A structural determinant that renders G alpha(i) sensitive to activation by GIV/girdin is required to promote cell migration

Abstract

Although several non-receptor activators of heterotrimeric G proteins have been identified, the structural features of G proteins that determine their interaction with such activators and the subsequent biological effects are poorly understood. Here we investigated the structural determinants in G alpha(i3) necessary for its regulation by GIV/girdin, a guanine-nucleotide exchange factor (GEF) that activates G alpha(i) subunits. Using G protein activity and in vitro pulldown assays we demonstrate that G alpha(i3) is a better substrate for GIV than the highly homologous G alpha(o). We identified Trp-258 in the G alpha(i) subunit as a novel structural determinant for GIV binding by comparing GIV binding to G alpha(i3)/G alpha(o) chimeras. Mutation of Trp-258 to the corresponding Phe in G alpha(o) decreased GIV binding in vitro and in cultured cells but did not perturb interaction with other G alpha-binding partners, i.e. G betagamma, AGS3 (a guanine nucleotide dissociation inhibitor), GAIP/RGS19 (a GTPase-activating protein), and LPAR1 (a G protein-coupled receptor). Activation of G alpha(i3) by GIV was also dramatically reduced when Trp-258 was replaced with Tyr, Leu, Ser, His, Asp, or Ala, highlighting that Trp is required for maximal activation. Moreover, when mutant G alpha(i3) W258F was expressed in HeLa cells they failed to undergo cell migration and to enhance Akt signaling after growth factor or G protein-coupled receptor stimulation. Thus activation of G alpha(i3) by GIV is essential for biological functions associated with G alpha(i3) activation. In conclusion, we have discovered a novel structural determinant on G alpha(i) that plays a key role in defining the selectivity and efficiency of the GEF activity of GIV on G alpha(i) and that represents an attractive target site for designing small molecules to disrupt the G alpha(i)-GIV interface for therapeutic purposes.

FIGURE 1.

GIV is a bona fide GEF for Gα_i3. A, His-GIV-CT (aa 1623–1870) and His-GIV-CTs (aa 1660–1870) are equally efficient in increasing the steady-state GTPase activity of Gα_i3. The steady-state GTPase activity of purified His-Gα_i3 (50 nm) was determined in the presence of the indicated amounts (0, 0.1, 0.25, 0.5, 1, and 2 μm) of purified His-GIV-CT (aa 1623–1870, closed circles) or His-GIV-CTs (aa 1660–1870, open circles) by quantification of the amount of [γ-³²P]GTP (0.5 μm, ∼50 cpm/fmol) hydrolyzed in 10 min. Data are expressed as % of GTP hydrolyzed by the G protein alone (0 μm His-GIV-CT or His-GIV-CTs). Results are shown as mean ± S.E. of n = 3 independent experiments. B, His-GIV-CTs does not affect the rate of GTP hydrolysis by Gα_i3. Single-turnover GTPase assays for Gα_i3 (50 nm) were performed as described under “Experimental Procedures” in the presence of His-GIV-CTs (2 μm, open circles), GST-GAIP (1 μm, “x”), or buffer (closed circles). GST-GAIP increases the rate of GTP hydrolysis by Gα_i3, whereas His-GIV-CTs has no effect. One representative experiment of four is shown (C) His-GIV-CTs increases GTPγS binding to Gα_i3. Nucleotide binding activity of purified His-Gα_i3 (50 nm) was determined in the presence of the indicated amounts (0, 0.1, 0.5, 1, and 2 μm) of purified wild-type His-GIV-CTs (closed circles) or His-GIV-CTs F1685A mutant (open circles) by quantification of the amount of [³⁵S]GTPγS (0.5 μm, ∼50 cpm/fmol) bound in 15 min. Data are expressed as % of GTPγS bound by the G protein alone in the absence of His-GIV-CTs. His-GIV-CTs increases GTPγS binding up to 2.2-fold over basal binding, whereas His-GIV-CTs F1685A has no significant effect. Results are shown as mean ± S.E. of n = 3 independent experiments.

FIGURE 2.

GIV binds to and activates Gα_i3 more efficiently than Gα_o. A, GST-Gα_i3·GDP (lane 1) binds ∼15- to 20-fold more endogenous GIV than GST-Gα_o·GDP (lane 3). No binding of either Gα_i3 or Gα_o is detected in the presence of AlF₄⁻ (lanes 2 and 4). COS7 cell lysates (∼250 μg of protein) were incubated with ∼10 μg of purified GST-Gα_i3 (lanes 1 and 2) or GST-Gα_o (lanes 3 and 4) pre-loaded with GDP (lanes 1 and 3) or GDP·AlF₄⁻ (lanes 2 and 4) immobilized on glutathione beads. After extensive washing, bound proteins were separated by SDS-PAGE and analyzed by immunoblotting (IB) for GIV. Equal loading of GST proteins was confirmed by Ponceau S staining (lower panel). B, GST-Gα_i3·GDP (lane 1) binds ∼10-fold more His-GIV-CT than GST-Gα_o·GDP (lane 3). His-GIV-CT binding to both GST-Gα_i3 and GST-Gα_o is dramatically reduced in the presence of AlF₄⁻ (lanes 2 and 4). ∼3 μg of purified His-GIV-CT (aa 1623–1870) was incubated with ∼10 μg of purified GST-Gα_i3 (lanes 1 and 2) or GST-Gα_o (lanes 3 and 4) pre-loaded with GDP (lanes 1 and 3) or GDP·AlF₄⁻ (lanes 2 and 4) immobilized on glutathione beads. After extensive washing, bound proteins were separated by SDS-PAGE and analyzed by immunoblotting (IB) for His. Equal loading of GST proteins was confirmed by Ponceau S-staining (lower panel). C, His-GIV-CTs increases steady-state GTP hydrolysis of His-Gα_i3 at least 3-fold more than His-Gα_o at all concentrations tested. Results are shown as mean ± S.E. of nine independent experiments. The steady-state GTPase activity of purified His-Gα_i3 (closed circles, 50 nm) or His-Gα_o (open circles, 50 nm) was determined in the presence of the indicated amounts (0, 0.1, 0.5, and 2 μm) of purified His-GIV-CTs (aa 1660–1870) by quantification of the amount of [γ-³²P]GTP (0.5 μm, ∼50 cpm/fmol) hydrolyzed in 10 min. Data is expressed as % of GTP hydrolyzed by the G protein alone in the absence of His-GIV-CTs.

FIGURE 3.

Amino acids 178–270 of Gα_i3 are responsible for the preferential binding of GIV to Gα_i3versus Gα_o. A, schematic showing the Gα_i3/o chimeras (1–4) used in B. In B: Upper panel, GDP-loaded GST-Gα_i3/o chimera 1 (lane 6) and chimera 3 (lane 10) bind as much full-length GIV as GST-Gα_i3 (lane 4), whereas GST-Gα_i3/o chimera 2 (lane 8) and chimera 4 (lane 12) show dramatically reduced GIV binding. Binding of GIV to GDP·AlF₄⁻-loaded G proteins is absent in all cases (lanes 3, 5, 7, 9, 11, and 13). COS7 cell lysates were incubated with purified GST (lanes 2 and 3), GST-Gα_i3 (lanes 4 and 5) or the indicated GST-Gα_i3/o chimeras (lanes 6–13) pre-loaded with GDP (lanes 2, 4, 6, 8, 10, and 12) or GDP·AlF₄⁻ (lanes 3, 5, 7, 9, 11, and 13) immobilized on glutathione beads and analyzed as in Fig. 2A. A higher exposure of the same immunoblot (middle panel) shows the weak binding observed for GST-Gα_i3/o chimera 2 (lane 8) and 4 (lane 12). Equal loading of GST proteins was confirmed by Ponceau S staining (lower panel).

FIGURE 4.

Trp-258 of Gα_i3 is responsible for GIV binding and activation of Gα_i3. A, sequence alignment of Gα_o, Gα_i1, Gα_i2, and Gα_i3 indicating the Gα_i3 mutants studied. Rat Gα_o, Gα_i1, Gα_i2, and Gα_i3 sequences corresponding to Gα_i3 aa 178–270 were obtained form the NCBI data base and aligned using ClustalW. Conserved identical residues are in black; similar residues are shaded in gray. The secondary structure elements (α = α-helix, β = β-sheet) indicated below the alignment are named according to their crystal structures (29, 33). Residues conserved among Gα_i1, Gα_i2, and Gα_i3 but different in Gα_o within the 178–270 region were mutated in GST-Gα_i3 to the corresponding residue in Gα_o (indicated above with arrows). B, mutations W258F/T260I and W258F, in the α3/β5 loop, impair endogenous GIV binding to GDP-loaded GST-Gα_i3, whereas mutations G217D, L232Q, A235H/E239T/M240T, and K248M do not affect binding (upper panels). GIV binding to GDP·AlF₄⁻-loaded G proteins is virtually absent in all cases (lower panels). COS7 cell lysates were incubated with ∼5 μg of purified GST-Gα_i3 or the indicated GST-Gα_i3 mutants pre-loaded with GDP (upper panels) or GDP·AlF₄⁻ (lower panels) immobilized on glutathione beads and analyzed as in Fig. 2A. Equal loading of GST proteins was confirmed by Ponceau S staining. C, binding of GST-GIV-CT to His-Gα_i3 W258F (lane 6) is reduced (∼80%) compared with wt His-Gα_i3 (lane 3). ∼3 μg of purified wt His-Gα_i3 (lanes 1–3) or His-Gα_i3 W258F (lanes 4–6) pre-loaded with GDP were incubated with ∼9 μg of GST (lanes 2 and 5) or GST-GIV-CT (aa 1623–1870, lanes 3 and 6) immobilized on glutathione beads. Bound proteins were analyzed by immunoblotting (IB) for His. Equal loading of GST proteins was confirmed by Ponceau S staining (lower panel). D, mutation of Trp-258 to Phe reduces (∼60–70%) activation of His-Gα_i3 by His-GIV-CTs at all concentrations tested. The steady-state GTPase activity of purified His-Gα_i3 (closed circles, 50 nm) or His-Gα_i3 W258F (open circles, 50 nm) was determined as in Fig. 2C. Results are shown as mean ± S.E. of n = 9 (Gα_i3) or n = 5 (Gα_i3 W258F) independent experiments. E, mutation of Phe-259 to Trp increases (∼2-fold) activation of His-Gα_o by His-GIV-CTs at all concentrations tested. The steady-state GTPase activity of purified His-Gα_o (closed circles, 50 nm) or His-Gα_o F259W (open circles, 50 nm) was determined as in Fig. 2C. Results are shown as mean ± S.E. of three independent experiments.

FIGURE 5.

Gα_i3 activation by GIV is reduced when Trp-258 is replaced by Tyr, Leu, Ser, His, Asp, or Ala. A, all the Gα_i3 Trp-258 mutants investigated show reduced activation by His-GIV-CT ranging from a ∼60% (W258Y) to ∼95% (W258D) reduction. Trp-258 of His-Gα_i3 was mutated to amino acids of different nature, i.e. tyrosine (aromatic), leucine (aliphatic), serine (polar), histidine (basic), aspartate (acidic), and alanine (small). The steady-state GTPase activity of purified His-Gα_i3 (closed circles, 50 nm) or the indicated His-Gα_i3 mutants (open circles, 50 nm) was determined as described in Fig. 1C. Results are shown as mean ± S.E. of 4–9 independent experiments. B, binding of GST-GIV CT to His-Gα_i3 mutants W258F (lane 3), W258A (lane 9), and W258D (lane 12) is reduced compared to wt His-Gα_i3 (lane 6). ∼3 μg of purified His-Gα_i3 (lanes 4–6) or the indicated His-Gα_i3 mutants (W258F: lanes 1–3, W258A: lanes 7–9, W258D: lanes 10–12) pre-loaded with GDP were incubated with ∼9 μg of GST (lanes 2, 5, 8, and 11) or GST-GIV-CT (aa 1623–1870, lanes 3, 6, 9, and 12) immobilized on glutathione beads. Bound proteins were analyzed by immunoblotting (IB) for His. Equal loading of GST proteins was confirmed by Ponceau S staining (lower panel).

FIGURE 6.

Mutations at Trp-258 do not affect the trypsin sensitivity of Gα_i3 after activation by AlF₄⁻. Gα_i3 wt and all the Gα mutants tested are digested when pre-loaded with GDP, and all adopt the trypsin-resistant conformation after incubation with GDP·AlF₄⁻. His-Gα_i3 and the indicated His-Gα_i3 mutants or His-Gα_o (0.5 mg/ml) were incubated in the presence of GDP or GDP·AlF₄⁻ and treated or not with trypsin as described under “Experimental Procedures.” A Coomassie Blue-stained gel of a representative experiment is shown. The arrowhead denotes the position of the non-trypsinized full-length proteins loaded, and the asterisk arrowhead denotes the trypsin-resistant form of the active, GDP·AlF₄⁻-loaded His-Gα subunit.

FIGURE 7.

Mutation of Trp-258 to Asp, Ala, or Phe impairs the interaction between Gα_i3 and endogenous GIV in cultured cells. Upper panel: co-immunoprecipitation of GIV, Gβγ, and AGS3 with FLAG-tagged Gα_i3 mutants W258D (lane 2) or W258A (lane 3) is dramatically reduced compared with wt Gα_i3 (lane 4). By contrast, with Gα_i3 W258F (lane 5) only co-immunoprecipitation of GIV, but not Gβγ and AGS3 is reduced compared to controls. COS7 cells were transfected with empty vector (lane 1) or plasmids encoding FLAG-tagged Gα_i3 W258D (lane 2), Gα_i3 W258A (lane 3), wt Gα_i3 (lane 4), or Gα_i3 W258F (lane 5). 48 h after transfection cells were harvested, and lysates were used for immunoprecipitation (IP) with anti-FLAG IgG (∼2 μg) as described under “Experimental Procedures.” IP was followed by immunoblotting (IB) for FLAG (Gα_i3), GIV, AGS3, and Gβ (pan-Gβ). Equal IgG loading was confirmed by Ponceau S staining. Lower panel: aliquots of the lysates (10%) were analyzed by immunoblotting (IB) to confirm the equal loading of Gα_i3, GIV, AGS3, and Gβ and the expression of Gα_i3-FLAG constructs in the different transfected samples.

FIGURE 8.

Gα_i3 W258F binds GAIP and LPAR1 as efficiently as wt Gα_i3. A, mutation of Trp-258 to Phe does not affect Gα_i3 interaction with GAIP. GDP·AlF₄⁻-preloaded Gα_i3 W258F (lane 6, upper panel) binds GAIP to the same extent as wt Gα_i3 (lane 3, upper panel). Neither of the GDP-loaded G proteins binds GAIP (lanes 3 and 6, lower panel). ∼2 μg of purified wt His-Gα_i3 (lanes 1–3) or His-Gα_i3 W258F (lanes 4–6) pre-loaded with GDP·AlF₄⁻ (upper panel) or GDP (lower panel) were incubated with ∼5 μg of GST (lanes 2 and 5) or GST-GAIP (lanes 3 and 6) immobilized on glutathione beads. Bound proteins were analyzed by immunoblotting (IB) for His. 10% of the input His-Gα proteins were loaded in lanes 1 and 4. Equal loading of GST proteins was confirmed by Ponceau S staining. In B: Upper panel, FLAG-tagged LPAR1 (lanes 4–6) co-immunoprecipitates with both exogenously expressed wt Gα_i3 (lane 5) and Gα_i3 W258F mutant (lane 6). Endogenous Gα_i3 (lane 4) is not detected in FLAG-LPAR1 immunoprecipitates, and no Gα_i3 is present in FLAG-immunoprecipitates from controls (lanes 1–3). 48 h after transfection cells were harvested and lysates used for immunoprecipitation (IP) with anti-FLAG IgG (∼2 μg) as described under “Experimental Procedures.” IP was followed by immunoblotting (IB) for FLAG (LPAR1) and Gα_i3. Equal IgG loading was confirmed by Ponceau S staining. Lower panel, aliquots of cell lysates (∼5%) were analyzed for expression of FLAG (LPAR1), Gα_i3, and α-tubulin by immunoblotting (IB) to confirm the expression of the analyzed proteins.

FIGURE 9.

Gα_i3 W258F fails to rescue Akt activation and cell migration defects observed upon depletion of endogenous Gα_i3. A and B, HeLa cells treated with scrambled (Scr) or hGα_i3 siRNA oligonucleotides and the indicated DNA plasmids (empty vector, rGα_i3 WT, or rGα_i3 W258F) were serum-starved for 6 h and stimulated with 10 μm LPA (A) or 100 nm insulin (B) for 5 min. Cell lysates were analyzed by immunoblotting (IB) for total (tAkt) and S473 phospho-Akt (pAkt), Gα_i3, and α-tubulin. Depletion of endogenous Gα_i3 reduces LPA-stimulated (A) and insulin-stimulated (B) Akt activation by ∼70%, which is restored upon transfection of wt rGα_i3 but not rGα_i3 W258F. C, in controls (Scr siRNA), HeLa cells cover the majority of the experimental wound area after 24 h, whereas in Gα_i3-depleted cells wound closure is greatly impaired. The ability to migrate and close the wound area at 24 h is restored by transfection of rGα_i3 wt but not rGα_i3 W258F. HeLa cell monolayers treated with the indicated siRNA oligonucleotides, and DNA plasmids were scratch-wounded and examined by light microscopy immediately (0 h) or 24 h after wounding. Scale bar = 500 μm. D, bar graph showing quantification of the wound area covered by cells in C. The area covered by cells was determined by calculating the difference between the wound area at 0 and 24 h expressed as percent of the wound area at 0 h. Results are shown as mean ± S.D. of 8–12 randomly chosen fields from three independent experiments. E, cell lysates from cells treated as in C were immunoblotted to assess the efficiency of siRNA depletion of Gα_i3 (∼95%) and the expression of rGα_i3 WT or rGα_i3 W258F.

FIGURE 10.

Sequence comparison and three-dimensional view of the newly identified structural determinant in Gα_i3 required for its activation by GIV. A, representative members of different Gα subfamilies were aligned to compare the sequence corresponding to the newly identified structural determinant in Gα_i3. Alignment of rat Gα_o, Gα_i1, Gα_i2, Gα_i3, Gα₁₂, Gα₁₃, Gα_q, and Gα_s sequences obtained form the NCBI data base was performed using ClustalW. Conserved identical residues are in black; similar residues are shaded in gray. The secondary structure elements (α = α-helix, β = β-sheet) corresponding to these sequences are named according to previously reported crystal structures (29, 33) and are indicated below the alignment. The arrow denotes the position corresponding to the Trp-258 of Gα_i3, and the asterisk denotes the previously identified binding site for GIV (11). B, three-dimensional view of the GEF motif of GIV bound to Gα_i3 is shown to depict the relative location of the newly identified structural determinant (Trp-258 in the α3/β5 loop), which is positioned C-terminal to the GEF motif. The homology model of GDP·Gα_i3 in complex with the GEF motif of GIV generated as described previously (11) using the structure of the synthetic peptide KB-752 bound to Gα_i1 (PDB: 1Y3A) as a template (18). The “Ras-like” domain of Gα_i3 is shown in blue, the “switch II” region in green, and Trp-258 in yellow. The GEF motif of GIV is shown in red.