in Fig. 10, with the exception of Gβ₅, the expression of all other Gβ subunits, in particular Gβ₁ and Gβ₂, increased in cells transfected with PhLP. Also, cells expressing PhLP-M1-G149 or PhLP-Q94-E283 showed a slight effect compared with the respective controls.

FIGURE 10.

Effect of PhLP, PhLP-M1-G149, or PhLP-Q94-E283 on the expression of different Gβγ heterodimers. Because it has been recently reported that PhLP acts as a chaperone contributing to the expression of Gβ (31), we tested if PhLP, PhLP-M1-G149, or PhLP-Q94-E283 affected the expression of different Gβγ heterodimers. The indicated FLAG-tagged Gβsubunits were cotransfected with Gγ₂ in HEK-293T cells, and their expression was detected in total cell lysates using FLAG antibodies that recognized this tag fused at the amino-terminal of Gβ. Untransfected cells or cells transfected with GST, and the different Gβγ heterodimers were used as negative controls. Total AKT was detected in the same lysates as a loading control.

Effect of PhLP-M1-G149 on the Activation of AKT and ERK in Response to the Overexpression of Different Gβγ Heterodimers

To test the effect of PhLP-M1-G149 on the action of different Gβγ heterodimers, COS7 cells were transfected with different Gβ subunits and Gγ₂. The results shown in Fig. 11A indicated that, by overexpression, only Gβ₁γ₂ and Gβ₂γ₂ were able to provoke a significant increase in the activation of both AKT and ERK. Thus, we tested the effect of PhLP-M1-G149 or full-length PhLP on the phosphorylation of AKT and ERK in response to Gβ₁γ₂ or Gβ₂γ₂ overexpression. As shown in Fig. 11 (B and C), PhLP-M1-G149 significantly attenuated the ability of Gβ₁γ₂ or Gβ₂γ₂ to promote the phosphorylation of AKT, whereas full-length PhLP showed just a, nonsignificant, slight attenuation on the effect of Gβγ overexpression on AKT phosphorylation. Neither construct showed a significant reduction on the activation of ERK in response to Gβγ overexpression.

FIGURE 11.

Effect of PhLP-M1-G149 on the activation of AKT and ERK in response to the overexpression of different Gβγ heterodimers. A, the effect of overexpressing different Gβγ heterodimers on the activation of AKT and ERK in COS7 cells was detected by Western blot using antibodies that recognized the phosphorylated forms of these kinases. The expression of total AKT and ERK was detected with antibodies that recognize these kinases regardless of their phosphorylation status. B and C, the effect of PhLP or PhLP-M1-G149 on the activation of AKT and ERK in response to Gβ₁γ₂ or Gβ₂γ₂ overexpression (those that were able to activate both AKT and ERK in the overexpression experiments shown in A) was determined by Western blot using phosphospecific antibodies as described in A. Cells transfected with EGFP and the indicated Gβγ heterodimers were used as controls. Bars represent the average results of eight independent experiments, vertical lines represent the ± S.E. (*, p < 0.05 respect to control).

DISCUSSION

Differential inhibitors of Gβγ could help to determine the cellular roles of different Gβγ effectors, thus providing the basis for future therapeutic strategies. Here, we demonstrate that PhLP-M1-G149, a construct corresponding to the amino-terminal region of PhLP1, has a differential effect in endothelial cells stimulated with S1P, attenuating the activation of AKT without affecting signaling to ERK, or the activation of both pathways in response to HGF. Both angiogenic factors known to activate AKT, ERK, and endothelial cell migration (41–45). The effect of PhLP-M1-G149 seems to be due to a blockade of Gβγ signaling by a direct interference with the effector interface of Gβγ involved in the activation of PI3Kγ. Accordingly, this construct interfered with the interaction between Gβγ and PI3Kγ and the recruitment of this phosphoinositide kinase to the membrane in response to Gβγ overexpression. This effect is diminished when the amount of transfected Gβγ increases, supporting the idea that the mechanism of inhibition is due to a competition for the effector interface. Considering these findings, an inherent limitation on the use of PhLP-M1-G149 as an inhibitor of Gβγ is the fact that this heterodimer may utilize additional mechanisms to activate AKT in a cell-specific fashion. These include an effect mediated by PI3Kβ (46), growth factor receptor transactivation (47, 48), and a recently described β-arrestin-dependent mechanism (49). In this regard, PhLP-M1-G149 prevents the activation of AKT in response to S1P in endothelial cells, but it has no activity in the response to this agonist in S1P1-transfected HEK293 cells (not shown), in which Gβγ mainly uses PI3Kβ to lead to AKT activation (46). A similar, cell-specific effect for a Gβγ inhibitor has been reported by Hamm's group showing the inhibitory potential of RACK1, a Gβγ partner that is unable to inhibit CXCR-2-dependent cell migration in HEK293 (50) but attenuates CXCR-4-dependent migration of Jurkat cells (51).

Our findings suggest that the effector interface of Gβγ linking to PI3Kγ lies at the region of Gβγ interacting with the amino-terminal part (amino acids M1-K102) of the PhLP-M1-G149 construct. This possibility comes from the different effects of PhLP-M1-G149 and PhLP-Q94-E283; both constructs share the amino acids Q94-G149, but they do not have the same inhibitory effects. According to the available structures of Phd-Gβγ (25, 32, 33) the residues of Gβ that interact with the amino-terminal region of Phd, within a distance of 3 Å, are Lys-57, Asn-230, Thr-274, and Asp-290. At this point, the hypothesis linking these residues to PI3Kγ activation is speculative. However, because the crystal structure of this phosphoinositide kinase in complex with Gβγ is not available, it will be interesting to explore this possibility in future studies. In agreement with these possibilities, Hamm's group recently published that RACK1 interferes in in vitro assays with the ability of Gβγ to activate PI3Kγ (51). The residues of Gβγ interacting with RACK1 are not known at the structural level, however, the ability of RACK1 to interact with both Gβ₁γ₂ and Gβ₅γ₂ (52) suggests that the interacting residues correspond to those shared between these Gβ subunits. In addition, because Gβ₅γ₂, contrary to the action of other heterodimers, is not able to activate PI3Kγ (53), it can be speculated that, although the RACK1-Gβ interface corresponds to residues shared between Gβ₁ and Gβ₅ (52), the effector interface linking Gβ₁ to PI3Kγ (putatively recognized by both RACK1 (51) and PhLP-M1-K102) corresponds to non-conserved residues. In this regard, it is interesting to notice that the amino acids of Gβ₁ that interact with PhLP-M1-K102 are either not conserved between Gβ₁ and Gβ₅ (Thr-274 and Asp-290) or located in a region of Gβ₅ in which the adjacent residues are not conserved (Lys-57 and Asn-230 are conserved in Gβ₅ but Ala-56, Ile-58, and Ala-231 are not). The putatively critical residues for the interaction between Gβγ and PI3Kγ are conserved in Gβ₁, Gβ₂, Gβ₃, and Gβ₄, all of them able to activate PI3Kγ (supplemental Fig. S2) (53).

Our results also indicate that the central region of PhLP (amino acids 94–149) is important to support a stable interaction between PhLP and Gβγ. Independent evidences pointing to this conclusion are derived from the characterization, in transfected cells, of the interaction between PhLP-derived constructs and Gβγ. The shortest PhLP-derived construct that shows an important interaction with Gβγ corresponds to this central region, which also competes in the interaction between PhLP-M1-G149 and Gβγ. In addition, according to the structure of the complex between Gβ₁γ₂ and Phd (25, 32, 33), Gβ₁(Ser-98) and Gβ₁(Trp-332) are residues involved in the interaction between Gβγ and the central region of phosducin. In agreement, we found that Gβ₁(S98A)γ₂ does not interact with PhLP and decreases its interaction with PhLP-M1-G149, whereas it interacts with PhLP-Q94-E283 as effectively as wild-type Gβ₁γ₂. These data also suggest that the PhLP-derived constructs have conformational adjustments that allow them to interact with Gβ mutants or isoforms with a different affinity compared with full-length PhLP. Another interesting difference regarding the interacting properties of PhLP-derived constructs is related to their ability to recognize different Gβγ heterodimers. Although both full-length PhLP and PhLP-M1-G149 interact with heterodimers containing Gβ₁, Gβ₂, Gβ₃, and Gβ₄, they lack the ability to interact with Gβ₅. In contrast, PhLP-Q94-E283 recognizes all the heterodimers, including Gβ₅γ₂. These data also suggest the existence of conformational differences between Gβ5 and other Gβ subunits, which, as mentioned, share the ability to activate PI3Kγ (53). In addition, the ability of PhLP-M1-G149 to interact with different Gβγ heterodimers containing the Gβ subunits known to be able to activate PI3Kγ (53), and interfere with signaling to AKT by Gβ₁γ₂ or Gβ₂γ₂, support the possibility that PhLP-M1-G149 can be used to characterize the signaling from different G protein-coupled receptors that utilize these different Gβγ heterodimers to activate the PI3Kγ/AKT signaling pathway.

Intriguingly, whereas PhLP-M1-G149 inhibits S1P signaling to AKT, full-length PhLP is not inhibitory. Based on our results, two alternative possibilities can be proposed. First, we observed that full-length PhLP has a positive effect contributing to the expression of different Gβγ heterodimers, in particular those containing Gβ₁ or Gβ₂, suggesting that cells that express full-length PhLP also express a higher content of Gβγ heterodimers that are then available to interact with PhLP but also with PI3Kγ and other effectors. Previous reports assigning an inhibitory role to phosducin and PhLP on Gβγ signaling were mainly based on in vitro assays or overexpression experiments (54). However, recent findings assign a positive role for PhLP in Gβγ function (31). In this scenario, Gβγ folding and heterodimer formation is positively influenced by PhLP (28, 55). Our results confirm these findings and extend the observations to different Gβγ heterodimers, suggesting that, excluding Gβ5, the expression of all other Gβ subunits is, to some extent, positively regulated by full-length PhLP. The second possibility that helps to explain why full-length PhLP is not inhibitory emerges from the observed differences in the interacting properties of full-length PhLP and PhLP-M1-G149 with Gβ mutants or different heterodimers. These differences can be interpreted as an indication of conformational differences that, in theory, can confer to the PhLP-M1-G149 construct a better ability to recognize and interfere with the Gβγ-PI3Kγ effector interface.

All together, our results indicate that PhLP-M1-G149 interacts with Gβγ thereby inhibiting its effector region involved in the activation of PI3Kγ/AKT, cell migration, and tubulogenesis, whereas the effector region leading to ERK activation remains available but does not participate in these S1P-dependent cellular events. In conclusion, PhLP-M1-G149 may represent a suitable tool to differentially modulate Gβγ in the pathway linking this heterodimer to AKT signaling, polarized cell migration, and in vitro angiogenesis.