G Protein Subunit Gα13 Binds to Integrin αIIbβ3 and Mediates Integrin “Outside-In” Signaling

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Science  15 Jan 2010:
Vol. 327, Issue 5963, pp. 340-343
DOI: 10.1126/science.1174779

Integrin G Protein

Adhesion molecules, known as integrins, are found on the surface of cells. When integrins adhere to components of the extracellular matrix, they act as receptors and initiate signaling events within the cell. Gong et al. (p. 340) show that they do so in part by partnering with a signal-transducing protein called Gα13. Such α subunits of heterotrimeric guanine nucleotide-binding proteins are well known for transducing signals from the large class of G protein–coupled receptors, but were not known to work with integrins. Gα13 appears to interact directly with the integrin αIIbβ3 and to transmit signals that regulate cell spreading.


Integrins mediate cell adhesion to the extracellular matrix and transmit signals within the cell that stimulate cell spreading, retraction, migration, and proliferation. The mechanism of integrin outside-in signaling has been unclear. We found that the heterotrimeric guanine nucleotide–binding protein (G protein) Gα13 directly bound to the integrin β3 cytoplasmic domain and that Gα13-integrin interaction was promoted by ligand binding to the integrin αIIbβ3 and by guanosine triphosphate (GTP) loading of Gα13. Interference of Gα13 expression or a myristoylated fragment of Gα13 that inhibited interaction of αIIbβ3 with Gα13 diminished activation of protein kinase c-Src and stimulated the small guanosine triphosphatase RhoA, consequently inhibiting cell spreading and accelerating cell retraction. We conclude that integrins are noncanonical Gα13-coupled receptors that provide a mechanism for dynamic regulation of RhoA.

Integrins mediate cell adhesion and transmit signals within the cell that lead to cell spreading, retraction, migration, and proliferation (1). Thus, integrins have pivotal roles in biological processes such as development, immunity, cancer, wound healing, hemostasis, and thrombosis. The platelet integrin αIIbβ3 typically displays bidirectional signaling function (2, 3). Signals from within the cell activate binding of αIIbβ3 to extracellular ligands, which in turn triggers signaling within the cell initiated by the occupied receptor (so-called “outside-in” signaling). A major early consequence of integrin “outside-in” signaling is cell spreading, which requires activation of the protein kinase c-Src and c-Src–mediated inhibition of the small guanosine triphosphatase (GTPase) RhoA (47). Subsequent cleavage of the c-Src binding site in β3 by calpain allows activation of RhoA, which stimulates cell retraction (7, 8). The molecular mechanism coupling ligand-bound αIIbβ3 to these signaling events has been unclear.

Heterotrimeric guanine nucleotide–binding proteins (G proteins) consist of Gα, Gβ, and Gγ subunits (9). G proteins bind to the intracellular side of G protein–coupled receptors (GPCRs) and transmit signals that are important in many intracellular events (911). Gα13, when activated by GPCRs, interacts with Rho guanine-nucleotide exchange factors (RhoGEF) and thus activates RhoA (1114), facilitating contractility and rounding of discoid platelets (shape change). To determine whether Gα13 functions in signaling from ligand-occupied integrin, we investigated whether inhibition of Gα13 expression with small interfering RNA (siRNA) affected αIIbβ3-dependent spreading of platelets on fibrinogen, which is an integrin ligand. We isolated mouse bone marrow stem cells and transfected them with lentivirus encoding Gα13 siRNA. The transfected stem cells were transplanted into irradiated C57/BL6 mice (15). Four to six weeks after transplantation, nearly all platelets isolated from recipient mice were derived from transplanted stem cells, as indicated by the enhanced green fluorescent protein (EGFP) encoded in lentivirus vector (Fig. 1A and fig. S1). Platelets from Gα13 siRNA-transfected stem cell recipient mice showed >80% decrease in Gα13 expression (Fig. 1B). When platelets were allowed to adhere to immobilized fibrinogen [αIIbβ3 binding to immobilized fibrinogen does not require prior “inside-out” signaling activation (16)], platelets depleted of Gα13 spread poorly as compared with control platelets (Fig. 1A and fig. S2). The inhibitory effect of Gα13 deficiency is unlikely to be caused by its effect on GPCR-stimulated Gα13 signaling because (i) washed resting platelets were used and no GPCR agonists were added, and (ii) prior treatment with 1 mM aspirin [which abolishes thromboxane A2 (TXA2) generation (17)] did not affect platelet spreading on fibrinogen (fig. S2), making unlikely the endogenous TXA2-mediated stimulation of Gα13. Furthermore, Gα13 siRNA inhibited spreading of Chinese hamster ovary (CHO) cells expressing human αIIbβ3 (123 cells) (18), which was rescued by an siRNA-resistant Gα13 (fig. S3). Thus, Gα13 appears to be important in integrin “outside-in” signaling leading to cell spreading.

Fig. 1

The role of Gα13 in integrin “outside-in” signaling. (A) Confocal microscopy images of spreading scrambled siRNA control platelets or Gα13-depleted platelets (Gα13-siRNA) on fibrinogen, without or with Y27632. Merged EGFP (green) fluorescence and Alex Fluor 546-conjugated phalloidin (red) fluorescence. (B) Western blot comparison of Gα13 abundance in platelets from mice inoculated with control siRNA- or Gα13-siRNA–transfected bone marrow stem cells. (C to E) Mouse platelets from scrambled siRNA- or Gα13 siRNA–transfected stem cells were allowed to adhere to immobilized fibrinogen, solubilized, and analyzed for c-Src Tyr416 phosphorylation and RhoA activation.

To determine whether Gα13 serves as an early signaling mechanism that mediates integrin-induced activation of c-Src, we measured phosphorylation of c-Src at Tyr416 (which indicates activation of c-Src) in control and fibrinogen-bound cells. Depletion of Gα13 in mouse platelets or 123 cells abolished phosphorylation of c-Src Tyr416 (Fig. 1C and fig. S3), indicating that Gα13 may link integrin αIIbβ3 and c-Src activation. Because c-Src inhibits RhoA (7, 19), we also tested the role of Gα13 in regulating activation of RhoA. RhoA activity was suppressed to baseline 15 min after platelet adhesion and became activated at 30 min (Fig. 1C), which is consistent with transient inhibition of RhoA by c-Src (7). The integrin-dependent delayed activation of RhoA was not inhibited by depletion of Gα13, indicating its independence of the GPCR-Gα13-RhoGEF pathway (Fig. 1C). In contrast, depletion of Gα13 accelerated RhoA activation (Fig. 1C). Thus, Gα13 appears to mediate inhibition of RhoA. The inhibitory effect of Gα13 depletion on platelet spreading was reversed by Rho-kinase inhibitor Y27632 (Fig. 1A), which suggests that Gα13-mediated inhibition of RhoA is important in stimulating platelet spreading. These data are consistent with Gα13 mediating integrin “outside-in” signaling inducing c-Src activation, RhoA inhibition, and cell spreading.

The integrin αIIbβ3 was coimmunoprecipitated by antibody to Gα13, but not control immunoglobulin G (IgG), from platelet lysates (Fig. 2A). Conversely, an antibody to β3 immunoprecipitated Gα13 with β3 (Fig. 2B). Coimmunoprecipitation of β3 with Gα13 was enhanced by guanosine triphosphate γS (GTP-γS) or AlF4 (Fig. 2A and fig. S4). Thus, β3 is present in a complex with Gα13, preferably the active GTP-bound Gα13. To determine whether Gα13 directly binds to the integrin cytoplasmic domain, we incubated purified recombinant Gα13 (20) with agarose beads conjugated with glutathione S-transferase (GST) or a GST-β3 cytoplasmic domain fusion protein (GST-β3CD). Purified Gα13 bound to GST-β3CD, but not to GST (Fig. 2C). Purified Gα13 also bound to the β1 integrin cytoplasmic domain fused with GST (GST-β1CD) (Fig. 2D). The binding of Gα13 to GST-β3CD and GST-β1CD was detected with GDP-loaded Gα13, but enhanced by GTP-γS and AlF4 (Fig. 2, C and D), indicating that the cytoplasmic domains of β3 and β1 can directly interact with Gα13 and that GTP enhances the interaction. The Gα133 interaction was enhanced in platelets adherent to fibrinogen, and by thrombin, which stimulates GTP binding to Gα13 via GPCR (Fig. 2E). Hence, the interaction is regulated by both integrin occupancy and GPCR signaling.

Fig. 2

Binding of Gα13 to β3 and the inhibitory effect of mSRI peptide. (A) Proteins from platelet lysates were immunoprecipitated with control IgG or antibody to Gα13 with or without 1 μM GDP, 1 μM GTP-γS, or 30 μM AlF4. Immunoprecipitates were immunoblotted with antibody to Gα13 or β3 [monoclonal antibody 15 (mAb15)]. See fig. S4 for quantitation. (B) Proteins from platelet lysates were immunoprecipitated with control mouse IgG, antibody to αIIbβ3 [D57 (25)], or an antibody to the glycoprotein Ibα (GPIb). Immunoprecipitates were immunoblotted with antibodies to Gα13, β3, or GPIb. (C and D) Purified GST-β3CD (C) or GST-β1CD (D) bound to glutathione beads was mixed with purified Gα13 with or without 1 μM GDP, 1 μM GTP-γS, or 30 μM AlF4. Bound proteins were immunoblotted with antibody to Gα13. Quantitative data are shown as mean ± SD and P value (t test). (E) Lysates of control platelets or platelets adherent to fibrinogen in the absence or presence of 0.025 U/ml thrombin were immunoprecipitated with antibody to Gα13 and then immunoblotted with mAb15. Quantitative data are shown as mean ± SD and P value (t test). (F) Lysates from 293FT cells transfected with Flag-tagged wild-type Gα13 or indicated truncation mutants (see fig. S5) were precipitated with GST-β3CD- or GST-bound glutathione beads. Bead-bound proteins were immunoblotted with antibody to Flag (Bound). Flag-tagged protein amounts in lysates are shown by anti-Flag immunoblot (Input). (G) Protein from platelet lysates treated with 0.1% dimethyl sulfoxide (DMSO), 250 μM scrambled control peptide (Ctrl), or mSRI were immunoprecipitated with antibody to Gα13. Immunoprecipitates were immunoblotted with antibody to Gα13 or β3. See fig. S4 for quantitation.

To map the β3 binding site in Gα13, we incubated cell lysates containing Flag-tagged wild type or truncation mutants of Gα13 (fig. S5) with GST-β3CD beads. GST-β3CD associated with wild-type Gα13 and the Gα13 1 to 212 fragment containing α-helical region and switch region I (SRI), but not with the Gα13 fragment containing residues 1 to 196 lacking SRI (Fig. 2F). Thus, SRI appears to be critical for β3 binding. To further determine the importance of SRI, Gα133 binding was assessed in the presence of a myristoylated synthetic peptide, Myr-LLARRPTKGIHEY (mSRI), corresponding to the SRI sequence of Gα13 (197 to 209) (21, 22). The mSRI peptide, but not a myristoylated scrambled peptide, inhibited Gα13 binding to β3 (Fig. 2G), indicating that mSRI is an effective inhibitor of β3-Gα13 interaction. Therefore, we further examined whether mSRI might inhibit integrin signaling. Treatment of platelets with mSRI inhibited integrin-dependent phosphorylation of c-Src Tyr416 and accelerated RhoA activation (Fig. 3A). The effect of mSRI is unlikely to result from its inhibitory effect on the binding of RhoGEFs to Gα13 SRI because Gα13 binding to RhoGEFs stimulates RhoA activation, which should be inhibited rather than promoted by mSRI (22). Thus, these data suggest that β3-Gα13 interaction mediates activation of c-Src and inhibition of RhoA. Furthermore, mSRI inhibited integrin-mediated platelet spreading (Fig. 3B), and this inhibitory effect was reversed by C3 toxin (which catalyzes the ADP ribosylation of RhoA) or Y27632, confirming the importance of Gα13-dependent inhibition of RhoA in platelet spreading. Thrombin promotes platelet spreading, which requires cdc42/Rac pathways (23). However, thrombin-promoted platelet spreading was also abolished by mSRI (Fig. 3B), indicating the importance of Gα133 interaction. Thus, Gα13-integrin interaction appears to be a mechanism that mediates integrin signaling to c-Src and RhoA, thus regulating cell spreading.

Fig. 3

Effects of mSRI on integrin-induced c-Src phosphorylation, RhoA activity, and platelet spreading. (A) Washed human platelets pretreated with DMSO, mSRI, or scrambled control peptide were allowed to adhere to fibrinogen and then solubilized at indicated time points. Proteins from lysates were immunoblotted with antibodies to c-Src phosphorylated at Tyr416, c-Src, or RhoA. GTP-bound RhoA was measured by association with GST–Rhotekin rho-binding domain (GST-RBD) beads (26). See fig. S4 for quantitative data. (B) Spreading of platelets treated with 0.1% DMSO, scrambled control peptide, or mSRI, in the absence or presence of C3 toxin, Y27632, or 0.01 U/ml thrombin. Platelets were stained with Alexa Fluor 546–conjugated phalloidin.

To further determine whether Gα13 mediates inhibition of integrin-induced RhoA-dependent contractile signaling, we investigated the effects of mSRI and depletion of Gα13 on platelet-dependent clot retraction (shrinking and consolidation of a blood clot requires integrin-dependent retraction of platelets from within) (7, 8). Clot retraction was accelerated by mSRI and depletion of Gα13 (Fig. 4, A and B, and fig. S6), indicating that Gα13 negatively regulates RhoA-dependent platelet retraction and coordinates cell spreading and retraction. The coordinated cell spreading-retraction process is also important in wound healing, cell migration, and proliferation (24).

Fig. 4

The role of Gα13 in clot retraction and dynamic RhoA regulation. (A) Effect of 250 μM mSRI peptide on clot retraction of human platelet-rich plasma compared with DMSO and scrambled peptide. Clot sizes were quantified using Image J (mean ± SD, n = 3, t test). (B) Comparison of clot retraction (mean ± SD, n = 3, t test) mediated by control siRNA platelets and Gα13-depleted platelets. (C to F) Platelets were stimulated with thrombin with or without 2 mM RGDS and monitored for turbidity changes of platelet suspension caused by shape change and aggregation (C). The platelets were then solubilized at indicated time points and analyzed for amount of β3 coimmunoprecipitated with Gα13 (D) and amount of GTP-RhoA bound to GST-RBD beads (E) by immunoblot. (F) Quantitative data (mean ± SD) from three experiments. (G) A schematic illustrating Gα13-dependent dynamic regulation of RhoA and crosstalk between GPCR and integrin signaling.

The function of Gα13 in mediating the integrin-dependent inhibition of RhoA contrasts with the traditional role of Gα13, which is to mediate GPCR-induced activation of RhoA. However, GPCR-mediated activation of RhoA is transient, peaking at 1 min after exposure of platelets to thrombin, indicating the presence of a negative regulatory signal (Fig. 4, D and F). Furthermore, thrombin-stimulated activation of RhoA occurs during platelet shape change before substantial ligand binding to integrins (Fig. 4, C, D, and F). In contrast, after thrombin stimulation, β3 binding to Gα13 was diminished at 1 min when Gα13-dependent activation of RhoA occurs, but increased after the occurrence of integrin-dependent platelet aggregation (Fig. 4, E and F). Thrombin-stimulated binding of Gα13 to αIIbβ3 and simultaneous RhoA inhibition both require ligand occupancy of αIIbβ3 and are inhibited by the integrin inhibitor Arg-Gly-Asp-Ser (RGDS) (Fig. 4, D to F). Thus, our study demonstrates not only a function of integrin αIIbβ3 as a noncanonical Gα13-coupled receptor but also a new concept of Gα13-dependent dynamic regulation of RhoA, in which Gα13 mediates initial GPCR-induced RhoA activation and subsequent integrin-dependent RhoA inhibition (Fig. 4G). These findings are important for our understanding of how cells spread, retract, migrate, and proliferate, which is fundamental to development, cancer, immunity, wound healing, hemostasis, and thrombosis.

Supporting Online Material

Materials and Methods

Figs. S1 to S6


References and Notes

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  27. This work was supported by grants HL080264, HL062350, and HL068819 from the National Heart, Lung, and Blood Institute (X.D.) and GM061454 and GM074001 from the National Institute of General Medical Sciences (T.K.). We thank G. Nucifora for help with bone marrow transplantation and K. O’Brien and M. K. Delaney for proofreading.

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