Characterisation of a GNAS variant linked to cortisol-producing adrenocortical adenoma

Figures

• 

  Figure 1. Predicted effects of the Lys58Gln variant on the Gαs protein. (A) Multiple protein sequence alignment of Gαs orthologs (top) and G protein paralogs (bottom) showing that the Lys58 residue and surrounding residues are highly conserved across five orthologs and representative members of each G protein subfamily. The wild-type Lys58 and mutant Gln58 (mut) are shown in bold blue text. Conserved residues are shaded grey. Numbering shows amino acid number in Gαs. (B) Predicted three-dimensional structure of Gαs in the inactive conformation (PDB ID: 6AU6) showing the helical domain in light blue and the GTPase domain in green, with the bound GDP in black. The wild-type Lys58 residue is shown in dark blue and forms a contact with Leu197 (yellow) across the interdomain interface. The Arg201 residue that is frequently mutated in adrenocortical adenoma is also shown adjacent to the switch 1 region. (C) The Gln58 mutant disrupts the contact with Leu197. (D and E) Predicted three-dimensional structure of Gαs in the active conformation (PDB: 1AZS, pink) overlaid over the structure of inactive Gαs (green) showing the consequences of activation on the positioning of the amino acid residue at…

• 

  Figure 2. Establishment of cell lines stably expressing Gαs wild-type or the Lys58Gln variant. (A) RT-PCR showing expression of SNAP-tagged GNAS in the wild-type and Lys58Gln stable cell lines. The parental HEK Gαs/l knockout cells were used as a negative control. GAPDH was used as a loading control. (B) Western blot showing stable overexpression of the SNAP-Gαs plasmids in the WT and Lys58Gln cell lines. The parental HEK Gαs/l knockout cells were used as a negative control. HEK293 cells were used to illustrate absence of the SNAP-Gαs and endogenous expression of Gαs/l. HEK293 transiently transfected with the SNAP-Gαs were used to illustrate the presence of the transfected SNAP-Gαs and endogenous Gαs/l. Calnexin was used as a loading control.

• 

  Figure 3. The Gαs-Lys58Gln variant enhances constitutive activity of the ACTH receptor. (A) ACTH-mediated cAMP responses measured by GloSensor in Gαs wild-type and Gαs-Lys58Gln variant cell lines transiently expressing MC2R and MRAP1. (B) Area under the curve was used to generate cAMP concentration–response curves. (C) Potency of ACTH in cells expressing either WT or Lys58Gln Gαs transfected with the ACTH receptor. n = 5 biological replicates. (D) ACTH-mediated phospho-CREB (pCREB) responses measured by AlphaLISA in Gαs wild-type and Gαs-Lys58Gln variant cell lines transiently expressing MC2R and MRAP1, with (E) pEC50. n = 4 biological replicates. (F) ACTH-mediated CRE luciferase activity in Gαs wild-type and Gαs-Lys58Gln variant cell lines transiently expressing MC2R and MRAP1, with (G) pEC50. n = 4 biological replicates. Statistical analyses were performed by two-way ANOVA with Bonferroni’s multiple-comparisons test in B and D, two-way ANOVA with Sidak’s multiple-comparisons test in F and unpaired t-test in C, E, G. ****P < 0.0001, ***P < 0.001, ns = not significant.

• 

  Figure 4. The Lys58Gln Gαs variant enhances cell growth and apoptosis. (A) Cell viability in Gαs wild-type and Gαs-Lys58Gln variant cell lines transiently expressing MC2R and MRAP1 measured over 96 h. Data were expressed corrected to the total protein, then normalized to hour 0. (B) Apoptosis measured over 96 h was expressed corrected to the total protein, then normalised to hour 0. Statistical analyses were performed by unpaired t-test in n = 5 replicates. **P < 0.01, *P < 0.05.