Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin

Abstract

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein-protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein-protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV-Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs.

FIGURE 1:

The C-terminus of GIV directly binds autophosphorylated cytoplasmic tails of multiple RTKs. (a) Recombinant cytoplasmic tails of EGFR, InsR, and VEGFR were autophosphorylated and subsequently used in binding assays with His-GIV-CT (aa 1660–1870) or His-Sec22 (a negative control) prebound to cobalt beads. Bound proteins were analyzed for His-tagged proteins by Ponceau staining (bottom) and autophosphorylated receptors by IB with anti-pTyr mAb (top). (b) Schematic representation of GST-tagged, phospho–EGFR-tail peptides used in this work. (c, d) Equal aliquots (25 μg) of GST and GST-EGFR peptides containing the indicated tyrosines were autophosphorylated in vitro using recombinant EGFR kinase and subsequently used in pull-down assays with His-GIV-CT as in a. (c) An aliquot of the GST proteins were analyzed for GST and pTyr by IB. Single-channel images for GST and pTyr are displayed in grayscale. Yellow pixels in the overlay images (merge panels) confirm that Y1068, 86, Y1148, and Y1173 are autophosphorylated on tyrosine(s) by EGFR (lanes 2, 4, 5), whereas GST (lane 1) and GST-Y1114 (lane 3) are not. (d) Bound proteins were visualized by IB for His. His-GIV-CT bound strongly to GST-Y1148 (lane 7) and with GST-Y1173 (lane 8) but not to GST alone or other GST-EGFR peptides (lanes 4–6).

FIGURE 2:

GIV's C-terminus folds into a SH2-like domain in the presence of phosphotyrosine ligand. (a) Schematic representation of the domain organization of GIV. The putative SH2-like domain (dark blue) is located in the C-terminus, ∼15 aa downstream of the GEF motif (black), within the Akt and actin-binding domains (light blue). Other domains include a microtubule-binding hook domain (red), a coiled-coil homodimerization domain (yellow), a Gα-binding domain (GBD, green), and a PI4P-binding domain (purple). The numbers denote the amino acids marking the boundaries of each domain. (b) The sequence corresponding to the C-terminal domain of human (BAE44387, aa 1623–1870) GIV/Girdin was used to align with 19 other known SH2 domains in various proteins by pfam alignment using the MolSoft molecular modeling platform. Pfam contains multiple alignments and hidden Markov model–based profiles (HMM profiles) of complete protein domains. The GXFXXR motif that is conserved among all SH2-like domains and conserved in GIV is highlighted (see also Supplemental Figure S1). (c) The sequence corresponding to the C-terminal domain of human GIV (BAE44387, aa 1623–1870) was used to identify homologues by BLAST search. The phosphotyrosine recognizing GXFXXR motif of GIV is absent in fish, birds, and platypus (egg-laying mammals) but highly conserved in higher mammals (see also Supplemental Figure S2 for an extended alignment). (d) A structural model of GIV's C-terminal sequence (aa 1714–1815; depicted as a ribbon, colored blue to red from N- to C-terminus) was built based on the alignment with 43 known SH2 templates using ICM software (MolSoft). Superimposition of the model onto an established SH2 domain complex structure (C-terminal SH2 domain of SOCS3 [black] bound to LR63 phosphopeptide derived [teal blue], PDB 2hmh) illustrates fold similarity. (e) Predicted atomic environment of the polar residues in the 1740-GDFYDR-1745 motif in the GIV CT in its folded SH2-like state. Asp1741, Tyr1743, and Asp1745 are shown in yellow. These residues are polar, unlike the corresponding residues in typical SH2 domains. In the predicted folded SH2 state, residues in gray make favorable polar interactions (cyan) with the residues in yellow. (f) Molecular modeling of the interface between GIV's SH2 domain and EGFR-derived phosphotyrosine peptide (purple) corresponding to pTyr1148 and its flanking residues, a high-affinity binding site for GIV on the EGF receptor. The acidic, neutral, and basic potentials are displayed in red, white, and blue, respectively. The electrostatic surface potential of the phosphotyrosine-recognizing and -binding pocket of GIV's SH2 domain is mostly basic. (g) Equal aliquots of His-GIV CT were first incubated with 10-fold molar excess of commercially synthesized pY1173EGFR or dephosphorylated control EGFR peptides before subjecting it to limited tryptic proteolysis on ice. Reactions were analyzed for GIV-CT (His) by IB. A trypsin-resistant product of ∼15 kDa is seen (asterisk) exclusively when His-GIV-CT is preincubated with phosphorylated EGFR peptide. Using His-GIV-CT-RL as substrate, it was confirmed that addition of pY1173EGFR peptide alone did not inhibit trypsin's protease activity (unpublished data).

FIGURE 3:

Validation of the homology model of GIV's SH2 domain. (a, b) Based on the generated three-dimensional model shown in Figure 2, d and e, a series of GIV C-terminal mutations were predicted to decrease, increase, or have no effect on the recognition and binding to the phosphotyrosine 1148 on the cytoplasmic tail of EGFR. These residues are highlighted in red, green, and gold, and listed along with the preferred amino acid substitution in b. R1745 of GIV corresponds to the invariant Arg residue at the βB5 position within the conserved GXFXXR motif that is characteristic of the entire SH2 family of adaptors (Songyang et al., 1993, 1994; Schlessinger, 1994). The βC-βD loop, which is predicted to not affect phosphotyrosine binding, was either deleted or replaced with a neutral flexible linker, SGS. F1765 was mutated to Thr (T) to resemble the mouse sequence; this substitution is predicted to increase the depth of the binding pocket and improve binding. α, helix; β, β-sheets. (c, d) Equal aliquots (25 μg) of GST and GST-pY1148 were phosphorylated in vitro using recombinant EGFR kinase and used in pull-down assays with purified WT or various mutants of His-GIV-CT listed in b. (c) An aliquot of the GST proteins was analyzed for GST and pTyr by IB. Yellow pixels in the overlay images (merge panels) confirm that GST-pY1148 is autophosphorylated on tyrosine(s) by EGFR kinase in vitro. (d) Bound GIV CT was visualized by IB for His. Equal loading of GST and GST-pY1148 was confirmed by Ponceau S staining. A representative experiment is shown; n = 4. (e) Schematic representation of EGFR-VC and VN-GIV-SH2 constructs used for BiFC assay. (f) Cos7 cells were cotransfected with indicated complementary pairs of probes, grown in 10% FBS, fixed, and analyzed for fluorescence by confocal microscopy. Images representative of each condition are shown. Fluorescence is observed at the PM (arrowheads) and on vesicles (arrows; likely endolysosomal compartments) exclusively when complementary VN-GIV-SH2 WT, but not the SH2-deficient RL mutant probe, was cotransfected with EGFR-VC. Paired transfection of other complementary VN- and VC-control probes did not show discernible fluorescence (∼400 cells/experiment; n = 4).

FIGURE 4:

GIV's SH2 and GEF domains are required for the recruitment of Gαi3 to ligand-activated EGFR and activation of G protein after EGF stimulation. (a) Equal aliquots (15 μg) of GST-EGFR-T (aa 1064–1210) were either phosphorylated in vitro using 5 ng of recombinant EGFR kinase (GST-pY EGFR-T; lanes 5–7) or mock treated (GST-EGFR-T; lane 4) and subsequently used in pull-down assays with equal amounts of purified WT, FA (GEF-deficient), or RL (SH2-deficient) mutants of His-GIV-CT (aa 1660–1870; lanes 1–3, inputs). Bound proteins were analyzed for His-GIV-CT and phosphorylation of GST-EGFR-T by IB. As anticipated, GST-pY EGFR-T (lane 5) but not GST-EGFR-T (lane 4) directly binds His-GIV-CT WT. The FA (lane 6) but not RL (lane 7) mutant of GIV-CT binds GST-pY EGFR-T. (b) WT, FA, and RL mutants of His-GIV-CT were incubated with 5 μg of GST-Gαi3 or GST preloaded with GDP immobilized on glutathione beads. Bound proteins were analyzed by IB for GIV-CT using anti-His mAb. His-GIV-CT WT (lane 3) and RL mutant (lane 9) bound GST-Gαi3 equally, and, as anticipated, the FA mutant did not (lane 6). (c) The amount of GTP hydrolyzed in 10 min by His-Gαi3 was determined in the presence of the indicated amounts of WT (∆), FA (Δ), and RL mutant (•) His-GIV-CT. Both WT and RL mutants increased the steady-state GTPase activity of His-Gαi3 efficiently and equally in a dose-dependent manner, whereas the FA mutant did not. The basal steady-state GTPase activity of His-Gαi3 is 0.021 ± 0.04 mol Pi mol Gαi3⁻¹ min⁻¹ and the results are displayed as percentage of basal activity. (d) Equal aliquots (15 μg) of GST-EGFR-T (aa 1064–1210) were either phosphorylated in vitro with recombinant EGFR kinase (GST-pY EGFR-T; lanes 2 and 4–7) or mock treated (GST-EGFR-T; lanes 1 and 3) and subsequently used in pull-down assays with purified His-Gαi3 alone (lanes 1 and 2), His-GIV-CT alone (lanes 3 and 4), or a mix of His-Gαi3 and WT, FA, or RL mutants of His-GIV-CT as indicated. Bound proteins were analyzed for His-GIV-CT and phosphorylation of GST-EGFR-T by IB. Gαi3 binds GST-pY EGFR in the presence of WT (lane 5) but not FA (lane 6) or RL (lane 7). (e) HeLa GIV-WT, HeLa GIV-FA, and HeLa GIV-RL cells stably expressing the indicated siRNA-resistant GIV constructs were depleted of endogenous GIV by siRNA, serum starved for 16 h, and then stimulated with EGF for the indicated durations before lysis. Equal aliquots of lysates (bottom) were incubated with anti-EGFR mAb (#225 IgG). Immune complexes (top) were analyzed for total (t-EGFR) and Gαi3 by IB. (f) Equal aliquots of lysates (bottom) from starved or EGF-treated HeLa GIV-WT and GIV-RL cells were incubated with anti-Gαi:GTP mAb. Immune complexes (top) were analyzed for Gαi3 by IB. (g) HeLa-GIV-WT and GIV-RL cells were depleted of endogenous GIV as in e, serum starved (0.2% FBS), and subsequently stimulated with EGF and analyzed for cAMP by RIA (see Materials and Methods). Results are displayed as fold change in cAMP (y-axis) in each cell line normalized to their respective starved state. (h) HeLa-GIV-WT and GIV-RL cells were stimulated with EGF before lysis as in e. Top, equal aliquots of whole-cell lysates were analyzed for phosphorylated CREB (pCREB) and tubulin by IB. Bottom, bar graphs display the quantification of pCREB/tubulin by band densitometry. All values are normalized to the starved HeLa-GIV-WT cells. Results are shown as mean ± SEM; n = 3.

FIGURE 5:

The SH2-like domain of GIV is required for enhanced tyrosine phosphorylation of GIV and subsequent binding to RTKs. (a) Homology model of GIV's SH2 domain is depicted as a ribbon as in Figure 2d. The positions and the orientations of the two tyrosines are displayed in two views: from the “front” (pTyr-binding interface) and “back.” Both tyrosines are well exposed to solvent, consistent with the fact that multiple receptor and nonreceptor tyrosine kinases can readily access and phosphorylate them and that they can directly bind and activate PI3K (Lin et al., 2011). (b) Cos7 cells expressing FLAG-tagged wild-type GIV (GIV-WT FLAG), SH2-deficient R1745L mutant GIV (GIV-RL FLAG), a tyrosine phosphorylation–deficient mutant (GIV-YF FLAG; Lin et al., 2011), or vector alone were serum starved, pretreated with Src inhibitor (PP2), and subsequently stimulated with EGF before lysis. GIV was immunoprecipitated from equal aliquots of lysates (right) with FLAG mAb, and immunoprecipitates (left) were analyzed for GIV (red) and pTyr (green) by IB and dual-color imaging. The merge confirms that tyrosine-phosphorylated GIV (yellow) was immunoprecipitated exclusively from EGF-treated cells expressing GIV-WT (lane 3) but not from cells expressing GIV-RL (lane 4). As shown previously (Lin et al., 2011), the negative control GIV-YF was not phosphorylated (lane 5). The lysates (right) were analyzed for FLAG (GIV-FLAG), phospho-Akt (pAkt), and tubulin by IB. (c) Equal aliquots of His-GIV-CT proteins were first incubated with 10-fold molar excess of either phosphorylated (left lane) or unphosphorylated (right lane) EGFR tail peptides (sequence flanking Y1173) before in vitro kinase assays with recombinant EGFR kinase. GIV-CT is phosphorylated only in the presence of dephosphorylated EGFR peptide (which it cannot bind) but not in the presence of phospho-EGFR ligand. (d) EGFR was immunoprecipitated from lysates of HeLa cells starved and stimulated with EGF with anti-EGFR (528) mAb or control IgG. Immune complexes were analyzed for the presence of tyrosine-phosphorylated GIV using anti-pY1764GIV and ligand-activated EGFR (pY1173 EGFR) by IB. (e) InsR was immunoprecipitated from lysates of Cos7 cells starved and stimulated with insulin with anti-pYInsR mAb. Immune complexes were analyzed for the presence of tyrosine phosphorylated GIV using anti-pY1764GIV and ligand-activated InsR (pYInsR) by IB.

FIGURE 6:

Interaction of GIV with EGFR determines the profile of other SH2 adaptors recruited to the ligand-activated receptor. (a) Equal aliquots (25 μg) of GST and GST-EGFR peptide containing Y1173 were autophosphorylated in vitro using recombinant EGFR kinase as in Figure 1c and subsequently used in pull-down assays with q constant amount (6 μg) of His-GIV-CT and increasing amounts of His-SHP-1 as indicated. Bound proteins were analyzed for His-SHP1 and His-GIV-CT by IB with His. (b) Equal aliquots (25 μg) of GST and GST-EGFR peptide containing Y1148 were autophosphorylated in vitro using recombinant EGFR kinase as in Figure 1c and subsequently used in pull-down assays with a constant amount (2 μg) of His-Shc1 and increasing amounts of His-GIV-CT as indicated. Bound proteins were analyzed for His-Shc1 and His-GIV-CT by IB with His. (c) HeLa GIV-WT and HeLa GIV-RL cells were depleted of endogenous GIV, starved, and stimulated with EGF as in Figure 4e before lysis. EGFR was immunoprecipitated from these lysates as in Figure 5d. Immunoprecipitates (left) and lysates (right) were analyzed for receptor phosphorylation and various SH2 adaptors by IB. Compared to HeLa GIV-WT cells, in HeLa GIV-RL cells, recruitment of SHP1 and Shc1 to ligand-activated EGFR is enhanced, autophosphorylation of EGFR is reduced, and recruitment of p85α (PI3K) and Grb2 is suppressed. (d) HeLa GIV-WT and HeLa GIV-RL cells were depleted of endogenous GIV, starved, and stimulated with EGF as in Figure 4e before lysis. Equal aliquots of whole-cell lysates were analyzed for phospho-Akt (pAkt), ERK (pERK1/2), and tubulin by IB. (e) Working model. A schematic summarizing the sequence of events triggered by the binding of GIV's SH2-like domain to EGFR. Left, in the absence of GIV, upon ligand stimulation, EGFR autophosphorylation is triggered at Y1148 and Y1173, which serve as sites of adaptor recruitment for Shc and SHP-1, respectively. Shc triggers activation of the MAPK/ERK pathway, and activated SHP-1 dephosphorylates EGFR and down-regulates receptor signaling. Right, in the presence of GIV, EGFR autophosphorylation sites pY1148 and pY1173 are recognized and approached by a partially structured SH2-like domain in GIV's C-terminus. Close proximity to EGFR facilitates efficient phosphorylation of GIV on critical tyrosines within a partially structured SH2 domain before/simultaneously with folding into a SH2-like module that stably docks onto autophosphorylated EGFR tail. Such docking competes with Shc for Y1148 and with SHP-1 for Y1173, thereby displacing and antagonizing signaling via both adaptors. Once GIV-SH2 is recruited to activated EGFR, GIV triggers two parallel pathways that were previously shown to synergistically activate Akt: a) GIV's phosphotyrosines bind p85α (class Ia PI3K) and activate PI3K/Akt signaling (Lin et al., 2011), and b) GIV's GEF motif activates Gαi in close proximity to activated EGFR and releases “free” Gβϒ, which directly binds p110 (class 1b PI3K) and activates PI3K/Akt signaling (Garcia-Marcos et al., 2009).

FIGURE 7:

EGFR degradation is delayed and actin remodeling and cell migration are impaired in the absence of an intact SH2 domain in GIV. (a) HeLa control, HeLa GIV-WT, HeLa GIV-RL, and HeLa GIV-FA cells stably expressing siRNA-resistant GIV constructs were either treated with control (Scr) siRNA or GIV siRNA, starved, and stimulated with EGF as in Figure 4e before fixation. Cells were costained for EEA1 (green), tEGFR (red; anti-EGFR cytoplasmic tail), and the nucleus/DAPI (blue). At 60 min after ligand stimulation, EGFR is virtually undetectable in GIV-WT cells (e, j), but significant staining is seen in GIV-RL cells (t) compared with controls (e). Images are representative of four independent repeats. Bar, 10 μm. (b) Serum-starved control, GIV-WT, GIV-RL, and GIV-FA HeLa cells were stimulated with EGF as in a before lysis. Equal aliquots of lysates were analyzed for total EGFR (tEGFR, anti-EGFR cytoplasmic tail) and tubulin by immunoblotting. (c) The amount of tEGFR present at 30 min in b was quantified by Odyssey infrared imaging, normalized to tubulin, and expressed as percentage remaining compared with 0 min. Results are shown as mean ± SEM (n = 4). (d) Control HeLa cells or HeLa GIV-WT, GIV-FA, and GIV-RL cells expressing various siRNA-resistant GIV constructs were treated with scrambled or GIV siRNA as indicated. Fixed cells were costained with phalloidin–Texas red (F-actin, red) and DAPI (DNA, blue) and visualized by confocal microscopy. Stress fibers were reduced when endogenous GIV was depleted in control, HeLa GIV-FA (f), and HeLa GIV-RL (h) cells but not in HeLa GIV-WT cells. Both HeLa GIV-FA and GIV-RL cells show a paucity of stress fibers even without depletion of endogenous GIV (e, g), indicating that these GIV mutants have a dominant negative effect on actin remodeling. Bar, 10 μM. (e) HeLa control or HeLa GIV-WT and GIV-RL cells expressing various siRNA-resistant GIV constructs were treated with scrambled or GIV siRNA as in d. Cell migration was determined as described in Materials and Methods. Depletion of GIV inhibited migration in control and HeLa GIV-RL cells but not in HeLa GIV-WT cells.

FIGURE 8:

Multimodular cooperation allows GIV, a class 1 SH2-like adaptor, to link GEF activity for trimeric Gαi to cytoplasmic tails of ligand-activated RTKs. Schematic illustration of the modular structure of GIV from amino- to carboxyl-terminus: phosphoinositide-binding domain (PI4P-BD, brown), which binds membrane lipid and helps localize GIV to the PI4P-enriched plasma (PM), where ligand-activated receptors and G proteins are located; a GEF motif (green), which binds and activates Gαi; a SH2 domain (blue), which recognizes and docks onto the autophosphorylation sites on cytoplasmic tails of ligand-activated EGFR and other RTKs; a pair of phosphotyrosines (P), which directly bind p85α (PI3K) and activate class 1A PI3Ks; and an actin-binding domain (actin-BD, purple), which binds and remodels actin at the leading edge of migrating cells. These binding events cooperatively maintain GIV at the PM such that Gαi and PI3K are activated, and actin is remodeled in close proximity to RTKs. Recruitment of GIV-SH2 to RTKs and activation of Gαi in the vicinity of activated receptors enable GIV to modulate signaling programs downstream of the receptor. The plasticity of GIV's SH2-like domain between a stably folded and partially folded state is likely to regulate GIV's ability to engage with activated RTKs.