Figure 1
Effector binding and conformational activation of myc-tagged, constitutively activated Gα12. Molecular weight markers (in kDa) are indicated at right of panels where applicable. All results shown are representative of two or more independent experiments. (A) Expression and solubilization of Gα12QL (12QL) and myc-tagged Gα12QL (myc-12QL) transiently expressed in HEK293 cells. Cells transfected with the vector pcDNA3.1 are included as a negative control (vector). Detergent-soluble extracts were prepared by high-speed centrifugation and subjected to SDS-PAGE and immunoblotting, using either anti-myc (Zymed) or anti-Gα12 (Santa Cruz Biotechnology) antibodies as described in Methods. (B) In vitro binding of myc-tagged and untagged Gα12QL by p115RhoGEF. HEK293 cells extracts containing myc-Gα12QL were subjected to protein interaction assays (see Methods) using an immobilized GST fusion of the RH domain of p115RhoGEF (RGS) or GST alone (GST). Samples were washed, separated by SDS-PAGE, and analyzed by immunoblotting using antibodies described above. (C) Specificity of myc-Gα12QL detection in interaction assays. HEK293 cells transfected with either myc-Gα12QL (myc-12QL) or the empty pcDNA3.1 plasmid (vect) were lysed and assayed for binding to GST fusions of the RH domain of p115RhoGEF (p115) or LARG, or GST alone (GST). Immunoblot analysis was performed using anti-Gα12 antibody as described above. (D) Serum response element (SRE) luciferase activation by myc-Gα12QL. HEK293 cells grown in 12-well plates were co-transfected with the plasmids SRE-L (0.2 μg) and pRL-TK (0.02 μg), plus 0.1 μg of the plasmid indicated on the X-axis. Y-axis values show firefly luciferase signal normalized for Renilla luciferase signal within each sample. (E) Trypsin protection assays of myc-tagged Gα12. Lysates from HEK293 cells transfected with myc-Gα12QL (myc-12QL) or the constitutively GDP-bound Gly228Ala mutant of wildtype Gα12 (myc-12G228A) were subjected to trypsin digests as described in Methods. Immunoblot analysis was performed using J169 antibody [25] at 1:700 dilution.
Figure 2
Residues replaced in Gα12 cassette mutants. For each mutant, designated in italics (A-Z, AA-ZZ, AAA-KKK), the native amino acid sextet replaced by the sequence Asn-Ala-Ala-Ile-Arg-Ser is shown. An arrow between Pro139 and Val140 indicates the site of myc tag insertion. Mutant W was not produced. The dashed box indicates the native Gln229 mutated to Leu to render Gα12 constitutively active. The native residues replaced in mutant KKK are Lys-Asp-Ile-Met-Leu-Gln and thus partially overlap with mutant JJJ. All cassette mutants contain the activating Q229L mutation, except mutant LL due to its cassette substitution.
Figure 3
In vitro interaction of Gα12 mutants with LARG. Immunoblot results for all LARG binding-impaired Gα12 cassette mutants and selected other mutants are shown. HEK293 cells were transfected with the indicated plasmids (7.0 μg per 10-cm plate) and lysates were prepared for co-precipitation assays as described in Methods. Prior to this step, 5% of each lysate was set aside as starting material (load). Pulldown experiments were performed on 7–9 mutants per experiment, plus myc-Gα12QL as a positive control, using equal amounts of GST-LARG-RH (LARG) immobilized on glutathione-sepharose. Immobilized GST was utilized in parallel as a negative control. For all experimental samples, 20% of the volume was analyzed by SDS-PAGE and Coomassie blue staining to verify equal amounts of GST-LARG-RH and GST proteins in the precipitates (data not shown). Immunoblots displayed in this figure are representative of at least three trials per cassette mutant, except for mutants A-D, F-H, V, and KKK that showed minimal impairment in LARG binding after two trials. (Inset) Coomassie blue analysis of GST-fusion constructs expressed in bacteria and immobilized on glutathione-sepharose: GST-LARG-RH (LARG), GST-p115-RH (p115), and GST alone. Molecular weight standards (in kDa) are indicated at right.
Table 1
Gα 12 cassette mutants impaired in binding LARG-RH
| N/D | W CC |
Cassette substitution mutants of myc-Gα12QL (see Figure 2 for alphabetical designations) were expressed in HEK293 cells and subjected to protein interaction assays using a GST-fusion of the RH domain of LARG as described in Methods, and for each mutant a pulldown:load ratio was determined and calculated as a percent (left column) of the same ratio for unmodified myc-Gα12QL assayed in parallel. Each Gα12 mutant was analyzed in three independent experiments, except for mutants that appeared in the 70-100% category in two independent experiments.
Table 2
Gα 12 cassette mutants corresponding to rgRGS contact points within Gα 13
| Gα 13 residues in contact with p115-RH | myc-Gα 12 QL NAAIRS mutant |
|---|---|
| Arg335 | DDD |
Gα13-native residues previously identified as providing contact points with the RH domain of p115RhoGEF [20,21] are indicated in the left column. Cassette mutants (“NAAIRS” substitution) in which the homologous residue(s) within Gα12 have been altered are indicated in the right column. See Figure 2 for Gα12 mutant designations.
Figure 4
Activation of serum response element mediated transcription by Gα12 mutants. (A) Luciferase reporter assay results of selected cassette mutants. HEK293 cells grown in 12-well plates were co-transfected with the plasmids SRE-L (0.2 μg) and pRL-TK (0.02 μg), plus 1.0 μg of the plasmid encoding each cassette mutant indicated on the X-axis. Firefly luciferase values were normalized for Renilla luciferase values within each sample, and values are presented as a percent of the value calculated for myc-Gα12QL (Y-axis) within the same experiment. Mutationally active (12QL) and inactive (G228A) samples were analyzed in parallel. Results shown are a representative of two experiments performed per Gα12 variant. (B) Expression level of Gα12 mutants. A sample of each lysate was set aside prior to luminometry and analyzed by SDS-PAGE and immunoblotting using anti-Gα12 antibody (Santa Cruz Biotechnology). For all samples, densitometric intensity was determined as described in Methods, then divided by positive control myc-Gα12QL levels within the same experiment, and SRE-L/Renilla values were adjusted to reflect this normalization for protein levels.
Figure 5
Conformational efficacy of N-terminal and C-terminal Gα12 mutants uncoupled from RhoGEFs. (A) Trypsin protection of selected Gα12 mutants. HEK293 cell lysates expressing the indicated variants of myc-Gα12QL, or unmodified myc-Gα12QL (12QL), or the G228A variant of myc-Gα12 (12G228A) were subjected to trypsin protection assays as described in Methods. Samples were incubated 20 min at 30°C in the presence (+) or absence (−) of TPCK-treated trypsin, and were analyzed by SDS-PAGE and immunoblotting using J169 antibody (1:700 dilution). Small horizontal arrows indicate position of the trypsin-protected fragment in selected lanes. Data presented are representative of two or more independent experiments per sample. (B) Specificity of uncoupling in selected Gα12 variants. For each cassette mutant of myc-Gα12QL (indicated at top), interaction with each Gα12 target (indicated at left) was quantified as a pulldown:load ratio as described in Methods, and was calculated as a percent of the identical ratio determined for myc-Gα12QL within the same experiment. Values are indicated as follows: (++) = >60%, (+) = 20 to 60%, (−) = 0 to 20%. Interacting proteins are GST fusions of the following: RH domain of LARG (LARG), C-terminal 107 amino acids of heat shock protein-90 alpha (Hsp90), protein phosphatase-5 (PP5), scaffolding Aα subunit of protein phosphatase-2A (PP2A), C-terminal 98 amino acids of E-cadherin (E-cad). Values presented indicate the mean of two or more trials per interaction sample.
Figure 6
Structural position of Gα12 C-terminal determinants of RhoGEF binding. The structure of N-terminally Gαi1-substituted Gα12 (PDB accession code 1ZCA, [22]) as a GDP•AlF4¯ activated complex was analyzed using PyMOL software. The native Gα12 region substituted for the sequence “NAAIRS” in the C-terminal mutants EEE, FFF, and GGG is highlighted in orange, and the sextet substituted in mutant HHH is highlighted in black. The bound GDP molecule is highlighted in blue. Figure was rendered in The PyMOL Molecular Graphics System, Version 1.5.0.1 Schrödinger, LLC.
Figure 7
Selective RhoGEF uncoupling by N-terminal charge substitutions in Gα12. (A) Luciferase reporter gene assays. Cassette mutant E (see Figure 2) and the double charge substitution mutant Glu31/33Arg were compared to myc-Gα12QL (12QL) in SRE-luciferase assays under the cell transfection conditions described in Figure 4. A constitutively GDP-bound variant of wildtype myc-Gα12 (12G228A) was assayed in parallel as a negative control. Results shown are the mean of three independent experiments, and error bars indicate range. (B) Protein-protein interaction assays. Detergent-soluble extracts from transfected HEK293 cells transfected with myc-Gα12QL, the Glu31/33Arg mutant, or empty pcDNA3.1 plasmid (vector) were subjected to co-precipitation assays as described in Methods, using GST-fusions of either LARG-RH (LARG), p115RhoGEF-RH (p115), the N-terminal domain of the Gα12 target radixin [46], or no adduct (GST). Prior to the precipitation step, 5% of each lysate was set aside as starting material (load). Table values show the pulldown:load ratio for Glu31/33Arg as a percent of the positive control value (12QL), with mean +/- range presented for three independent experiments. (C) Trypsin protection of the Glu31/33Arg mutant, in comparison to constitutively GTP- and GDP-bound Gα12. Assays were performed as described in Methods. Results shown are representative of two independent experiments.