DAPLE protein inhibits nucleotide exchange on Gαs and Gαq via the same motif that activates Gαi

Abstract

Besides being regulated by G-protein-coupled receptors, the activity of heterotrimeric G proteins is modulated by many cytoplasmic proteins. GIV/Girdin and DAPLE (Dvl-associating protein with a high frequency of leucine) are the best-characterized members of a group of cytoplasmic regulators that contain a Gα-binding and -activating (GBA) motif and whose dysregulation underlies human diseases, including cancer and birth defects. GBA motif-containing proteins were originally reported to modulate G proteins by binding Gα subunits of the G_i/o family (Gα_i) over other families (such as G_s, G_q/11, or G_12/13), and promoting nucleotide exchange in vitro However, some evidence suggests that this is not always the case, as phosphorylation of the GBA motif of GIV promotes its binding to Gα_s and inhibits nucleotide exchange. The G-protein specificity of DAPLE and how it might affect nucleotide exchange on G proteins besides Gα_i remain to be investigated. Here, we show that DAPLE's GBA motif, in addition to Gα_i, binds efficiently to members of the G_s and G_q/11 families (Gα_s and Gα_q, respectively), but not of the G_12/13 family (Gα₁₂) in the absence of post-translational phosphorylation. We pinpointed Met-1669 as the residue in the GBA motif of DAPLE that diverges from that in GIV and enables better binding to Gα_s and Gα_q Unlike the nucleotide-exchange acceleration observed for Gα_i, DAPLE inhibited nucleotide exchange on Gα_s and Gα_q These findings indicate that GBA motifs have versatility in their G-protein-modulating effect, i.e. they can bind to Gα subunits of different classes and either stimulate or inhibit nucleotide exchange depending on the G-protein subtype.

Keywords: G protein; G-protein–coupled receptor (GPCR); GTPase; Gα-binding and -activating (GBA); cell signaling; guanine nucleotide dissociation inhibitor (GDI); guanine nucleotide–exchange factor (GEF); protein–protein interaction.

Figure 1.

DAPLE binds efficiently to Gα_s and Gα_q through its GBA motif. A, bar diagrams depicting the domains of DAPLE (left) and GIV (right) and the fragments of each one fused to GST used for experiments shown in this figure. B, DAPLE binds efficiently to Gα_i3, Gα_s, and Gα_q but not to Gα₁₂, whereas GIV only binds efficiently to Gα_i3 among the G proteins tested. Lysates of HEK293T cells transfected with Gα_i3–FLAG, Gα_s, Gα_q–HA, and Gα₁₂–MYC were incubated with GST, GST–DAPLE, or GST–GIV immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. C, DAPLE WT but not DAPLE F1675A (FA) binds to purified Gα_i3, Gα_s, and Gα_q. His–Gα_i3, His–Gα_s, or His–Gα_q* were incubated with GST, GST–DAPLE (WT or FA mutant), or GST–GIV immobilized on GSH-agarose beads, and bead-bound proteins were detected by Ponceau S staining or IB as indicated. D, DAPLE WT, but not DAPLE FA mutant, co-immunoprecipitates with Gα_i3, Gα_s, and Gα_q. Lysates of HEK293T cells co-expressing full-length MYC–DAPLE (WT or FA mutant) with the indicated FLAG-tagged G proteins (or no tagged G protein as negative control) were subjected to IP with a FLAG antibody, and bound proteins were detected by IB as indicated. The lower immunoblot panels (lysates) correspond to aliquots of the starting material used for IPs shown in the upper panels (IP: FLAG). All results presented in this figure are representative of at least three independent experiments (n ≥3).

Figure 2.

DAPLE binds preferentially to inactive versus active Gα_s or Gα_q. A, binding of DAPLE to the constitutively-active Gα_s mutant R201C is diminished compared with Gα_s WT. Lysates of HEK293T cells expressing Gα_s WT, or Gα_s R201C were incubated with GST or GST–DAPLE immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. B, binding of DAPLE to Gα_s loaded with GDP·AlF₄⁻ or with GTPγS is diminished compared with binding to Gα_s loaded with GDP. Lysates of HEK293T cells expressing Gα_s were incubated with nucleotides as indicated under “Experimental procedures” and incubated with GST or GST–DAPLE immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. C, binding of DAPLE to the constitutively-active Gα_q mutant Q209L is diminished compared with Gα_q WT. Lysates of HEK293T cells expressing Gα_q–HA WT or Gα_q–HA Q209L were incubated with GST or GST–DAPLE immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. D, binding of DAPLE to Gα_q loaded with GDP·₄⁻ is diminished compared with binding to Gα_q loaded with GDP. Lysates of HEK293T cells expressing Gα_q were incubated with nucleotides as indicated under Experimental procedures” and incubated with GST or GST–DAPLE immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. All results presented in this figure are representative of two independent experiments (n = 2).

Figure 3.

Met-1669 in DAPLE is responsible for its enhanced binding to Gα_s and Gα_q compared with GIV. A, DAPLE binding to Gα_s or Gα_q, but not to Gα_i3, is reduced upon replacing its GBA motif with that of GIV. Upper panel, diagram depicting the alignment of the GBA motifs of DAPLE and GIV and the sequence of the GBA motif of the DAPLE/GIV GBA chimera 1 (ch1) containing GIV's GBA motif residues (red) grafted into DAPLE's sequence (black). Lower panel, purified His–Gα_i3, His–Gα_s, or His–Gα_q* was incubated with GST, GST–DAPLE (WT or ch1), or GST–GIV immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. The vertical dotted lines indicate that the images were assembled by splicing lanes from the same experiment and membrane. B, mapping of residues involved in the differential G-protein selectivity of DAPLE versus GIV. Upper panel, sequences of DAPLE/GIV GBA chimeras (ch1–7, M1669V, and S1666G), with the GIV residues replaced in DAPLE indicated in red. Lower panel, purified His–Gα_i3, His–Gα_s, or His–Gα_q* was incubated with GST or GST–DAPLE (WT or mutants) immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. C, validation of the effects of DAPLE F1675A (FA) and M1669V (MV) mutations on binding to different G proteins using a shorter DAPLE-purified protein. Upper panel, diagram depicting the GST–DAPLE (short) construct used in this panel along with a diagram of the previously used GST–DAPLE construct. Lower panel, purified His–Gα_i3, His–Gα_s, or His–Gα_q* was incubated with GST or GST–DAPLE (WT or mutants) immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. All results presented in this figure are representative of at least three independent experiments (n ≥3).

Figure 4.

M1669V mutation in full-length DAPLE disrupts binding to Gα_s or Gα_q but not to Gα_i3. A, co-immunoprecipitation experiments comparing the effect of DAPLE M1669V and F1675A mutations on G-protein binding, which show that the former disrupts binding to Gα_s and Gα_q, but not to Gα_i3, whereas the latter disrupts binding to all G proteins tested. Lysates of HEK293T cells co-expressing full-length MYC–DAPLE (WT or mutants) with the indicated FLAG-tagged G proteins (or no tagged G protein as negative control) were subjected to IP with a FLAG antibody, and bound proteins were detected by IB as indicated. The lower immunoblot panels (Lysates) correspond to aliquots of the starting material used for IPs shown in the upper panels (IP: FLAG). One representative experiment of four is shown for Gα_s and Gα_q (n = 4), or one representative experiment of two is shown for Gα_i3 (n = 2). B, comparison of DAPLE Met-1669 and GIV Val-1679 in the context of their respective Gα_i3/GBA motif complex structures. Left panel, homology model of DAPLE GBA motif (green, ribbon representation) in complex with Gα_i3 (blue, space-filling representation) was generated using the X-ray crystal structure of the Gα_i3/GIV GBA motif complex (PDB code 6MHF). The area of the Gα_i3/DAPLE structure model within the dotted box is shown enlarged in the middle panel to illustrate that Met-1669 is largely solvent-exposed. Right panel, detail of the structure of Gα_i3 in complex with GIV GBA motif (brown) showing that Val-1669 is also largely solvent-exposed. C, proposed model for the structural basis of DAPLE's G-protein selectivity. Much like GIV Val-1679, DAPLE Met-1669 does not make direct contact with Gα_i3. In contrast, DAPLE Met-1669 is required for binding to Gα_s or Gα_q, suggesting that it makes a contact with these proteins that is not allowed by the shorter chain of the valine located in the corresponding position in GIV.

Figure 5.

DAPLE inhibits nucleotide exchange on Gα_s via its GBA motif. A and B, DAPLE GBA peptide decreases the steady-state GTPase activity of Gα_s. A representative time course of the steady-state GTPase activity of His–Gα_s alone (black), in the presence of DAPLE GBA peptide (30 μm, red), or control peptide (30 μm, blue) is shown in A, and quantification of the dose-dependent effect of the peptides is shown in B (mean ± S.E., n = 3). Results are presented as raw production of free [³²P]P_i (pmol) in A or percent change relative to the production of free [³²P]P_i by Gα_s alone at 10 min (% of control) in B. Average IC₅₀ value was determined as described under “Experimental procedures.” C and D, DAPLE GBA peptide decreases the rate of GTPγS binding to Gα_s. A representative time course of [³⁵S]GTPγS binding to His–Gα_s in the absence (black) or presence of DAPLE GBA peptide (30 μm, red) is shown in C, and quantification of the dose-dependent effect of the DAPLE GBA peptide is shown in D (mean ± S.E., n = 3). Results are presented as raw [³⁵S]GTPγS binding (picomoles) in C or percent change relative to [³⁵S]GTPγS binding to Gα_s alone at 10 min (% of control) in D. Rate constants and average IC₅₀ values were determined as described under “Experimental procedures,” E and F, purified DAPLE WT (amino acids 1650–2028), but not F1675A mutant, decreases GTPγS binding to Gα_s. A representative time course of [³⁵S]GTPγS binding to His–Gα_s in the absence (black) or presence of purified His–DAPLE (9 μm, red) is shown in E, and quantification of the effect of His–DAPLE WT (3.3 μm, red) compared with His–DAPLE F1675A (3.3 μm, blue) is shown in D (mean ± S.E., n = 3). Results are presented as raw [³⁵S]GTPγS binding (picomoles) in E or percent change relative to [³⁵S]GTPγS binding to Gα_s alone at 10 min (% of control) in F. Rate constants determined as described under “Experimental procedures.”

Figure 6.

DAPLE inhibits nucleotide exchange on Gα_q via its GBA motif. A and B, DAPLE GBA peptide decreases the rate of GTPγS binding to Gα_q. A representative time course of [³⁵S]GTPγS binding to Gα_q in the absence (black) or presence of DAPLE GBA peptide (30 μm, red) using a buffer that contains 0.2 m (NH₄)₂SO₄ is shown in A, and quantification of the dose-dependent effect of DAPLE GBA peptide (red) or control peptide (blue) in a buffer that contains 0.2 m (NH₄)₂SO₄ is shown in B (mean ± S.E., n = 3). Results are presented as raw [³⁵S]GTPγS binding (picomoles) in A or percent change relative to [³⁵S]GTPγS binding to Gα_q alone at 45 min (% of control) in B. Rate constants and average IC₅₀ values were determined as described under “Experimental procedures.” C, purified DAPLE WT (amino acids 1650–2028), but not F1675A mutant, decreases GTPγS binding to Gα_q. Quantification of the effect of His–DAPLE WT (3.3 μm, red) compared with His–DAPLE F1675A (3.3 μm, blue) in a buffer that contains 0.2 m (NH₄)₂SO₄ is presented as percent change relative to [³⁵S]GTPγS binding to Gα_q alone at 45 min (% of control, mean ± S.E., n = 3).

Figure 7.

Proposed model. Top, DAPLE (green) binds to Gα_i (blue) to stabilize a G-protein conformation that favors nucleotide exchange. DAPLE Met-1669 is not required for efficient binding to Gα_i. Bottom, Met-1669 in DAPLE allows its efficient physical engagement to Gα_s and Gα_q (red), which in turn stabilizes a G-protein conformation that prevents nucleotide exchange.