G protein subunit Galpha13 binds to integrin alphaIIbbeta3 and mediates integrin "outside-in" signaling

Abstract

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) Galpha13 directly bound to the integrin beta3 cytoplasmic domain and that Galpha13-integrin interaction was promoted by ligand binding to the integrin alphaIIbbeta3 and by guanosine triphosphate (GTP) loading of Galpha13. Interference of Galpha13 expression or a myristoylated fragment of Galpha13 that inhibited interaction of alphaIIbbeta3 with Galpha13 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 Galpha13-coupled receptors that provide a mechanism for dynamic regulation of RhoA.

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 innoculated with control siRNA- or Gα₁₃-siRNA-transfected bone marrow stem cells. (C, D, 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.

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 or 30 μM AlF₄^-. Immunoprecipitates were immunoblotted with anti-Gα₁₃ or anti-β₃ (MAb15). See Fig. S4 for quantitation. (B) Proteins from platelet lysates were immunoprecipitated with control mouse IgG, anti-α_IIbβ₃ (D57 (24)) or an antibody to the glycoprotein Ibα (GPIb). Immunoprecipitates were immunoblotted with anti-Gα₁₃, anti-β₃, or anti-GPIb antibodies. (C, 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 GTPγS or 30 μM AlF₄^-. Bound proteins were immunoblotted with anti-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 anti-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 anti-Flag (Bound). Flag-tagged protein amounts in lysates are shown by anti-Flag immunoblot (Input). (G) Protein from platelet lysates treated with 0.1% DMSO, 250 μM scrambled control peptide (Ctrl) or mSRI were immunoprecipitated with anti-Gα₁₃. Immunoprecipitates were immunoblotted with anti-Gα₁₃ or anti-β₃. See Fig. S4 for quantitation.

Fig. 3. Effects of mSRI on integrin-induced c-Src phosphorylation, RhoA activity and platelet spreading

(A) Washed human platelets pre-treated 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-RBD beads (25). 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.

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, D, E, 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 GSTRBD beads (E) by immunoblot. (F) quantitative data (mean±SD) from 3 experiments. (G) A schematic illustrating Gα₁₃-dependent dynamic regulation of RhoA and crosstalk between GPCR and integrin signaling.