G Protein binding sites on Calnuc (nucleobindin 1) and NUCB2 (nucleobindin 2) define a new class of G(alpha)i-regulatory motifs

Abstract

Heterotrimeric G proteins are molecular switches modulated by families of structurally and functionally related regulators. GIV (Gα-interacting vesicle-associated protein) is the first non-receptor guanine nucleotide exchange factor (GEF) that activates Gα(i) subunits via a defined, evolutionarily conserved motif. Here we found that Calnuc and NUCB2, two highly homologous calcium-binding proteins, share a common motif with GIV for Gα(i) binding and activation. Bioinformatics searches and structural analysis revealed that Calnuc and NUCB2 possess an evolutionarily conserved motif with sequence and structural similarity to the GEF sequence of GIV. Using in vitro pulldown and competition assays, we demonstrate that this motif binds preferentially to the inactive conformation of Gα(i1) and Gα(i3) over other Gα subunits and, like GIV, docks onto the α3/switch II cleft. Calnuc binding was impaired when Lys-248 in the α3 helix of Gα(i3) was replaced with M, the corresponding residue in Gα(o), which does not bind to Calnuc. Moreover, mutation of hydrophobic residues in the conserved motif predicted to dock on the α3/switch II cleft of Gα(i3) impaired the ability of Calnuc and NUCB2 to bind and activate Gα(i3) in vitro. We also provide evidence that calcium binding to Calnuc and NUCB2 abolishes their interaction with Gα(i3) in vitro and in cells, probably by inducing a conformational change that renders the Gα(i)-binding residues inaccessible. Taken together, our results identify a new type of Gα(i)-regulatory motif named the GBA motif (for Gα-binding and -activating motif), which is conserved across different proteins throughout evolution. These findings provide the structural basis for the properties of Calnuc and NUCB2 binding to Gα subunits and its regulation by calcium ions.

FIGURE 1.

Identification of a putative Gα-binding motif in Calnuc and NUCB2. A, a sequence at the end of the Calnuc (aa 309–324) and NUCB2 (aa 311–326) second EF-hand shows a similarity to the GEF motif of GIV (aa 1678–1693) and the synthetic KB-752 and GSP peptides. Rat Calnuc, rat NUCB2, and human GIV sequences were obtained from the NCBI and aligned with the KB-752 (15) and GSP (33) sequences using ClustalW. Conserved identical residues are shaded in black and similar residues in gray. A consensus sequence of 7 aa based on this alignment and the phylogenetic analysis of Calnuc (Fig. S1) and GIV (14) is indicated. B, the Calnuc putative Gα_i-binding motif has a structural similarity to the KB-752 peptide and the GEF motif of GIV. The coordinates of the Calnuc putative Gα-binding sequence (aa 309–320 (red)) were extracted from the Protein Data Bank (ID code: 1SNL) and the coordinates for the GEF motif of GIV (aa 1678–1689 (green)) from a previously described homology model (14). Both were threaded over the structure of KB-752 (yellow) in complex with Gα_i1 (blue) (Protein Data Bank ID code: 1Y3A) using ICM-Browser-Pro. The Calnuc putative Gα_i-binding motif, the GEF motif of GIV, and KB-752 form a helix in which the side chains of hydrophobic residues corresponding to positions 3, 6, and 7 from the consensus sequence depicted in A dock onto the α3/SwII hydrophobic cleft of the Gα_i subunits (cyan surface).

FIGURE 2.

Calnuc and NUCB2 bind inactive but not active Gα_i3. A, His-Gα_i3·GDP (lane 4) but not His-Gα_i3·GDP·AlF₄⁻ (lane 5) or His-Gα_i3·GTPγS (lane 6) binds to GST-Calnuc. No binding of Gα_i3 to GST was detected under any of the conditions tested (lanes 1–3). 6 μg of His-Gα_i3 preloaded with GDP (lanes 1 and 4), GDP and AlF₄⁻ (lanes 2 and 5), or GTPγS (30 μm, lanes 3 and 6) were incubated with ∼20 μg of purified GST (lanes 1–3) or GST-Calnuc (lanes 4–6) 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 (middle panel), and equal loading of His-Gα_i3 by His immunoblotting (lower panel). B, GST-Gα_i3·GDP (lane 5) but not GST-Gα_i3·GDP·AlF₄⁻ (lane 3) or GST (lanes 2 and 4) binds His-NUCB2. 10 μg of His-NUCB2 was incubated with ∼15 μg of purified GST (lanes 2 and 4) or GST-Gα_i3 (lanes 3 and 5) preloaded with GDP (lanes 4 and 5) or GDP·AlF₄⁻ (lanes 2 and 3) immobilized on glutathione beads and analyzed as described in A. Input (lane 1), 1 μg of His-NUCB2. C, His-Gα_i3·GDP binds to GST-Calnuc and GST-NUCB2-(173–333) with a K_d of 3.7 ± 1.2 μm (n = 4) and 1.0 ± 0.3 μm (n = 3), respectively. Increasing concentrations of His-Gα_i3·GDP (0.3, 0.5, 1, 1.5, 3, 5, 10, and 20 μm) were incubated with 20 μg of GST-Calnuc (closed circles) or GST-NUCB2 (open circles) and analyzed as in A. His-Gα_i3 binding was determined by quantitative immunoblotting using an Odyssey infrared imaging system, and data were fitted to a nonlinear, one-site binding hyperbola (solid lines) using Prism 4.0.

FIGURE 3.

Calnuc and NUCB2 compete with KB-752 and GIV for binding to inactive Gα_i3. A, the KB-752 peptide but not a control peptide (see “Experimental Procedures”) competes with GST-Calnuc for binding to His-Gα_i3·GDP. Increasing concentrations of the KB-752 peptide (0, 0.1, 1, 3, 10, and 30 μm) or control peptide (30 μm) were incubated with 20 μg (∼0.9 μm) of GST-Calnuc in the presence of 6 μg (∼0.5 μm) of His-Gα_i3 preloaded with GDP and analyzed as described in the legend for Fig. 2A. No binding of His-Gα_i3·GDP GST was detected. Input, 0.2 μg of His-Gα_i3·GDP. IB, immunoblot. B, a peptide corresponding to the Gα-binding motif of Calnuc 309–324 peptide, but not a control peptide, competes with His-GIV-CTs (aa 1660–1870, containing the GIV GEF motif) for binding to GST-Gα_i3·GDP. Increasing concentrations of Calnuc 309–324 peptide (0, 0.3, 1, 3, 10, 30, and 100 μm) or a control peptide (100 μm) were incubated with 3 μg (∼250 nm) of GST-Gα_i3 preloaded with GDP immobilized on glutathione beads in the presence of 2 μg (∼250 nm) of His-GIV-CTs and analyzed as described in the legend for Fig. 2A. No binding of His-GIV-CTs to GST is detected. Input, 0.1 μg of His-GIV-CTs. C, the KB-752 peptide but not a control peptide (see “Experimental Procedures”) competes with GST-NUCB2 for binding to His-Gα_i3·GDP. Increasing concentrations of the KB-752 peptide (0, 0.1, 1, 3, 10, and 30 μm) or the control peptide (30 μm) were incubated with 20 μg (∼1.7 μm) of GST-NUCB2 in the presence of 6 μg (∼0.5 μm) of His-Gα_i3 preloaded with GDP and analyzed as described in the legend for Fig. 2A. No binding of His-Gα_i3·GDP to GST is detected. Input, 0.2 μg of His-Gα_i3·GDP. D, His-GIV-CTs WT but not the Gα_i binding-deficient His-GIV-CTs F1685A (FA) (14) competes with GST-NUCB2 for binding to His-Gα_i3·GDP. Increasing concentrations of His-GIV-CTs WT (∼0, 0.26, 0.5, 0.8, 1.3, and 2 μm) or His-GIV-CTs F1685A (2 μm) were incubated with 20 μg (∼1.7 μm) of GST-NUCB2 in the presence of 5 μg (∼0.4 μm) of His-Gα_i3 preloaded with GDP and analyzed as described in the legend for Fig. 2A. No binding of His-Gα_i3·GDP to GST was detected. Input, 0.15 μg of His-Gα_i3·GDP.

FIGURE 4.

Calnuc binds to different Gα_i subunits but not to Gα_o or Gα_s. A, Upper panel, GST-Calnuc binds Gα_i3 (lane 3) but not Gα_o (lane 7) or Gα_s (lane 11) from rat brain membrane lysates in the presence of GDP but not GDP·AlF₄⁻ (lanes 4, 8, and 12). Solubilized proteins from 750 μg of rat brain membranes were incubated with ∼20 μg of purified GST (lanes 2, 6, and 10) or GST-Calnuc (lanes 3, 4, 7, 8, 11, 12, and 13) immobilized on glutathione beads in the presence of GDP (30 μm; lanes 2, 3, 6, 7, 10, and 11) or GDP and AlF₄⁻ (AlCl₃, 30 μm; NaF, 10 mm; lanes 4, 8, and 12). An additional control without rat brain membrane lysate was performed to validate Gα_s antibody specificity (lane 13). Input (lanes 1, 5, and 9), 10% of the membrane lysate. No binding of Gα_i3, Gα_o, or Gα_s to the negative control GST was detected (lanes 2, 6, and 10). The arrows (lanes 1, 3, 5, and 9) denote the specific bands corresponding to the different Gα subunits (including the long and short splice forms of Gα_s, lane 9), and the star (lanes 11, 12, and 13) denotes a nonspecific band recognized by the Gα_s antibody. IB, immunoblot. Lower panel, equal loading of GST proteins was confirmed by Ponceau S staining. B, His-CalnucΔN binds to GST-Gα_i1·GDP (lane 3) and GST-Gα_i3·GDP (lane 5) to a greater extent (∼20-fold) than to GST-Gα_i2·GDP (lane 4) and binds only marginally to either of the Gα_i subunits preloaded with GDP·AlF₄⁻ (lanes 6, 7, and 8) or GST (lane 2). 10 μg of His-CalnucΔN was incubated with 15 μg of GST (lane 2), GST-Gα_i1 (lanes 3 and 6), GST-Gα_i2 (lanes 4 and 7), or GST-Gα_i3 (lanes 5 and 8) preloaded with GDP (lanes 2–5) or GDP·AlF₄⁻ (lanes 6–8), immobilized on glutathione beads, and analyzed as described for Fig. 2A. Input (lane 1), 1 μg of His-CalnucΔN.

FIGURE 5.

Lys-248 but not Trp-258 is responsible for preferential binding of Calnuc to Gα_i3versus Gα_o. A, sequence alignment of Gα_o, Gα_i1, Gα_i2, and Gα_i3 indicating the Gα_i3 and Gα_o mutants studied. Rat Gα_o, Gα_i1, Gα_i2, and Gα_i3 sequences corresponding to the SwII and the α3 helix were obtained from the NCBI database and aligned using ClustalW. Conserved identical residues are shaded in black and similar residues in gray. The secondary structure elements (α = α-helix, β = β-sheet) indicated below the alignment are named according to their crystal structures. Residues within this region that are conserved among Gα_i1, Gα_i2, and Gα_i3 but are different in Gα_o were mutated in Gα_i3 to the corresponding residues in Gα_o (indicated below with arrows) or in Gα_o to the corresponding residues in Gα_i3 (indicated above with arrows). B, wild-type His-Gα_i3·GDP (lane 1) and His-Gα_i3·GDP W258F (lane 5) but not wild-type His-Gα_o·GDP (lane 3) or His-Gα_o·GDP F259W (lane 7) bind to GST-Calnuc. No binding of any of the Gα subunits loaded with GDP·AlF₄⁻ to GST-Calnuc was detected (lanes 2, 4, 6, and 8). 6 μg of His-Gα_i3 (lanes 1 and 2), His-Gα_o (lanes 3 and 4), His-Gα_i3 W258F (lanes 5 and 6), or His-Gα_o F259W (lanes 7 and 8) preloaded with GDP (lanes 1, 3, 5, and 7) or GDP and AlF₄⁻ (lanes 2, 4, 6, and 8) was incubated with ∼20 μg of purified GST-Calnuc immobilized on glutathione beads and analyzed as described in the legend for Fig. 2A. C, His-CalnucΔN binding to GST-Gα_i3 K248M·GDP (Gα_i3 K_m (lane 4)) is reduced ∼80% compared with wild-type GST-Gα_i3·GDP (Gα_i3 WT, lane 3). No binding of His-CalnucΔN to GST (lane 2) or GDP·AlF₄⁻-loaded Gα_i3 (lanes 5 and 6) is detected. 10 μg of His-CalnucΔN was incubated with purified GST (lane 2), wild-type GST-Gα_i3 (lanes 3 and 5), or GST-Gα_i3 K248M (lanes 4 and 6) preloaded with GDP (lanes 2–4) or GDP·AlF₄⁻ (lanes 5 and 6) immobilized on glutathione beads and analyzed as described in the legend for Fig. 2A. Input (lane 1), 1 μg of His-CalnucΔN. IB, immunoblot. D, wild-type His-Gα_i3·GDP (Gα_i3 WT (lane 4)) and His-Gα_o M249K·GDP (Gα_o MK (lane 6)) but not wild-type His-Gα_o (Gα_o WT (lane 5)) bind to GST-Calnuc. No binding of any of the Gα subunits to GST is detected (lanes 1–3). 6 μg of each His-Gα subunit preloaded with GDP was incubated with ∼20 μg of purified GST (lanes 1–3) or GST-Calnuc (lanes 4–6) immobilized on glutathione beads and analyzed as in the legend for Fig. 2A.

FIGURE 6.

Identification of critical residues in Calnuc required for binding Gα_i3. A, His-Gα_i3·GDP binds to wild-type GST-Calnuc (lane 3) but not to GST-Calnuc mutants L313A (lane 4), F316A (lane 5), L317A (lane 6), or L313A/L317A (lane 7) or GST alone (lane 2). 6 μg of His-Gα_i3 preloaded with GDP (30 μm) was incubated with ∼20 μg of purified GST (lane 2), wild-type GST-Calnuc (lane 3), or GST-Calnuc mutants L313A (lane 4), F316A (lane 5), L317A (lane 6), or L313A/L317A (lane 7) immobilized on glutathione beads and analyzed as described in the legend for Fig. 2A. Input (lane 1), 0.1 μg of His-Gα_i3. IB, immunoblot. B, His-Gα_i3·GDP binds to wild-type GST-NUCB2-(173–333) (lane 3) but not to GST-NUCB2 mutants F318A (lane 4) or L315A/L319A (lane 5) or to GST alone (lane 2). 6 μg of His-Gα_i3 preloaded with GDP (30 μm) was incubated with ∼20 μg of purified GST (lane 2), wild-type GST-NUCB2 (lane 3), or GST-Calnuc mutants L313A (lane 4) or L315A/L319A (lane 5) immobilized on glutathione beads and analyzed as described in the legend for Fig. 2A. Input (lane 1), 0.1 μg of His-Gα_i3.

FIGURE 7.

Calnuc and NUCB2 activate Gα_i3. A, His-CalnucΔN WT but not the L313A/L317A (LL/AA) mutant increases the steady-state GTPase activity of His-Gα_i3. The steady-state GTPase activity of purified His-Gα_i3 (50 nm) was determined in the absence (closed circles) or presence of 60 μm purified wild-type His-CalnucΔN (open circles) or His-CalnucΔN L315A/L319A mutant (inverted triangles) by quantifying the amount of [γ-³²P]GTP (0.5 μm, ∼50 cpm/fmol) hydrolyzed at the indicated time points. Results are shown as mean ± S.D. of one representative experiment of three performed in duplicate. B, WT GST-NUCB2-(173–333) but not the L315A/L319A mutant increases the steady-state GTPase activity of His-Gα_i3. The steady-state GTPase activity of purified His-Gα_i3 (100 nm) was determined exactly as described in A except that 50 μm NUCB2 was used. C, dose-dependent activation of His-Gα_i3 by His-CalnucΔN and GST-NUCB2. The steady-state GTPase activity of purified His-Gα_i3 was determined in the presence of the indicated amounts of purified wild-type His-CalnucΔN (closed circles) or GST-NUCB2 (open circles) by quantification of the amount of [γ-³²P]GTP (0.5 μm, ∼50 cpm/fmol) hydrolyzed in 10 min. Data expressed as percent of GTP hydrolyzed by the G protein alone (0 μm) were fitted to a nonlinear, one-site hyperbola (solid line) using Prism 4.0. Results are shown as mean ± S.E. of the indicated number of experiments (n). D, His-CalnucΔN increases the rate of GTPγS binding of His-Gα_i3. Nucleotide exchange activity of purified His-Gα_i3 (50 nm) was determined in the absence (closed circles) or presence of 25 μm purified wild-type His-CalnucΔN (open circles) by quantification of the amount of [³⁵S]GTPγS (0.5 μm, ∼50 cpm/fmol) bound at the indicated time points. No significant binding of GTPγS binding was detected in the absence of His-Gα_i3 or the presence of His-CalnucΔN alone (not shown). Results are shown as mean ± S.D. of one representative experiment of four performed in duplicate.

FIGURE 8.

Effect of Ca²⁺ on Calnuc and NUCB2 binding to Gα_i3. A, structural view of the Gα_i-binding motif of Calnuc in the calcium-bound conformation. The coordinates of the NMR-resolved structure of Calnuc were extracted from the Protein Data Bank (ID code: 1SNL) and visualized using ICM-Browser-Pro. Residues of the Gα_i-binding motif of Calnuc required for the interaction with Gα_i3 (Fig. 6) are not solvent-exposed in the calcium-bound conformation of Calnuc because they were utilized to make an intramolecular contact. B, His-Gα_i3 binding to GST-Calnuc is virtually abolished in the presence of CaCl₂. 6 μg of His-Gα_i3 preloaded with GDP (30 μm) was incubated with ∼20 μg of purified GST (lanes 1 and 3) or GST-Calnuc (lanes 2 and 4) immobilized on glutathione beads in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 8 mm CaCl₂ (∼3 mm free Ca²⁺). Subsequent steps were performed as described in the legend for Fig. 2A. C, binding of GST-Gα_i3 to His-NUCB2 is virtually abolished in the presence of CaCl₂. 10 μg of His-NUCB2 was incubated with purified GST (lanes 1 and 3) or GST-Gα_i3 (lanes 2 and 4) immobilized on glutathione beads in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 8 mm CaCl₂ (∼3 mm free Ca²⁺). Subsequent steps were performed as described in the legend for Fig. 2B. Input (lane 5), 1 μg of His-NUCB2. D, activation of His-Gα_i3 by His-CalnucΔN is inhibited by CaCl₂ in a dose-dependent manner. Nucleotide exchange activity of purified His-Gα_i3 (50 nm) at the indicated concentrations of CaCl₂ was determined in the absence (closed circles) or presence of 25 μm purified wild-type His-CalnucΔN (open circles) by quantification of the amount of [³⁵S] GTPγS (0.5 μm, ∼50 cpm/fmol) bound at 20 min. Results are shown as mean ± S.D. of one representative of three independent experiments performed in duplicate. CaCl₂ does not affect the basal activity of Gα_i3 alone (closed circles). E, stimulation of COS-7 cells with thapsigargin or ATP inhibits the interaction of Calnuc with Gα_i3. Upper panels, co-immunoprecipitation of ΔSS-Calnuc-CFP but not Gβγ with Gα_i3-FLAG is dramatically reduced after stimulation with thapsigargin (TG) (lane 3) or ATP (lane 4) compared with unstimulated cells (lane 2). No ΔSS-Calnuc-CFP or Gβγ was detected in FLAG immunoprecipitates of cells transfected with vector control (lane 1). COS-7 cells were transfected with empty vector (lane 1) or plasmids encoding Gα_i3-FLAG and ΔSS-Calnuc-CFP (lanes 2, 3, and 4), stimulated with thapsigargin (lane 3) or ATP (lane 4) and immunoprecipitated (IP) as described under “Experimental Procedures.” Immunoprecipitation was followed by immunoblotting (IB) for FLAG (Gα_i3), GFP (ΔSS-Calnuc-CFP), and Gβ. Equal IgG loading was confirmed by Ponceau S staining. Lower panels, aliquots of the lysates (10%) were analyzed by immunoblotting to confirm equal loading of Gα_i3, Calnuc, Gβ, and α-tubulin. F, schematic illustration of how Ca²⁺ binding to Calnuc mediates a molecular rearrangement that prevents Gα_i3 binding. Left, in the absence of Ca²⁺, the Calnuc calcium-binding domain (black line with red semicircles) is disordered (34) allowing the exposure of the Gα_i-binding motif (aa 309–324 (blue box)). Right, in the presence of Ca²⁺, Calnuc undergoes a conformational change such that residues in the Gα_i-binding motif (blue box) make an intramolecular interaction (dotted lines) and binding to Gα_i3 is hindered because the Gα_i-binding motif is not exposed. Presumably the same occurs for the NUCB2 Gα_i-binding motif (aa 311–326) upon calcium binding.