ric 8a  (Proteintech)

Journal: The Journal of Biological Chemistry

Article Title: The guanine nucleotide exchange factor Ric-8A regulates the sensitivity of constitutively active Gαq to the inhibitor YM-254890

doi: 10.1016/j.jbc.2025.108426

Figure Lengend Snippet: Loss of Ric-8A leads to YM-sensitivity of αqAG-QL and increases sensitivity of αqQL to YM. A , TEAD luciferase assay in RIC-8A KO cells transfected with either pcDNA3 (negative control), αqQL, αqAG-QL, and increasing amounts of RIC-8A as indicated. TEAD luciferase 8X-GTIIC reporter and renilla luciferase were cotransfected. Two hours after transfection, media were changed to serum-free and 1 μM YM was added overnight where indicated. Cells were lysed the following day, and luciferase experiments were performed. TEAD luciferase values were normalized to renilla luciferase values for the same well and averaged for each condition. Results are shown as mean ± SD and statistical significance is indicated. (n = 3, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001, two-way ANOVA, Šidák's multiple comparison’s test). αq protein expression was assessed by immunoblotting. B , immunoblot for Ric-8A demonstrating endogenous levels in q/11 KO cells in comparison to exogenously expressed levels in RIC-8A KO cells. Endogenous Ric-8A migrates slightly slower than the plasmid-expressed Ric-8A, and this is indicated by the two lines at the Ric-8A immunoblot. This difference may be due to alternative methionine initiation or slight proteolysis of the exogenously expressed Ric-8A. C , YM IC50 analysis readout by the TEAD luciferase reporter comparing RIC-8A KO and q/11 KO cells transfected with αqQL and treated with YM concentrations ranging from 0.0001 to 1 μM. YM treatments were overnight in serum-free media (n = 3).

Article Snippet: The pERK1/2 (Cat. #9101S) and ERK1/2 (Cat. #4696S) antibodies were from Cell Signaling Technologies. αq (Cat. #13927-1-AP), GAPDH (Cat. #60004-1-Ig), and Ric-8A (Cat. #66625-1-Ig) were from Proteintech.

Techniques: Luciferase, Transfection, Negative Control, Expressing, Western Blot, Comparison, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: The guanine nucleotide exchange factor Ric-8A regulates the sensitivity of constitutively active Gαq to the inhibitor YM-254890

doi: 10.1016/j.jbc.2025.108426

Figure Lengend Snippet: Ric-8A only exerts its effects on YM-sensitivity in the context of CA αq, not αq WT. A and B , SRE luciferase reporter assays in RIC-8A KO cells transfected with pcDNA3 (negative control), αqQL, or αq WT, and increasing amounts of RIC-8A where indicated, along with SRE and renilla luciferase plasmids. For αq WT experiments ( B ), m3AChR was coexpressed. Two hours after transfection, media were changed to serum-free and 1 μM YM was added overnight where indicated. Cells were lysed the following day, and luciferase experiments were run. B , to stimulate αq WT, 100 μM carbachol was added 1 h after YM-treatment. SRE values are normalized to respective renilla values for the same well and averaged for each condition. Results are shown as mean ± SD and statistical significance is indicated. (n = 3, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001, two-way ANOVA, Šidák's multiple comparison’s test). C , lysates were immunoblotted to assess αq protein levels. m3AChR, muscarinic acetylcholine m3 receptor; SRE, serum response element.

Article Snippet: The pERK1/2 (Cat. #9101S) and ERK1/2 (Cat. #4696S) antibodies were from Cell Signaling Technologies. αq (Cat. #13927-1-AP), GAPDH (Cat. #60004-1-Ig), and Ric-8A (Cat. #66625-1-Ig) were from Proteintech.

Techniques: Luciferase, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The guanine nucleotide exchange factor Ric-8A regulates the sensitivity of constitutively active Gαq to the inhibitor YM-254890

doi: 10.1016/j.jbc.2025.108426

Figure Lengend Snippet: αqAG-QP YM sensitivity is directly altered by Ric-8A. A , TEAD luciferase assay in RIC-8A KO cells transfected with either pcDNA3 (negative control), αqQP, αqAG-QP, and increasing amounts of RIC-8A as indicated. TEAD luciferase 8X-GTIIC reporter and renilla luciferase were cotransfected. Two hours after transfection, media were changed to serum-free and 1 μM YM was added overnight where indicated. Cells were lysed the following day and luciferase experiments were performed. TEAD luciferase values were normalized to renilla luciferase values for the same well and averaged for each condition. B and C , SRE-luciferase reporter assays in q/11 KO cells transfected with pcDNA3 (negative control), αqQL/P, or αqAG-QL/P, and SRE and renilla luciferase plasmids. Where indicated, increasing amounts of RIC-8A were added . The same protocol was followed as in A. Lysates were immunoblotted to assess αq protein levels ( bottom , B ). SRE values are normalized to their respective renilla value and averaged for each condition. Results are shown as mean ± SD and statistical significance is indicated. (n = 3, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.005, two-way ANOVA, Šidák's multiple comparison’s test). SRE, serum response element.

Article Snippet: The pERK1/2 (Cat. #9101S) and ERK1/2 (Cat. #4696S) antibodies were from Cell Signaling Technologies. αq (Cat. #13927-1-AP), GAPDH (Cat. #60004-1-Ig), and Ric-8A (Cat. #66625-1-Ig) were from Proteintech.

Techniques: Luciferase, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The guanine nucleotide exchange factor Ric-8A regulates the sensitivity of constitutively active Gαq to the inhibitor YM-254890

doi: 10.1016/j.jbc.2025.108426

Figure Lengend Snippet: BRET studies reveal that αqAG-QL remains GTP-bound and active in the presence of YM, and Ric-8A can increase the amount of activated αqAG-QL and αqQL. A , schematic depicting bioluminescence resonance energy transfer (BRET) sensors for activated GTP-bound venus-αq interaction with NLuc-GRK2-RH, which would lead to high BRET signal upon addition of luciferase substrate. B and C , HEK293 ( B ) or HEK293 RIC-8A KO cells ( C ) were transfected with 25 ng of Nano-luciferase fused GRK2-RH (donor) and 500 ng of Venus-fused αq (acceptor). 300 ng of Gβ and 100 ng of Gγ were coexpressed. Additionally, RIC-8A KO cells were transfected with 10 ng of RIC-8A where indicated ( C ). The following day, respective samples were treated with 1 μM YM for 2 h and BRET measurements were read 2 min after addition of Nano-glo (Promega). Venus-αq WT values were subtracted as background from all emission ratios (acceptor/donor). Results are shown as mean ± SD and statistical significance indicated (n = 4 in HEK293 and n = 3 in HEK293 RIC-8A KO cells, ∗∗ p < 0.01; ∗∗∗ p < 0.005; ∗∗∗∗ p < 0.0001, two-way ANOVA, Šidák's multiple comparison’s test). D , validation of expression of venus-αq constructs in RIC-8A KO cells with and without Ric-8A and YM. RH, RGS-homology.

Article Snippet: The pERK1/2 (Cat. #9101S) and ERK1/2 (Cat. #4696S) antibodies were from Cell Signaling Technologies. αq (Cat. #13927-1-AP), GAPDH (Cat. #60004-1-Ig), and Ric-8A (Cat. #66625-1-Ig) were from Proteintech.

Techniques: Bioluminescence Resonance Energy Transfer, Luciferase, Transfection, Biomarker Discovery, Expressing, Construct

Journal: The Journal of Biological Chemistry

Article Title: The guanine nucleotide exchange factor Ric-8A regulates the sensitivity of constitutively active Gαq to the inhibitor YM-254890

doi: 10.1016/j.jbc.2025.108426

Figure Lengend Snippet: αqQL and αqAG-QL bind GRK2-RH more strongly in the presence of Ric-8A. A and B , cells were transfected with FLAG-GRK2-RH and either αqQL or αqAG-QL with and without 10 ng of RIC-8A . As negative controls, RIC-8A KO cells were transfected with pcDNA3, αq WT, FLAG-GRK2-RH, αq WT + FLAG-GRK2-RH, or αq WT + FLAG-GRK2-RH + 10 ng of RIC-8A. Three hours after transfection, 1 μM YM was added where indicated overnight. A , cells were lysed the day after transfection, and FLAG beads were used to immunoprecipitate FLAG-GRK2-RH, and the pulldowns and inputs subsequently immunoblotted for the proteins indicated. B , the αq pull-down signal intensities were quantified and normalized to their respective input signal intensity. Results are shown as mean ± SD. Statistical significance is indicated (n = 4, ∗ p < 0.05; ∗∗ p < 0.01, two-way ANOVA, Šidák's multiple comparison’s test). RH, RGS-homology.

Article Snippet: The pERK1/2 (Cat. #9101S) and ERK1/2 (Cat. #4696S) antibodies were from Cell Signaling Technologies. αq (Cat. #13927-1-AP), GAPDH (Cat. #60004-1-Ig), and Ric-8A (Cat. #66625-1-Ig) were from Proteintech.

Techniques: Transfection

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Ric-8A P3 peptide (400-432) , GenicBio , N/A , .

Techniques: Recombinant, Plasmid Preparation, Residue, Construct, Mutagenesis, Phospho-proteomics, Software, Purification

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet: ( A ) Ribbon representation of NCS protein structures bound to their targets. NCS-1/D2R (PDB: 5AER, ), NCS-1/Grk1 (PDB: 5AFP, ), Frq1/Pik1 (PDB: 2JU0, ), KChIP1/Kv4.3 (PDB: 2I2R, ), Recoverin/RK25 (PDB: 2I94, ), Frq2 (PDB: 4BY4, ). The N and C-terminal pairs of EF-hands (EF-1–2 and EF-3–4) are shown in cyan and purple respectively. The C-terminal helix H10 is shown in pink and target proteins in yellow. Ca 2+ is shown as hot pink spheres. ( B ) Top: Cryo-EM structure of the rRic-8A/Gα i1 complex (PDB: 6UKT, ). Gα i1 is depicted in silver. Ric-8A ARM-HEAT repeats 1–8 in blue and repeat 9 in green and helices H10 (rH10) and H11 (rH11) in orange and gold respectively. The C-terminal coiled regions are shown in magenta. Phosphorylated residues S435 and T440 are depicted as spheres. Bottom: rRic-8A sequence from residue 400 to the end. Helices are squared following the same color code used above. Phosphorylation sites are indicated as red spheres. P1, P2, and P3 brackets indicate the boundaries of the synthesized Ric-8A peptides.

Article Snippet: Three highly pure (>95%) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Cryo-EM Sample Prep, Sequencing, Residue, Synthesized

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet: ( A ) Ribbon representation of the hNCS-1ΔH10/Ric-8A-P3 complex. Two views are displayed. The NCS-1 structure is shown in light purple, while Ric-8A-P3 is shown in light pink. The N- and C-termini are indicated. Ca 2+ , Na + , and Cl - ions are shown in hot pink, yellow, and cyan, respectively. R1 and R2 helices, and EF-hands 2, 3, and 4 are indicated. The orange square represents a zoomed view of the R1-R2 loop in stick mode, Cl - coordination and H-bonds are displayed as yellow and gray dashes, respectively. Residues participating in R1-R2 contacts are displayed in hot pink (triad 1: I407-T410-A415), magenta (F406-L418), and purple (triad 2: K408-Y409-N414). ( B ) rRic-8A sequence from 400 to 442 residues. The helix boundaries of Ric-8A sequence encompassing a9 and b9 in different structural contexts (NCS-1/Ric-8A-peptide [PDB: 8AHY], Ric-8A/Gα [PDB: 6UKT, ] and uncomplexed Ric-8A [PDB: 6NMG, ]) are indicated as pink boxes and labeled. Coiled regions are shown in pink. Disordered regions are shown in gray, while phosphorylated sites are shown as red spheres. The interacting residues shown in panel ( A ) are indicated with dots in the same color code as in A . ( C ) Electrostatic surface potential of rRic-8A-P3. NCS-1 is shown as yellow ribbons. Positive and negative potentials are represented in blue and red, respectively. On the right, the Ric-8A region that faces and contacts NCS-1 is shown with NCS-1 removed for proper visualization. ( D ) Representative co-immunoprecipitation assay in HEK293 cells transfected with full-length hNCS-1 and V5-tagged hRic-8A mutants. Mutations on NCS-1 and Ric-8A are indicated in blue and pink, respectively. The numbering of the rat Ric-8A sequence has been maintained for proper comparison with A and B. Quantifications of each lane from three independent experiments (mean ± SD) are shown on the right. Mean differences were analyzed by two-tailed, paired Student’s t-test, comparing with wild-type NCS-1 and Ric-8A. **p=0.01; *p=0.05. Figure 3—source data 1. Original WBs.

Article Snippet: Three highly pure (>95%) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Sequencing, Labeling, Co-Immunoprecipitation Assay, Transfection, Comparison, Two Tailed Test

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet: ( A ) Structure 1 showing the 2F o -F c electron density map (green) of Ric-8A-P2 (stick mode, pink). The molecular surface of NCS-1 is depicted. Squares represent magnifications of R1 and R2 regions. ( B ) Left: Superimposition of Structures 1 and 2. NCS-1 and Ric-8A peptide threads in blue and in pink tones, respectively. The gray arrow indicates the main differences found in Ric-8A peptide structures. ( C ) Temperature factor representation of Structure 2 (blue and red, high and low values, respectively) in two rotated views.

Article Snippet: Three highly pure (>95%) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques:

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet: ( A ) Ribbon representation of NCS-1. Helices are labeled and residues implicated in Ric-8A recognition are displayed as light purple sticks. ( B ) H-bonds (black dashes) between NCS-1 (gray) and Ric-8A (pink). Interacting residues are shown as pink sticks and light-purple sticks, respectively. Water molecules are displayed as red spheres. ( C ) H-bonds found in the R1-R2 loop. A rotated and zoomed view of that shown in B is depicted. ( D ) Ric-8A residues implicated in van der Waals interactions are displayed in sticks and labeled. Ric-8A is shown as pink ribbon and the molecular surface of NCS-1 is represented. Residues that were mutated to alanine to verify the protein-protein interaction (PPI) interface are indicated in purple in A–D.

Article Snippet: Three highly pure (>95%) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Labeling

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet: ( A ) Identification of Ca 2+ , Mg 2+ , and Na + ions in the hNCS-1ΔH10/Ric-8A-P3 complex (Structure 2, see ). Top: Electron density at EF-hands EF-2, -3 and -4. The 2F o -F c electron density map (contoured at 1.0σ) and the anomalous difference map (contoured at 6.0σ) are shown in green and blue, respectively. NCS-1 is shown in stick mode (light purple), Ca 2+ and Na + ions as hot pink and yellow spheres, respectively, and water molecules (w) as red spheres. Bottom: The Mg 2+ ion (green sphere) found in Structures 1 and 2 (see ). NCS-1 symmetry-related molecule is depicted in yellow. ( B ) The binding of Na + to hNCS-1 in solution. Representation of the normalized fluorescence emission (mean ± standard error of the mean [SEM]; n=3) of full-length hNCS-1 at increasing concentrations of NaCl or KCl. The curves are the least squares fitting of the experimental data to a 1:1 stoichiometry equilibrium. Na + and K + titrations are shown in blue and magenta, respectively. ( C ) Assembly of the NCS-1ΔH10/rRic-8A-452 complex in the presence of 200 mM Na + (blue) or K + (magenta). Size exclusion chromatograms indicating the elution of the assembled complexes ( C ). ( D ) Isothermal titration calorimetry (ITC) binding isotherm at 25°C for Ca 2+ to NCS-1 in 20 mM Tris pH 7.9 supplemented with 150 mM Na + (blue) or 150 mM K + (magenta). Solid lines show the best fits of the titration data in terms of a three-site sequential binding model using the thermodynamic parameters shown in . Θ is the fraction of sites available for each class of Ca 2+ sites. ( E ) The binding of full-length His-NCS-1 to Ric-8A-P3 peptide at increasing Ca 2+ concentrations. Representative biolayer interferometry (BLI) sensograms showing association and dissociation of Ric-8A-P3 over the time. Data are represented as the wavelength shift, △λ (nm), during the association and dissociation phases (s). Figure 4—source data 1. Raw chromatograms, nano-DSF and Bli data.

Article Snippet: Three highly pure (>95%) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Binding Assay, Fluorescence, Isothermal Titration Calorimetry, Titration

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet: ( A ) Nano-differential scanning fluorimetry (nano-DSF) curve of the His-NCS-1 sample used in the BLI assay. ( B ) Representative BLI sensogram of the binding of His-NCS-1 to the Ni-NTA biosensor and negative control showing that Ric-8A-P3 peptide does not bind to the biosensor in the absence of NCS-1. ( C ) BLI sensograms at increasing concentrations of Ric-8A-P3 peptide.

Article Snippet: Three highly pure (>95%) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Nano Differential Scanning Fluorimetry, Binding Assay, Negative Control

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet: ( A ) Co-IP protein-protein interaction assay of hNCS-1 and V5-tagged full-length hRic-8A wild-type (WT) (hRic-8A-WT) and a non-phosphorylatable mutant (Ric-8A-P-Mut; S436A, T441A) in HEK293 cells. ( B ) Anionic exchange chromatograms of casein kinase II (CK2)-treated samples eluted in a salt gradient. On the left, phosphorylated and unphosphorylated rRic-8A-452 prRic-8A-452 (orange) and urRic-8A-452 (green), respectively. On the right, CK2 treated (pink) or untreated (blue) NCS-1ΔH10/rRic-8A samples. Conductivity (mS/cm) is shown as gray lines. ( C ) Size exclusion chromatograms of the resulting samples after the assembly of NCS-1ΔH10 with unphosphorylated (green) and phosphorylated (orange) rRic-8A-452. C stands for assembled complex. Figure 5—source data 1. Original WBs and raw chromatogram data.

Article Snippet: Three highly pure (>95%) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Co-Immunoprecipitation Assay, Protein Protein Interaction Assay, Mutagenesis

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet: ( A ) The structure of the rRic-8A/G⍺ i1 complex (PDB: 6UKT, ). Electrostatic potential surface representation of ARM-HEAT domain (repeats 1–8). The repeat 9 is shown as ribbons. The Ric-8A region present in the NCS-1/Ric-8A-P crystal structure is shown in green and side chains of the corresponding residues in stick mode. Phosphorylated S435 and T440 are indicated. ( B ) Superposition of the structures of Ric-8A bound to G⍺ (magenta and green, PDB: 6UKT, ), uncomplexed rRic-8A (yellow, PDB: 6NMG, ), Ric-8A peptide (light purple) bound to hNCS-1. Ric-8A helix R2 of the complex with NCS-1 was superposed with helix b9 of uncomplexed Ric-8A. ( C ) Schematic representation of Ric-8A ARM/HEAT repeat domain (repeats 1–9 are indicated) explaining the detachment of 9 for NCS-1/Ric-8A assembly. The redistribution of charged (blue) and hydrophobic residues (green) in Ric-8A repeat 9 generates the platform for NCS-1 recognition. Repeats 1–8 have been colored in different gray tonalities since previous studies have shown global changes within the ARM-HEAT repeat domain of unphosphorylated Ric-8A compared to the phosphorylated version .

Article Snippet: Three highly pure (>95%) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques:

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet: ( A , B ) Superposition of NCS-1/Ric-8A-P (only Ric-8A is shown in pink ribbons) with other NCS-1 structures in complex with regulatory ligands, the protein-protein interaction (PPI) inhibitor FD-44 and the PPI stabilizer 3b . The molecular surface of NCS-1 is represented except the helix H10 (ribbon). FD-44 and 3b compounds are represented in stick mode and yellow arrows indicate their position. ( C ) Superposition of the structure of hNCS-1 (light purple) bound to Ric-8A-P (pink) with that of hNCS-1 (light blue; helix H10 in dark blue) bound to 3b regulator (cyan sticks). Yellow arrows indicate the NCS-1 regions that rearrange to accommodate Ric-8A. ( D ) Superposition of the hNCS-1/Ric-8A-P (light purple/pink) complex with that of the KChIP1/Kv4.3 (orange/green) complex (PDB: 2I2R (52)). N- and C-terminal end of the different polypeptide chains are indicated following the same color code.

Article Snippet: Three highly pure (>95%) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques:

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet: Step 1: At low Ca 2+ concentrations NCS-1 interacts with unphosphorylated Ric-8A (uRic-8A), at the plasma membrane. NCS-1 protects Ric-8A from phosphorylation or Gα subunit binding. Ric-8A ARM-HEAT repeat domain is labeled with a question mark since the structure in the context of the NCS-1 complex is unknown. Step 2: When Ca 2+ levels increase in the cytosol, NCS-1 binds Ca 2+ at EF-4 and the complex is disassembled. NCS-1 helix H10 inserts in the hydrophobic crevice (green) and would be ready for dopamine D2 receptor recognition. Inactive uRic-8A, free of NCS-1, repacks repeats 8 and 9 (helices a9 and b9 in light green) and S335 and T440 are phosphorylated (P) by casein kinase II (CK2) (Step 3). In this state, phosphorylated Ric-8A (pRic-8A) is now active, recognizes prefolded Gα subunit and allows GTP loading (Step 4).

Article Snippet: Three highly pure (>95%) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Membrane, Binding Assay, Labeling

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet:

Article Snippet: Three highly pure (>95%) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Recombinant, Plasmid Preparation, Residue, Construct, Mutagenesis, Software, Purification

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Ric-8A P2 peptide (400-429) , GenicBio , N/A , .

Techniques: Recombinant, Plasmid Preparation, Residue, Construct, Mutagenesis, Phospho-proteomics, Software, Purification

Journal: eLife

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.7554/eLife.86151

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Ric-8A P1 peptide (400-423) , GenicBio , N/A , .

Techniques: Recombinant, Plasmid Preparation, Residue, Construct, Mutagenesis, Phospho-proteomics, Software, Purification

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Ribbon representation of NCS protein structures bound to their targets. NCS-1/DR2 (PDB: 5AER ), NCS-1/Grk1 (PDB: 5AFP ), Frq1/Pik1 (PDB: 2JU0 ), KChIP1/Kv4.3 (PDB: 2I2R ), Recoverin/RK25 (PDB: 2I94, ), Frq2 (PDB: 4BY4, ). The N and C-terminal pairs of EF-hands (EF-1-2 and 3-4) are shown in cyan and purple respectively. The C-terminal helix H10 is shown in pink and target proteins in yellow. Ca 2+ is shown as hot pink spheres. B) Top: Cryo-EM structure of the rRic-8A/Gα i1 complex (PDB: 6UKT, ). Gα i1 is depicted in silver. Ric-8A Arm-HEAT repeats 1-8 in blue and repeat 9 in green and helices H10 (rH10) and H11 (rH11) in orange and gold respectively. The C-terminal coiled regions are shown in magenta. Phosphorylated residues S435 and T440 are depicted as spheres. Bottom: rRic-8A sequence from residue 400 to the end. Helices are squared following the same colour code used above. Phosphorylation sites are indicated as red spheres. P1, P2 and P3 brackets indicate the boundaries of the synthesized Ric-8A peptides.

Article Snippet: Three highly pure (>95 %) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Cryo-EM Sample Prep, Sequencing, Synthesized

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Ca 2+ dependency of the interaction of the rat complex. Size exclusion chromatograms after assemblies: (i) in Ca 2+ free conditions (grey), (ii) with Ca 2+ preloaded NCS-1ΔH10 (magenta) and (iii) with a dialysis from EGTA to Ca 2+ (green). 12 % SDS-PAGE gels analyzing elution of NCS-1ΔH10 (N) and rRic-8A-452 (R) after assembly (i) and the NCS-1/rRic-8A-452 complex (C) after assembly (iii). B) Nano-DSF curves of the different samples during assembly (iii). The ratio between the emission fluorescence at 350 and 330 nm is shown vs the temperature. Curves corresponding to the EGTA purified NCS-1 (NCS-1 2 mM EGTA), the fully Ca 2+ saturated protein (NCS-1 2 mM Ca 2+ ) and rRic-8A-452 are shown as references in yellow, brown and grey, respectively. The mixture of proteins before dialysis (0.6 mM EGTA) and afterwards (assembled complex, 2 mM CaCl 2 ) are shown in magenta and blue respectively. NCS-1 refers to NCS-1ΔH10, while Ric-8A to Ric-8A-452 construct. C) Co-IP protein-protein interaction assay in HEK293 cells of full-length human NCS-1 and V5-tagged hRic-8A constructs: full-length (hRic-8A-FL) and C-terminally truncated hRic-8A-424 (residue 1-424) and hRic-8A-433 (residue 1-433). D) Nano-DSF curves of hNCS-1 bound to different Ric-8A peptides. NCS-1 refers to NCS-1ΔH10, P2 and P3 refers to Ric-8A peptides P2 (purple) and P3 and (green).

Article Snippet: Three highly pure (>95 %) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: SDS Page, Fluorescence, Purification, Construct, Co-Immunoprecipitation Assay, Protein Protein Interaction Assay

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet:

Article Snippet: Three highly pure (>95 %) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques:

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Ribbon representation of the hNCS-1ΔH10/Ric-8A-P3 complex. Two views are displayed. The NCS-1 structure is shown in light purple, while Ric-8A-P3 is shown in light pink. The N- and C-termini are indicated. Ca 2+ , Na + and Cl - ions are shown in hotpink, yellow and cyan, respectively. R1 and R2 helices, and EF-hands 2, 3 and 4 are indicated. The orange square represents a zoomed view of the R1-R2 loop in stick mode, Cl - coordination and H-bonds are displayed as yellow and grey dashes, respectively. Residues participating in R1-R2 contacts are displayed in hot pink (Triad 1: I407-T410-A415), magenta (F406-L418) and purple (Triad 2: K408-Y409-N414). B) rRic-8A sequence from 400 to 442 residues. The helix boundaries of Ric-8A sequence encompassing a9 and b9 in different structural contexts (NCS-1/Ric-8A-peptide (PDB: 8AHY), Ric-8A/Gα (PDB: 6UKT, ) and uncomplexed Ric-8A (PDB: 6NMG, ) are indicated as pink boxes and labelled. Coiled regions are shown in pink. Disordered regions are shown in grey, while phosphorylated sites are shown as red spheres. The interacting residues shown in panel (A) are indicated with dots in the same color code as in (A). C) Electrostatic surface potential of rRic-8A-P3. NCS-1 is shown as yellow ribbons. Positive and negative potential are represented in blue and red. On the right, the Ric-8A region that faces and contacts NCS-1 is shown with NCS-1 removed for proper visualization.

Article Snippet: Three highly pure (>95 %) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Sequencing

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Identification of Ca 2+ , Mg 2+ and Na + ions in the hNCS-1ΔH10/Ric-8A-P3 complex (Structure 2, see ). Top: Electron density at EF-hands EF-2, 3 and 4. The 2F o -F c electron density map ( contoured at 1.0 σ) and the anomalous difference map (contoured at 6.0 σ) are shown in green and blue respectively. NCS-1 is shown in stick mode (light purple), Ca 2+ and Na + ions as hot-pink and yellow spheres, respectively, and water molecules (w) as red spheres. Bottom: The Mg 2+ ion (green sphere) found in Structure 1 and 2 (see ). NCS-1 symmetry related molecule is depicted in yellow. B) The binding of Na + to hNCS-1 in solution. Representation of the normalized fluorescence emission (mean + SEM; n =3) of full-length hNCS-1 at increasing concentrations of NaCl or KCl. The curves are the least squares fitting of the experimental data to a 1:1 stoichiometry equilibrium. Na + and K + titrations are shown in blue and magenta respectively. C) Assembly of the NCS-1ΔH10/rRic-8A-452 complex in the presence of 200 mM Na + (blue) or K + (magenta). Size exclusion chromatograms indicating the elution of the assembled complexes (C). D) ITC binding isotherm at 25 ºC for Ca 2+ to NCS-1 in 20 mM Tris pH 7.9 supplemented with 150 mM Na + (blue) or 150 mM K + (magenta). Solid lines show the best fits of the titration data in terms of a three-site sequential binding model using the thermodynamic parameters shown in . Θ is the faction of sites available for each class of Ca 2+ sites.

Article Snippet: Three highly pure (>95 %) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Binding Assay, Fluorescence, Titration

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: Front (A) and back (B) view of the cryo-EM map (grey) with the docked rRic-8A-400/Nbs (PDB: 6UKT, ) and hNCS-1/Ric-8A-P complexes (displayed as cartoons). The inset shows the N-terminus of Ric-8A-P protruding into the unassigned density. C) Top view of the complex showing a part of the HEAT repeat 8 (residue 356-400) protruding out of the cryo-EM map.

Article Snippet: Three highly pure (>95 %) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Co-IP protein-protein interaction assay of hNCS-1 and V5-tagged full-length hRic-8A WT (hRic-8A-WT) and a non-phosphorylatable mutant (Ric-8A-P-Mut; S436A, T441A) in HEK293 cells. B) Anionic exchange chromatograms of CK2 treated samples eluted in a salt gradient. On the left, phosphorylated and unphosphorylated rRic-8A-452 (prRic-8A-452 (orange) and urRic-8A-452 (green), respectively. On the right, CK2 treated (pink) or untreated (blue) NCS-1ΔH10/rRic-8A samples. Conductivity (mS/cm) is shown as grey lines. C) Size exclusion chromatograms of the resulting samples after the assembly of NCS-1ΔH10 with unphosphorylated (green) and phosphorylated (orange) rRic-8A-452. C stands for assembled complex.

Article Snippet: Three highly pure (>95 %) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Co-Immunoprecipitation Assay, Protein Protein Interaction Assay, Mutagenesis

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) The structure of the rRic-8A/Gα i1 complex (PDB: 6UKT, ). Electrostatic potential surface representation of ARM-HEAT domain (repeats 1 to 8). The repeat 9 is shown as ribbons. The Ric-8A region present in the NCS-1/Ric-8A-P crystal structure is shown in green and side chains of the corresponding residues in stick mode. Phosphorylated S435 and T440 are indicated. B) Superposition of the structures of Ric-8A bound to Gα (magenta and green, PDB: 6UKT, ), uncomplexed rRic-8A (yellow, PDB: 6NMG, ), Ric-8A peptide (light purple) bound to hNCS-1. Ric-8A helix R2 of the complex with NCS-1 was superposed with helix b9 of uncomplexed Ric-8A. C) Schematic representation of Ric-8A ARM/HEAT repeat domain (repeats 1 to 9 are indicated) explaining the detachment of repeats 8 and 9 for NCS-1/Ric-8A assembly. The redistribution of charged (blue) and hydrophobic residues (green) in Ric-8A repeat 9 generates the platform for NCS-1 recognition. D) Structural comparison between the rRic-8A/Gα complex (PDB: 6UKT, ) with the NCS-1/Ric-8A X-ray/cryo-EM model presented in this work. Ric-8A ARM-HEAT domains (from repeat 1 to repeat 8 helix A8) have been superposed. The helices B8 and C8 from repeat 8 (magenta) and a9 and b9 from repeat 9 (bright green), which rearrange for NCS-1 recognition, are shown in ribbons. Ric-8A helices a9 and b9 (complex with Gα i1 ) convert to helices R1 and R2 (light pink) when interacting with NCS-1. A magenta curved arrow represents the conformational change that Ric-8A repeat 8 (356-400) may undergo in order to connect with Ric-8A helices R1 and R2. The NCS-1 C-terminal helix is represented as a cartoon. On the right, a top view of the NCS-1/Ric-8A complex is displayed to shown how NCS-1 Ca 2+ binding sites are exposed to the solvent, and how Ric-8A ARM repeat 8 helices B8 and C8 (magenta cartoons) would occupy the unassigned extra density observed in the cryo-EM map to connect with Ric-8A helices R1 and R2. A dashed black line represents the C-terminus of Ric-8A which gets trapped between NCS-1 and the Ric-8A ARM-HEAT repeat domain.

Article Snippet: Three highly pure (>95 %) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: Step 1: At low Ca 2+ concentrations NCS-1 interacts with unphosphorylated Ric-8A (uRic-8A), at the plasma membrane. NCS-1 protects Ric-8A from phosphorylation or Gα subunit binding by inducing a conformational rearrangement of repeats 8 (helices B8 and C8 in magenta) and 9 (helices R1 and R2 in pink). Step 2: When Ca 2+ levels increase in the cytosol, NCS-1 binds Ca 2+ at EF-4 and the complex is disassembled. NCS-1 helix H10 inserts in the hydrophobic crevice (green) and would be ready for Dopamine D2 receptor recognition. Inactive uRic-8A, free of NCS-1, repacks repeats 8 and 9 (helices a9 and b9 in light green) and S335 and T440 are phosphorylated (P) by CK2 (Step 3). In this state, phosphorylated Ric-8A (pRic-8A) is now active, recognizes prefolded Gα subunit and allows GTP loading (Step 4).

Article Snippet: Three highly pure (>95 %) HPLC-verified Ric-8A peptides were purchased from GenicBio for structural studies.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Ca 2+ dependency of the interaction of the rat complex. Size exclusion chromatograms after assemblies: (i) in Ca 2+ free conditions (grey), (ii) with Ca 2+ preloaded NCS-1ΔH10 (magenta) and (iii) with a dialysis from EGTA to Ca 2+ (green). 12 % SDS-PAGE gels analyzing elution of NCS-1ΔH10 (N) and rRic-8A-452 (R) after assembly (i) and the NCS-1/rRic-8A-452 complex (C) after assembly (iii). B) Nano-DSF curves of the different samples during assembly (iii). The ratio between the emission fluorescence at 350 and 330 nm is shown vs the temperature. Curves corresponding to the EGTA purified NCS-1 (NCS-1 2 mM EGTA), the fully Ca 2+ saturated protein (NCS-1 2 mM Ca 2+ ) and rRic-8A-452 are shown as references in yellow, brown and grey, respectively. The mixture of proteins before dialysis (0.6 mM EGTA) and afterwards (assembled complex, 2 mM CaCl 2 ) are shown in magenta and blue respectively. NCS-1 refers to NCS-1ΔH10, while Ric-8A to Ric-8A-452 construct. C) Co-IP protein-protein interaction assay in HEK293 cells of full-length human NCS-1 and V5-tagged hRic-8A constructs: full-length (hRic-8A-FL) and C-terminally truncated hRic-8A-424 (residue 1-424) and hRic-8A-433 (residue 1-433). D) Nano-DSF curves of hNCS-1 bound to different Ric-8A peptides. NCS-1 refers to NCS-1ΔH10, P2 and P3 refers to Ric-8A peptides P2 (purple) and P3 and (green).

Article Snippet: Label-free thermal shift assays with hNCS-1 full-length, hNCS-1ΔH10, rRic-8A-452, NCS-1ΔH10/rRic-8A-452, NCS-1ΔH10/Ric-8A-P2 peptide and NCS-1ΔH10/Ric-8A-P3 peptide were performed using a Tycho NT.6 instrument (NanoTemper Technologies).

Techniques: SDS Page, Fluorescence, Purification, Construct, Co-Immunoprecipitation Assay, Protein Protein Interaction Assay

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Ribbon representation of the hNCS-1ΔH10/Ric-8A-P3 complex. Two views are displayed. The NCS-1 structure is shown in light purple, while Ric-8A-P3 is shown in light pink. The N- and C-termini are indicated. Ca 2+ , Na + and Cl - ions are shown in hotpink, yellow and cyan, respectively. R1 and R2 helices, and EF-hands 2, 3 and 4 are indicated. The orange square represents a zoomed view of the R1-R2 loop in stick mode, Cl - coordination and H-bonds are displayed as yellow and grey dashes, respectively. Residues participating in R1-R2 contacts are displayed in hot pink (Triad 1: I407-T410-A415), magenta (F406-L418) and purple (Triad 2: K408-Y409-N414). B) rRic-8A sequence from 400 to 442 residues. The helix boundaries of Ric-8A sequence encompassing a9 and b9 in different structural contexts (NCS-1/Ric-8A-peptide (PDB: 8AHY), Ric-8A/Gα (PDB: 6UKT, ) and uncomplexed Ric-8A (PDB: 6NMG, ) are indicated as pink boxes and labelled. Coiled regions are shown in pink. Disordered regions are shown in grey, while phosphorylated sites are shown as red spheres. The interacting residues shown in panel (A) are indicated with dots in the same color code as in (A). C) Electrostatic surface potential of rRic-8A-P3. NCS-1 is shown as yellow ribbons. Positive and negative potential are represented in blue and red. On the right, the Ric-8A region that faces and contacts NCS-1 is shown with NCS-1 removed for proper visualization.

Article Snippet: Label-free thermal shift assays with hNCS-1 full-length, hNCS-1ΔH10, rRic-8A-452, NCS-1ΔH10/rRic-8A-452, NCS-1ΔH10/Ric-8A-P2 peptide and NCS-1ΔH10/Ric-8A-P3 peptide were performed using a Tycho NT.6 instrument (NanoTemper Technologies).

Techniques: Sequencing

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Identification of Ca 2+ , Mg 2+ and Na + ions in the hNCS-1ΔH10/Ric-8A-P3 complex (Structure 2, see ). Top: Electron density at EF-hands EF-2, 3 and 4. The 2F o -F c electron density map ( contoured at 1.0 σ) and the anomalous difference map (contoured at 6.0 σ) are shown in green and blue respectively. NCS-1 is shown in stick mode (light purple), Ca 2+ and Na + ions as hot-pink and yellow spheres, respectively, and water molecules (w) as red spheres. Bottom: The Mg 2+ ion (green sphere) found in Structure 1 and 2 (see ). NCS-1 symmetry related molecule is depicted in yellow. B) The binding of Na + to hNCS-1 in solution. Representation of the normalized fluorescence emission (mean + SEM; n =3) of full-length hNCS-1 at increasing concentrations of NaCl or KCl. The curves are the least squares fitting of the experimental data to a 1:1 stoichiometry equilibrium. Na + and K + titrations are shown in blue and magenta respectively. C) Assembly of the NCS-1ΔH10/rRic-8A-452 complex in the presence of 200 mM Na + (blue) or K + (magenta). Size exclusion chromatograms indicating the elution of the assembled complexes (C). D) ITC binding isotherm at 25 ºC for Ca 2+ to NCS-1 in 20 mM Tris pH 7.9 supplemented with 150 mM Na + (blue) or 150 mM K + (magenta). Solid lines show the best fits of the titration data in terms of a three-site sequential binding model using the thermodynamic parameters shown in . Θ is the faction of sites available for each class of Ca 2+ sites.

Article Snippet: Label-free thermal shift assays with hNCS-1 full-length, hNCS-1ΔH10, rRic-8A-452, NCS-1ΔH10/rRic-8A-452, NCS-1ΔH10/Ric-8A-P2 peptide and NCS-1ΔH10/Ric-8A-P3 peptide were performed using a Tycho NT.6 instrument (NanoTemper Technologies).

Techniques: Binding Assay, Fluorescence, Titration

Journal: bioRxiv

Article Title: The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

doi: 10.1101/2022.12.09.519724

Figure Lengend Snippet: A) Co-IP protein-protein interaction assay of hNCS-1 and V5-tagged full-length hRic-8A WT (hRic-8A-WT) and a non-phosphorylatable mutant (Ric-8A-P-Mut; S436A, T441A) in HEK293 cells. B) Anionic exchange chromatograms of CK2 treated samples eluted in a salt gradient. On the left, phosphorylated and unphosphorylated rRic-8A-452 (prRic-8A-452 (orange) and urRic-8A-452 (green), respectively. On the right, CK2 treated (pink) or untreated (blue) NCS-1ΔH10/rRic-8A samples. Conductivity (mS/cm) is shown as grey lines. C) Size exclusion chromatograms of the resulting samples after the assembly of NCS-1ΔH10 with unphosphorylated (green) and phosphorylated (orange) rRic-8A-452. C stands for assembled complex.

Article Snippet: Label-free thermal shift assays with hNCS-1 full-length, hNCS-1ΔH10, rRic-8A-452, NCS-1ΔH10/rRic-8A-452, NCS-1ΔH10/Ric-8A-P2 peptide and NCS-1ΔH10/Ric-8A-P3 peptide were performed using a Tycho NT.6 instrument (NanoTemper Technologies).

Techniques: Co-Immunoprecipitation Assay, Protein Protein Interaction Assay, Mutagenesis