G Protein βγ Subunits Regulate Cell Adhesion through Rap1a and Its Effector Radil

The activation of several G protein-coupled receptors is known to regulate the adhesive properties of cells in different contexts. Here, we reveal that Gβγ subunits of heterotrimeric G proteins regulate cell-matrix adhesiveness by activating Rap1a-dependent inside-out signals and integrin activation. We show that Gβγ subunits enter in a protein complex with activated Rap1a and its effector Radil and establish that this complex is required downstream of receptor stimulation for the activation of integrins and the positive modulation of cell-matrix adhesiveness. Moreover, we demonstrate that Gβγ and activated Rap1a promote the translocation of Radil to the plasma membrane at sites of cell-matrix contacts. These results add to the molecular understanding of how G protein-coupled receptors impinge on cell adhesion and suggest that the Gβγ·Rap1·Radil complex plays important roles in this process.

G protein-coupled receptors (GPCRs) 3 form the largest family of cell surface signal transducing molecules in vertebrates. The regulation of cell migration as well as cell-cell and cellmatrix adhesion by GPCRs is essential for normal embryonic development (1)(2)(3) and is important for several cellular processes in adults such as immune response (4 -6), hemostasis (7,8), and vascular integrity (3,9). The activation of chemokine receptors, for example, has been well demonstrated to regulate directional cell migration and cell-matrix adhesion in the context of various immune functions (10) but also during several other instances of cell motility (11,12). The activation of platelets at sites of vascular wall injury is another context where GPCR signaling is involved in regulating cell adhesion with the extracellular matrix (13). Revealing the importance of these processes, defects in GPCR signaling downstream of pro-migratory or adhesive signals have been linked with different human diseases including inflammation (14 -17) and cancer metastasis (18 -20).
Because pertussis toxin-sensitive G␣ i -activated pathways are thought to play important roles downstream of GPCRs during migration and adhesion, the G␤␥ subunits of the heterotrimeric G proteins can be predicted to play integral signaling functions during these processes. Supporting this, G␤␥ subunits are known to associate and potentiate the activity of the p110␤ and -␥ isotypes of phosphoinositide 3-kinase that regulate directed cell migration (21,22) by facilitating the polarized recruitment of downstream signaling proteins, which leads to cytoskeleton remodeling. The discovery that PLIC-1 interferes with chemokine receptor-mediated chemotaxis by sequestering G␤␥ and hindering its normal cellular functions as well as evidence implicating G␤␥ in cell invasion and spreading further highlights the importance of G␤␥ in cell movement (23,24). However, the precise molecular mechanisms by which GPCRs and G␤␥ impinge on cell motility remain incompletely understood especially with respect to the dynamic control of cell adhesiveness.
In all instances of cell motility, cells constantly need to alter their interactive associations with the extracellular matrix in response to environmental cues. Central to this process is the regulation of integrin avidity and affinity for matrix protein components (11,25,26). The small GTP binding protein Rap1 is implicated in the "inside-out" signaling that rapidly leads to integrin activation. For example, overexpression of the constitutively active mutant Rap1aQ63E leads to increased cell adhesion (27) and integrin activation (28) by inducing a change in the conformation of the extracellular region of integrins. This unclasping of the inhibitory interaction between the ␣ and ␤ integrins switches their affinity for the extracellular matrix to a high affinity state. Although Rap1induced inside-out signaling has been deemed important in the context of GPCR signaling during platelet and leukocyte activation (13, 29 -33), how this signaling and integrin activation are regulated by GPCRs and whether it extends to other cellular contexts is not well understood.
Despite significant indirect evidence positioning G␤␥ subunits as functional regulators of cell motility and adhesion downstream of GPCR activation, our understanding of the molecular mechanisms underlying their roles in these processes is incomplete. In the present study we, thus, employed a functional proteomic approach to further interrogate how G␤␥ subunits mediate their actions. We identify Rap1a and its effector Radil as novel G␤␥-associated proteins and present evidence functionally implicating this protein complex downstream of GPCR activation for the control of cell-matrix adhesion.
Tissue Culture and Transfection-HEK293T and HT1080 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (Sigma) and penicillin/streptomycin (Sigma) in a 37°C humidified incubator with 5% CO 2 . Stable cell lines were generated by calcium phosphate transfection followed by puromycin selection (2 g/ml). cDNA transfections in HT1080 cells were done using Lipofectamine 2000 (Invitrogen) at a 1:2 ratio (cDNA:Lipofectamine 2000) or polyethyleneimine at a 1:3 ratio at 70% cells confluency with a typical 80 -90% transfection efficiency. siRNAs were delivered at a final concentration of 50 nM in HT1080 cells by reverse transfection using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's protocol. The siGenome Non-Targeting siRNA (control siRNA) and human Radil ON-TARGET plus SMARTpool siRNA (hRadil siRNA pool) were purchased from Thermo Scientific, and the single siRNA targeting human Radil (hRadil siRNA #1) was purchased from Ambion Inc. The sequences for each siRNA in the ON-TARGET plus SMARTpool (Thermo Scientific) for human Radil are GCACAGGAACCCACUACAA, GAGCGU-ACUCUUCCCUUGA, CGUCAUGGCGGGAAGAAGA, and GAAGGUUUAUCCCGGAGGU. The sequence for human Radil siRNA #1 (Ambion) is GGUUUAUCCCGGAGGUUUG. The sequence for siGENOME Non-Targeting siRNA #4 (Thermo Scientific) is AUGAACGUGAAUUGCUCAAUU.
Production of Radil Antibody-A rabbit polyclonal antibody was raised against full-length recombinant human Radil protein. Human Radil coding sequence was cloned into pGEX vector (Promega) downstream of GST. Proteins were expressed in BL-21 Escherichia coli strain upon stimulation with isopropyl 1-thio-␤-D-galactopyranoside (500 M; Sigma) in a shaker at room temperature (240 rpm) overnight. The bacterial pellet was lysed by sonication in buffer containing 20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and proteins purified on Glutathione-Sepharose 4B (GE Healthcare). The purity of the purified Radil protein was confirmed by SDS-PAGE and mass spectrometry analysis and provided to Covance Custom Immunology Services for antibody production. The final antisera was purified first by negative selection by running the crude antisera through GST proteins immobilized on a glutathione-Sepharose 4B column followed by affinity purification on a GST-Radilglutathione-Sepharose 4B column. The antibody was characterized for antigen specificity by enzyme-linked immunosorbent assay (Covance Custom Immunology Services) and by Western blotting lysates from Radil siRNA-treated cells.
Tandem Affinity Purification and Mass Spectrometry-HEK293T cells (2 ϫ 10 8 ) expressing tandem affinity purification (TAP)-tagged G␤ 2 , G␥ 2 , or Radil constructs were used for the TAP procedure as described previously (34). Briefly, cells were lysed and solubilized in TAP lysis buffer (0.1% Igepal CA630, 10% glycerol, 50 mM Hepes-NaOH, pH 8.0, 150 mM NaCl, 2 mM EDTA, 2 mM dithiothreitol, 10 mM NaF, 0.25 mM NaOVO 3 , 50 mM ␤-glycerophosphate, and protease inhibitor mixture (Calbiochem)), and extracts were incubated at 4°C with 100 l of packed streptavidin (GE Healthcare) or ␣-FLAG M2 (Sigma) resin. The resin was washed, and protein complexes were then eluted from the streptavidin resin in calmodulin binding buffer (50 mM Hepes-NaOH, pH 8.0, 150 mM NaCl, 10 mM ␤-mercaptoethanol, 1 mM magnesium acetate, 1 mM imidazole, 0.1% Nonidet P-40) supplemented with 2 mM biotin (Sigma). The second round of affinity purification was performed using 100 l of calmodulin resin (GE Healthcare) followed by elution in calmodulin elution buffer (25 mM EGTA, 50 mM ammonium bicarbonate). ␣-FLAG immunoprecipitation for mass spectrometry analysis-purified proteins were eluted from the beads using 500 mM ammonium hydroxide at pH 11.0. The proteins in the complex were reduced in 25 mM dithiothreitol and alkylated using 100 mM iodoacetamide (Sigma) and brought to 1 mM CaCl 2 . The proteins were then directly digested with sequencing-grade trypsin (Promega). The resulting peptide mixture was then analyzed by liquid chromatography-tandem mass spectrometry using a LTQ-XL Linear Ion Trap Mass spectrometer (Thermo Scientific). The acquired tandem mass spectra were searched against a FASTA file containing the human NCBI sequences using a normalized implementation of SEQUEST running on the Sorcerer platform (Sage-N Research). The resulting peptide identifications returned by SEQUEST were filtered and assembled into protein identifications using peptide and protein prophets (Institute of Systems Biology, Seattle).
Affinity Purification, Immunoprecipitation, and Western Blot-Cells were lysed (0.5% Igepal CA630, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, and protease inhibitor) and incubated at 4°C for 20 min to solubilize the proteins. Affinity purification and immunoprecipitations were performed using streptavidin resin (GE Healthcare) or FLAG-M2 beads (Sigma) for 1 h at 4°C. After extensive washes with lysis buffer, the beads were eluted with 2ϫ Laemmli sample buffer and heated at 95°C for 5 min in the presence of ␤-mercaptoethanol (Sigma). Whole cell lysates or purified protein samples were resolved by SDS-PAGE and transferred onto nitrocellulose transfer membranes (Pall Corp.). Western blotting was performed with antibodies as indicated in the figure legends (Figs. 1-3, 6, and 7) followed by chemiluminescent detection with SuperSignal West Pico (Thermo Scientific) and exposed on film.
Immunocytochemistry and Confocal Microscopy-HEK293T and HT1080 stably expressing Venus-Radil were transfected with HA-Rap1a, HA-Rap1aG12V, HA-Rap1aQ63E, or FLAG-G␤ 2 and G␥ 2 as indicated. For intracellular protein staining, cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) supplemented with 10% normal donkey serum. Samples were then immunostained with ␣-HA (HA.11, Covance) or ␣-FLAG (Sigma)-specific mouse monoclonal antibodies in PBS supplemented with 1% normal donkey serum. Cells were subsequently labeled with secondary goat-anti-mouse antibody conjugated to Alexa Fluor 594 (Invitrogen) to visualize HA-tagged Rap1a or FLAG-tagged G␤ 2 . The coverslips were mounted onto slides using Vectashield mounting media (Vector Laboratories). Cells were visualized, and images were acquired with a Carl Zeiss LSM510 confocal microscope using a Plan-Apochromat 63ϫ/ 1.4 oil immersion objective. Lasers at a 488-and 543-nm wavelength were fired independently using the multi-track function of LSM510. Uncompressed images were processed using Zeiss LSM Image browser Version 4.2. To magnify portions of the plasma membrane to depict co-localization, images were cropped as designated in Fig. 3A (in white boxes) using Adobe Photoshop CS3. For fMLP stimulation experiments, HT1080 cells stably expressing Venus-Radil were serum-starved for 24 h and stimulated with fMLP (1 M) for the indicated times at 37°C. After treatments, cells were immediately chilled on ice and washed once with ice-cold PBS followed by fixation in 4% paraformaldehyde for 30 min at room temperature.
Subcellular Fractionation-HT1080 cells transiently transfected with the indicated cDNAs in 100-mm tissue culture dishes were washed with PBS, pH 7.4, and lysed by Dounce homogenization (20 strokes) in hypotonic buffer (5 mM Tris-HCl, 2 mM EDTA). A small fraction was separated into aliquots from each sample and labeled as "inputs." The lysates were centrifuged at 1000 ϫ g for 5 min at 4°C to remove unbroken cells and nuclei. The post-nuclear supernatant was transferred to a new tube followed by centrifugation at 100,000 ϫ g (34,100 RPM in Ti90 rotor) for 30 min. The supernatant was removed, and the pellet was resuspended in the above buffer and subjected to a second round of centrifugation at 100,000 ϫ g for 30 min. The supernatant was discarded, and the pellet containing the membrane fraction was resuspended in the above lysis buffer. Equal amounts of proteins for each the input and membrane fractions were then resolved by SDS-PAGE for Western blot analysis. An antibody against Na-K-ATPase was used as internal loading control.
Cell Spreading Assay-HT1080 cells were plated on glass coverslips and transfected with GFP alone, Venus-Rap1GAP, or GFP together with different expression plasmids coding for HA-Rap1aQ63E, HA-Radil, or HA-G␤ and untagged G␥. 48 h after transfections, cells were fixed in 4% paraformaldehyde and immunostained with ␣-HA monoclonal antibody to label the overexpressed proteins followed by secondary detection with goat-anti-mouse conjugated Alexa 594. Cells were mounted on slides and visualized under a 63ϫ oil immersion lens using a Carl Zeiss LSM 510 confocal microscope. Spreading of cells co-expressing GFP and the overexpressed protein of interest was quantified by measuring the area of each cells using Image J software. To acquire more quantitative and real-time insights into cell adhesion and spreading, we employed a change in impedance as a measure of the degree of cell attachment and spreading using the xCELLigence system (Roche Applied Science). HT1080 cells were transfected with the different plasmids. 48 h after transfections, cells were trypsinized and resuspended in DMEM containing 10% fetal bovine serum. 25,000 cells from each condition were seeded onto a 96-well microtiter xCELLigence assay plate (E-Plate) (ACEA Biosciences Inc.) and placed on the Real-time xCELLigence Cell Analyzer (Roche Applied Science) platform at 37°C to measure the "cell index" every 5 min. The cell index unit is defined as (R n Ϫ R b )/15, where R n is the cell electrode impedance of the well when it contains cells, and R b is the background impedance of the well with the media alone.
Cell Adhesion Assay-96-well flat-bottom plates (BD Biosciences) were coated with 5 g/ml fibronectin from bovine plasma (Sigma) for 16 h at 4°C. Wells were washed and blocked with a solution of 2% filtered bovine serum albumin in PBS for 1 h at room temperature. 48 h after transfection with cDNA or siRNAs, HT1080 cells were dissociated from culture plates with trypsin, washed twice with PBS, and resuspended in serum free DMEM supplemented with 0.1% bovine serum albumin and 20 mM HEPES, pH 7.4. Before the adhesion assay the cells were incubated at room temperature for 1 h on a nutating shaker. Where indicated, cells were incubated at 37°C for 1 h with the indicated drugs. For pertussis toxin (PTX) treatment, cells were incubated overnight in 10% fetal bovine serum, DMEM media containing 100 ng/ml PTX. Cells were plated in triplicate at a density of 2.5 ϫ 10 4 per well and allowed to adhere for 8 min at 37°C followed by washing with warm 0.2% bovine serum albumin in PBS. For each condition, input controls were let to adhere for 30 min followed by aspiration of the media and fixation. Cells were fixed with glutaraldehyde (Sigma) for 20 min and stained with hematoxylin Gill's No.1 reagent (Sigma) followed by washing with acid alcohol (0.5% hydrochloric acid in 70% ethanol) to remove residual the stain and 0.04% ammonium hydroxide for color maturation. Cell adhesion was quantified by taking a minimum of three random pictures per well. Cells in each picture were then counted using an automated macro algorithm created in ImageJ Version 1.41 software.
Integrin Activation Assay-HT1080 cells were resuspended as described for adhesion assay and let to recover at 37°C for 45 min. Transfected or drug-treated cells were incubated for 1 h in the presence of ␣-CD29 (9EG7) antibody (BD Biosciences). Cells were washed in PBS three times and incubated in the presence of Dylight TM -488-conjugated donkey ␣-rat IgG (Jackson ImmunoResearch Laboratories) for 45 min followed by three washes in PBS. Cells were then fixed in 4% paraformaldehyde for 15 min and resuspended in PBS. Flow cytometry analysis was performed on a Beckman Coulter Cytomics FC500 MPL analyzer using the CXP 2.2 software. Post acquisition data analysis was done with FlowJo Version 7.2.5 software (Tree Star Inc).
Rap Activation Assay-HT1080 cells were serum-starved for 16 h followed by stimulation with 100 nM fMLP for different time points as indicated. After three washes with PBS, the cells were lysed in TAP lysis buffer containing protease and phosphatase inhibitors (10 mM NaF, 0.25 mM NaOVO 3 , 50 mM ␤-glycerophosphate). 800 g of proteins was then incubated with GST-RalGDS-RBD coupled to glutathione-Sepharose 4B beads for 1 h at 4°C. Beads were then washed 3 times in TAP lysis buffer, bound proteins were eluted in 15 l of 2ϫ Laemmli buffer containing ␤-mercaptoethanol (Sigma), and samples were analyzed by Western blot analysis using ␣-Rap1a antibodies. A fraction of whole cell lysate was also analyzed by Western blot to assess total Rap1a.
Statistical Analysis-Data reported as ϮS.E. were analyzed by analysis of variance (ANOVA) followed by Tukey's post hoc multiple comparison tests (Graphpad Prism 5 software). Statistical significance was measured at p Ͻ 0.05 for all analysis.

Proteomic Identification of Radil and Rap1a as Novel Effectors of G␤␥ Subunits of Heterotrimeric G Proteins-
To identify novel effectors of G␤␥ subunits of heterotrimeric G proteins, G␤ 2 and G␥ 2 cDNAs were cloned downstream of streptavidin (Strep)-and calmodulin-binding peptides in the pGLUE vector (35), and stable human embryonic kidney (HEK293T) cells expressing low levels of G␤ or G␥ fusion proteins were derived. Extracts from these cells were subjected to tandem affinity chromatography to isolate G␤ 2 and G␥ 2 protein complexes. These protein complexes were digested in solution with trypsin, and the resulting peptide mixture was analyzed by liquid chromatography-tandem mass spectrometry. The detailed list of proteins identified in the complexes is listed in the supplemental table and is schematically represented in Fig. 1A. Many peptides corresponding to several G␣ subunits were predictably found in both G␤-and G␥-purified complexes. Furthermore, most G␥ subunits and G␤ subunits were found to associate with G␤ 2 and G␥ 2 , respectively. Based on the total number of peptides identified and derived sequence coverage, the data also support previous reports that DnaJ (36,37), the t-complex polypeptide (TCP) complex (38), and phosducin-like proteins (39) associate with either fully formed or nascent G␤␥ subunits.
In terms of the total peptides identified, the recently identified Rap effector Radil (40) was one of the most abundant proteins found in both G␤ 2 and G␥ 2 protein complexes. Of note, a few peptides attributed to Rap1a were also present in the G␤ 2 pulldown experiment ( Fig. 1A and the supplemental table). The reciprocal analysis of Radil protein complexes by mass spectrometry agreed with the previous showing that Radil interacts with the Rap family of small GTPases (40) and confirmed the association of Radil with G␤␥ heterodimers as several peptides corresponding to the different Rap isoforms as well as G␤ and G␥ subunits were identified ( Fig. 1A and the supplemental  table). The Radil affinity purification/mass spectrometry experiments were performed in HEK293T and HT1080 fibrosarcoma cells with similar results (supplemental table).
Co-affinity purification and Western blot experiments were then performed to support and validate the mass spectrometry data. Lysates from control HEK293T cells or from cells stably expressing Strep-HA-G␥ 2 or Strep-HA-Radil were subjected to affinity purification using Sepharose-streptavidin followed by Western blotting using antibodies specific to Radil, HA, G␤, or Rap1. Confirming the mass spectrometry results, endogenous G␤ and Rap1 were detected in purified Radil complexes (Fig.  1B, left panel), and endogenous Radil and G␤ proteins were detected in G␥ 2 samples (Fig. 1B, right panel).
Radil is a protein of 1075 amino acids composed of a RA (Ras-associating) domain known to bind small GTP-binding proteins of the Ras family, a central DIL (Diluted) domain of unknown function, and a PDZ domain at the C terminus (Fig.  1C). To test for specificity of the association between Radil and G␤␥, the interaction between AF6 and G␤␥ was addressed (Fig.  1C). AF6 is a protein related to Radil sharing its domain architecture and has been similarly reported to interact with Rap1a and play important roles in cell adhesion (41)(42)(43). Whereas endogenous G␤ could be co-immunoprecipitated with Radil, it was absent from AF6 or control immunoprecipitates (Fig. 1C, compare lane 6 with lanes 4 and 5). We conclude that the Rap effector Radil is a novel G␤␥-associated protein.
Radil Recruits G␤␥ and Activated Rap1a in the Same Protein Complex-Because only a few peptides for Rap1a were identified in the G␤ and G␥ pulldown assays and several hundred in Radil complexes, the interaction between G␤ and Rap1 is likely indirect and mediated by Radil. To examine this possibility, G␤ 2 was purified from cells exogenously expressing Rap1 alone or from cells overexpressing both Rap1 and Radil. Whereas trace amounts of Rap1 were co-purified with G␤ 2 in cells expressing these proteins, the overexpression of Radil strongly promoted the association of Rap1 with G␤ 2 (Fig. 2A, compare lanes 2 and 3). Radil was previously shown to selectively interact with the activated GTPbound form of Rap1 and exhibit negligible affinity for its inactive GDP-bound form (40). To determine whether the activation state of Rap1 is important for the binding of G␤ to Radil, we first established that overexpression of Rap1GAP, a GTPase-activating protein (GAP) specific for Rap1 (44,45), could block the association of Radil with Rap1a (Fig. 2B, compare lanes 2 and 3). Similarly, whereas G␤ is efficiently co-purified with Radil in control cells, this association is markedly attenuated in Rap1GAP-overexpressing cells (Fig. 2B). We also performed the reciprocal experiment and showed that expression of the constitutively activated Rap1aQ63E promoted the association of G␤␥ with Radil (Fig. 2C). We conclude that after Rap1 activation, Radil interacts with Rap1 and can recruit G␤␥ into the complex.
G␤␥ and Rap1 Promote the Recruitment of Radil at Sites of Cell-Matrix Adhesion-The activation of several GPCRs has previously been shown to activate Rap1a in multiple contexts (29, 46 -51). Our results describing G␤␥ forming a protein complex with Rap1a, and its effector Radil is consistent with G␤␥ participating in Rap1a-dependent processes. Upon activation, Rap1 translocates from a cytoplasmic pool to the plasma membrane (52,53). Rap effectors are subsequently recruited to this activated membrane pool of Rap1 to in turn exert their biological functions (54,55). To test if the subcellular localization of Radil changes upon Rap1 activation and G␤␥ overex-pression, we performed subcellular localization microscopy experiments in HT1080 fibrosarcoma cells stably expressing a Venus-Radil fusion protein. We chose this model system because Radil expression is known to be induced after an oncogenic translocation in a pediatric sarcoma (40), and its func-tions may be particularly relevant in this cellular context. In the resting state, Radil appears to be mainly localized in a cytoplasmic pool with minimal plasma membrane localization (Fig. 3A,  panel c). In contrast, overexpression of wild-type Rap1a (Fig.  3A, panels e-g) or two different constitutively active Rap1a mutants ( Fig. 3A panels, h-m) led to the translocation of Radil to the plasma membrane with extensive co-localization with the activated pool of Rap1a. Note that although expressing wild-type Rap1a was sufficient to translocate Radil, this effect was significantly stronger with the constitutively active Rap1a mutants (data not shown). These results support previous data   FEBRUARY 26, 2010 • VOLUME 285 • NUMBER 9

The G␤␥⅐Rap1⅐Radil Complex Regulates Cell Adhesion
that Radil forms a complex with the GTP-bound form of Rap1a (Fig. 2B) (40). Interestingly, at the plasma membrane, Radil appears to be enriched in both lamellipodia and filopodia (Fig.  3A, insets) a subcellular localization compatible with a role in cell-matrix interactions and cell migration. We next asked if overexpression of G␤␥ impacts on the localization of Radil. G␤␥ is predominantly localized to the plasma membrane in these cells (Fig. 3A, panel o), and overexpression of G␤ 2 and G␥ 2 phenocopied Rap1a expression in that it resulted in translocation of Radil to the plasma membrane (Fig. 3A, panels n-p).
As control, we showed that overexpression of activated Rap1aQ63E did not lead to the membrane enrichment of wildtype GFP, which localizes to the cytosol and nucleus of these cells (supplemental Fig. 1). Also, we demonstrated that overex-

FIGURE 3. Rap1a-GTP and G␤␥ promote the translocation of Radil at cell-matrix contacts in HT1080 cells. A, shown is localization of GFP (a and b),
Venus-Radil (c and d), or Venus-Radil co-expressed with wild-type HA-Rap1 (e-g), HA-Rap1aQ63E (h-j), HA-Rap1G12V (k-m), or FLAG-G␤ 2 /G␥1 (n-p). HA-and FLAG-tagged Rap1a or G␤ 2 ϩ G␥ (untagged) were transiently transfected in HT1080 cells stably expressing Venus-Radil as indicated. Cells were fixed followed by immunodetection with ␣-HA (e-m) or ␣-FLAG (n-p) monoclonal antibody followed by secondary detection with goat-anti-mouse conjugated to Alexa 594 antibody. Cells were visualized using a Zeiss LSM 510 confocal microscope under 63ϫ oil immersion objective. White arrows depict plasma-membrane co-localization. White boxes indicate magnified sections cropped in Adobe Photoshop CS3. Images shown are representative of 10 -15 cells analyzed in three independent experiments. Bars, 50 m. B, membrane fractions show enrichment of Venus-Radil when Rap1aQ63E and G␤␥ are expressed. HT1080 cells stably expressing Venus-Radil were transiently transfected to express the indicated proteins. Cells were lysed in hypotonic buffer and subjected to subcellular fractionation as described under "Experimental Procedures." Membrane fractions and the inputs were analyzed by Western blot (WB) to determine the amount of Venus-Radil present in the membrane pool when HA-Rap1aQ63E or FLAG-G␤ 2 was co-expressed. The blot for the membrane fractions was stripped and re-probed using ␣-Na-K-ATPase antibody to provide for an internal loading control.
pression of an active form of Rac1, which is a Rho family GTPase with described roles in cell migration and spreading (56,57), did not promote the plasma membrane recruitment of Radil (supplemental Fig. 1). To quantitatively support our immunofluorescence data, we performed biochemical fractionation experiments. Although a modest amount of Venus-Radil was found in the membrane pool under resting conditions, expression of Rap1aQ63E and G␤␥ markedly promoted the association of Radil with the membrane fractions (Fig. 3B, compare lanes 2 and 3 with lane 1). We, thus, conclude that after G␤␥ and Rap1a activation, the Rap effector Radil is translocated to the plasma membrane at sites of cell-matrix contacts likely through the formation of the G␤␥⅐Rap1⅐Radil protein complex.
The G␤␥⅐Rap1⅐Radil Complex Regulates Cell Spreading and Cell-Matrix Adhesiveness-Research in the last 10 years has established Rap1a as an important factor in the regulation of cell-matrix and cell-cell adhesion (58 -60). Rap1a activation has been implicated in inside-out signaling leading to the activation of integrins and increases in cell-matrix adhesion (42, 58 -61). Radil was recently described as a Rap effector with a role in the control of cell-matrix adhesion (40). Consistent with these notions, we noted that overexpression of WT or constitutively active Rap1a or Radil led to increased cell spreading (Fig. 4, A and B), a phenotype commonly associated with increased cell-matrix adhesiveness (55). Interestingly, the overexpression of G␤␥ also led to an increased spreading of these cells (Fig. 4, A and B). To quantitatively monitor the effects of Rap1a, Radil, or G␤␥ overexpression on the spreading of HT1080 cells in real time, we employed the xCELLigence impedance system (62). This system provides a cell index that increases as a function of cell attachment, spreading, and cell growth. As Fig. 4C shows, the overexpression of Rap1WT, G␤␥, Radil, and Rap1aQ63E led to progressively higher cell indices compared with control-transfected cells over the first 2 h. We conclude that the induced formation of the G␤␥⅐Rap1⅐Radil complex increases the spreading of HT1080 cells.
These results led us to test the hypothesis that the G␤␥⅐ Rap1a⅐Radil protein complex may represent a cellular machinery regulating cell adhesion downstream of GPCR activity. To test this we optimized a cell-matrix adhesion assay to measure the adhesion of HT1080 cells to the extracellular matrix protein fibronectin. In this assay, overexpression of Rap1aQ63E predictably led (as it promotes inside-out integrin signaling) to a strong increase in the adhesive properties of these cells (Fig. 5A, quantified in Fig. 5B). Inhibiting Rap1a activation by expressing Rap1GAP on the other hand did not lead to any increase in cell adhesion.
After having determined the dynamic range of this assay, we tested whether the overexpression of the other members of the G␤␥⅐Rap1a⅐Radil complex was sufficient to promote adhesion of these cells. Both Radil or G␤␥ overexpression led to a strong potentiation of cell-matrix adhesion (Fig. 5, A and B) to levels similar to that observed with Rap1aQ63E expression (Fig. 5, A  and B). Consistent with the interaction data between G␤␥, Radil, and Rap1a (Fig. 2B), the increase in adhesion promoted by Radil or G␤␥ expression depended on Rap1a activity. Indeed, coexpression of Rap1GAP strongly inhibited both G␤␥-or Radil-promoted cell-matrix adhesion (Fig. 5, A and B). We conclude that the G␤␥⅐Rap1a⅐Radil protein complex is sufficient to promote cell-matrix adhesiveness. The bottom panels demonstrate the total cells input for each conditions. B, shown is a quantified representation of the adhesion assay as -fold stimulation compared with control. C, shown are representative pictures comparing the ability of ⌬RA and ⌬PDZ Radil mutants to promote HT1080 cells adhesion. D, shown is a quantified representation of C. The inset shows expression of the different Radil proteins in one representative experiment. Each experiment was done in triplicate, and several pictures from random fields were taken from each well of 96-well plate. Cells were counted (using an automated cell counting macro created in ImageJ software) from each field and expressed as -fold of control from each experiment. The results from three independent experiments were pooled for the quantification. Each dot on the graph represents cell counts from a single field. Error bars, ϮS.E. Statistics are one-way ANOVA followed by Tukey's multiple comparison test; p Ͻ 0.05. Asterisks indicate statistical significance compared with control.
As stated previously, Radil is composed of RA, DIL, and PDZ domains. We next studied the individual requirement of the RA and PDZ domains of Radil for their ability to promote cellmatrix adhesion. Whereas overexpression of full-length Radil led to a robust increase in adhesion (Fig. 5, C and D), equivalent expression (Fig. 5D, inset) of ⌬RA or ⌬PDZ mutant Radil proteins had no effect. Because the RA domain is known to interact with GTP-bound Rap1a, these results further support the requirement for the Rap1a-Radil interaction for this process and indicate that the PDZ domain is also required possibly through the interaction of an as yet unidentified PDZ ligandcontaining effector protein.
GPCR Activation Leads to Rap1 Activation, Radil Translocation to the Plasma Membrane, and Increased Cell-Matrix Adhesion-Having established that the formation of the G␤␥⅐ Rap1a⅐Radil protein complex was sufficient to promote cellmatrix adhesion, we next wanted to assess its requirement in the context of a GPCR known to modulate cell adhesion. A previous report described that HT1080 cells endogenously express functional formyl peptide receptors (FPRs) and that their activation with the formylated peptide fMLP strongly potentiates cell matrix adhesion (63). We first repeated this experiment and showed that fMLP treatment of these cells also caused an ϳ5-fold increase in adhesion using our experimental conditions (Fig. 6A). We next asked whether fMLP-promoted cell adhesion requires active Rap1a. To do this we overexpressed Rap1GAP and asked whether fMLP could still potentiate adhesion. As shown in Fig. 6A, expression of Rap1GAP strongly antagonized the ability of fMLP to induce cell adhesion. We conclude that FPRs utilize a Rap1a-dependent pathway to regulate cell adhesion to the fibronectin matrix. Supporting this, fMLP treatment of these cells led to a rapid and robust activation of Rap1a (Fig. 6B). This activation was sustained, peaked at 2 min, and slowly waned over time. Learning from the localization experiments (Fig. 3) that activated Rap1 and G␤␥ translocates Radil to the plasma membrane, we tested the prediction that fMLP treatment of the cells would also lead to a translocation of Radil to the plasma membrane. As shown  FEBRUARY 26, 2010 • VOLUME 285 • NUMBER 9

The G␤␥⅐Rap1⅐Radil Complex Regulates Cell Adhesion
in Fig. 6C, Venus-Radil was enriched at the plasma membrane within 2-3 min after FPR activation, a kinetic that mirrored Rap1a activation. We conclude that FRP activation by fMLP leads to Rap1a activation and to the recruitment of Radil to the plasma membrane. The G␤␥⅐Rap1a⅐Radil Signaling Axis Regulates Adhesion Downstream of GPCR Activation-Our next goal was to determine whether Radil was required downstream of FPR activation for the control of cell adhesion using a RNAi loss of function approach. We first validated the efficiency of a pool of four individual siRNA sequences targeting Radil to inhibit the expression of Venus-Radil (Fig. 7A, left blot compare lanes 2 and 1) and more importantly to target the levels of endogenous Radil proteins in HT1080 cells (Fig. 7A, right blot). Using these reagents we then assessed the ability of fMLP to promote cellmatrix adhesion when Radil levels are reduced. Whereas control and Radil siRNA-treated cells exhibited background levels of adhesion on their own, Radil siRNAs strongly inhibited the ability of fMLP to promote adhesion when compared with control siRNA-treated cells (Fig. 7B). Importantly, we also screened several individual Radil siRNAs and found one (Radil siRNA#1) that effectively targeted Radil expression (Fig. 7A, compare lanes 3 and 1). Similar results were obtained using this single siRNA in the functional adhesion assay (Fig. 7B). To evaluate the implication of G␣ i -containing heterotrimeric G proteins for the fMLP-promoted increase in adhesion, we pretreated the cells with 100 ng/ml of PTX for 16 h, a concentration known to inactivate G␣ i . Although fMLP induced the expected 5-fold increase in controltreated cells, PTX completely inhibited fMLP-mediated cell adhesion (Fig. 7C). We conclude from these results that a G␣ i -containing heterotrimeric G protein and Radil are required downstream of FPRs for the control of cell-matrix adhesion.
The G␤␥⅐Rap1a⅐Radil Complex Regulates Inside-out Signaling Leading to Integrin Activation-We were then interested in addressing the mechanism by which GPCR activation and the G␤␥⅐Rap1a⅐Radil complex modulate cell adhesion. Cellmatrix adhesiveness is a dynamic process regulated through the interaction of integrin receptors with extracellular matrix components. Both integrin availability and affinity for their ligands can be regulated. The latter has been shown to be controlled in part by signaling through the small GTPase Rap1a in a process known as inside-out signaling (64 -66). HT1080 cells are known to express several integrins including the ␣5␤1 subtypes that form the receptor for fibronectin. Because GPCRs have previously been shown to regulate integrin affinity (30,67,68), we investigated whether the G␤␥⅐ Radil⅐Rap1a protein complex regulates cell-matrix adhesion through inside-out signaling and integrin activation. To perform this experiment we took advantage of a conformationally sensitive monoclonal antibody (9EG7) that selectively recognizes ␤1-integrin when activated (69,70) and initially asked whether Rap1aQ63E, Radil, or G␤␥ overexpression or fMLP treatment led to integrin activation. When expressed in HT1080 cells, these proteins increased cell spreading (Fig.  4) and adhesion (Fig. 5). After expression of these proteins or treatment of the cells with fMLP, a substantial increase in ␤1-integrin activation was measured (Fig. 8, A and B) to levels approaching 50 -60% that obtained after treatment of the cells with the divalent cation Mn 2ϩ used as a positive control to maximally activate integrins. Importantly and consistent with the requirement of activated Rap1a for the FIGURE 7. Radil is required for the fMLP-promoted HT1080 cell adhesion on fibronectin matrix. A, shown is efficiency of Radil knockdown. HT1080 cells stably expressing Venus-Radil were treated with control, a pool of four Radil siRNAs (hRadil siRNA pool), or a single Radil siRNA (hRadil siRNA #1) (left panel), and Radil expression was monitored by Western blot (IP) using GFP antibodies. Immunoblotting with total Erk1/2 antibodies was performed as loading controls. Endogenous hRadil knockdown efficiency in HT1080 cells using the hRadil siRNA pool is shown in the right panel. Cells transfected with control or hRadil siRNA pool were lysed, and equivalent amounts of lysates were immunoprecipitated (IP) using ␣-Radil rabbit polyclonal antibodies followed by detection in Western blot using the same antibody. B, shown is fMLP-promoted cell adhesion in HT1080 cells treated with control or hRadil siRNAs (n ϭ 4). Each dot on the graph represents counts from a single field. The asterisk indicates statistical significance compared with control treatment. C, treatment of HT1080 cells with PTX (100 ng/ml) inhibits fMLP-mediated cell adhesion. Each experiment was done in triplicate, and several pictures from random fields were taken from each well of a 96-well plate. Cells were counted from each field and expressed as -fold of control from each experiment. Data from all experiments were pooled. Error bars, ϮS.E. Statistical significance was assessed using one-way ANOVA followed by post-hoc analysis using Tukey's multiple comparison test; p Ͻ 0.05.
G␤␥⅐Rap1a⅐Radil complex assembly and function, the overexpression of Rap1GAP inhibited integrin activation in each case (Fig. 8, A and B).
To address the requirement for Radil in fMLP-induced integrin activation, we treated HT1080 cells with control or two independent Radil siRNAs and compared integrin activation patterns upon fMLP stimulation. Although fMLP promoted integrin activation by ϳ3-fold in control siRNA-treated cells, depletion of Radil severely impaired the ability of fMLP to activate integrins (Fig. 8, C and D) but did not affect the Mn 2ϩ response (data not shown). These findings support the view that the G␤␥⅐Radil⅐Rap1a complex controls cell adhesiveness downstream of GPCR activation by regulating inside-out signaling leading to integrin activation.

DISCUSSION
Several GPCRs have been implicated in the control of cell motility and adhesion. However, the intracellular signaling pathways underlying these functions remain poorly defined. We present here the functional characterization of a novel protein complex functioning downstream of GPCR signaling to regulate cell-matrix adhesion. We have identified Radil, a Rap1 effector, as a multidomain protein linking GPCRs, G␤␥, and Rap1-GTP to integrin activation. First, G␤␥ or Radil overex-  FEBRUARY 26, 2010 • VOLUME 285 • NUMBER 9

The G␤␥⅐Rap1⅐Radil Complex Regulates Cell Adhesion
pression is sufficient to activate mesenchymal cell spreading and adhesion to fibronectin and to promote the active conformation of integrins. Second, G␤␥ and active Rap1a promote the localization of Radil to sites of cell-matrix adhesion within the plasma membrane. Third, the stimulation of cell adhesion by fMLP receptors was abrogated in Radil knockdown cells and when Rap1a function was inhibited with the overexpression of Rap1GAP. Our results, thus, unveil a new signaling axis downstream of Rap1 activation by GPCRs and implicate a function for the G␤␥⅐Rap1a⅐Radil protein complex in the inside-out signaling cascade leading to integrin activation.
Other Rap1 effectors have previously been identified and shown to be important in regulating cell adhesion. RAPL, which is enriched in lymphoid tissues, has been shown to be important for the polarized relocalization of LFA1, the major leukocyte integrin (33), to the leading edge. Another effector important for T cell functions is RIAM, which regulates adhesion by the modulation of actin dynamics through its interaction with Profilin and Ena/Vasp proteins (28). Similar to Radil, overexpression of RAPL or RIAM also leads to enhanced adhesion and integrin activation (29,33). Additional studies will be needed to understand how Rap1 selectively activates different effectors in different contexts. One possibility is that the expression of the different Rap1 effectors is restricted to specific cell types. Supporting this, RAPL and RIAM are expressed highly in lymphoid cells but are either absent or expressed at low levels in other cell types (28,33). Although Radil is expressed in all epithelial and fibroblast cell lines that we examined (data not shown) and is broadly expressed in zebrafish embryos (40), we do not know its level of expression in lymphocytes and whether it is functionally redundant with RAPL or RIAM in this setting. Alternatively, the recruitment of Rap1 and one of its effectors within a protein complex could account for the specificity of action. GPCR-activated G␤␥ subunits in this design could serve to insulate the activation of Radil from the other Rap1 effectors after Rap1 activation. Supporting this possibility, we found that G␤␥ interacts with Radil but not with its close homologue AF-6 ( Fig. 1C).
Because Rap1 regulates cell-cell and cell-matrix adhesion in different contexts, its role in tumorigenesis and cancer progression has been closely scrutinized (71)(72)(73)(74). Recent findings suggest a role for aberrant Rap1 activity in prostate cancer progression, as activation of Rap1 increased prostate cancer cell migration and invasion, and its inhibition had the opposite effect (75). Interestingly, in the same study integrins were shown to be involved in the mechanism of Rap1-mediated migration and invasion of prostate cancer cells. Further supporting a role for Rap1 activation for cancer dissemination, Rap1GAP expression has been found to be down-regulated in several cancers (76 -78). Roles for Rap1 effectors in the context of cancers have not been explored. As mentioned earlier, Radil was originally identified as a factor induced by a chromosomal translocation in a sarcoma (40). Interestingly, a recent analysis of prostate cancer biopsies demonstrated that Radil expression is up-regulated during prostate cancer progression (79). Together this suggests that Rap1 signaling through Radil may be important for cancer initiation/progression. Given Radil assembly in the G␤␥⅐Rap1⅐Radil complex, the role of Radil may, thus, be particularly important in the context of GPCR signal-ing during metastasis. Indeed, chemokine receptors for example are known to direct tumor cells metastasis and tropism to different secondary sites. Further studies examining the role of this protein complex during the progression of different cancers are, thus, needed.
This study unveils a previously unappreciated role for G␤␥ in regulating cell adhesion through its recruitment in the G␤␥⅐Rap1⅐Radil complex. Given that the formation of this protein complex requires the activation of Rap1, one function for this complex, which we characterized in the present study, occurs downstream of Rap1 activation by GPCRs. One important question that remains unclear and that was beyond the focus of our study is how GPCRs lead to Rap1 activation. Further studies are, thus, needed to precisely map the sequence of events leading to Rap1 activation by GPCRs and to evaluate whether context-dependent signaling pathways may contribute to Rap activation and engagement of the G␤␥⅐Rap1⅐Radil complex to regulate cell adhesion.
In conclusion, this study identifies the G␤␥⅐Rap1⅐Radil complex as an important component of GPCR signaling regulating cell-matrix adhesion. Although we found that Radil leads to integrins activation, how exactly Radil regulates this process and whether it has other functions is not known. Perhaps the identification of which PDZ ligand containing binding partners interacts with Radil will help to further understand how this protein complex works. Because Rap1 is also known to function in cell-cell adhesion and cell invasion, which are also known to be regulated by GPCR signaling, more work is also needed to examine the role of the G␤␥⅐Rap1⅐Radil complex in these processes.