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

Abstract

Objective: Adrenocortical adenomas are frequent in the general population and can be associated with autonomous cortisol excess, increasing morbidity and mortality. Altered cAMP/PKA signalling is common in sporadic cortisol-producing adenomas, typically due to somatic activating mutations in the catalytic subunit α of PKA (PRKACA) or the G-protein α subunit, Gα_s (GNAS), which activate cAMP signalling. We previously identified a novel p.Lys58Gln GNAS somatic variant in a patient with a 5.3 cm adenoma and overt Cushing's syndrome. This novel mutation was not charactersised before but provided enough evidence to warrant further investigation.

Design and methods: Using HEK293 cells depleted of GNAS, we established wild-type (WT) Gα_s and Gα_s-Lys58Gln stable cell lines and evaluated adrenocorticotropic hormone (ACTH) receptor signalling using a cAMP GloSensor assay, measured CREB transcription factor phosphorylation (pCREB) by AlphaLISA and assessed CRE luciferase reporter activity. Cell viability and apoptosis were also assessed over 5 days.

Results: The Gα_s-Lys58Gln variant showed a significantly higher basal cAMP, pCREB and CRE luciferase reporter concentration and a greater response to ACTH (0-10 nM, P < 0.001) compared to WT Gα_s. The variant had no effect on ligand potency. There was also significantly enhanced cell viability and apoptosis in cells with the Gα_s-Lys58Gln variant.

Conclusions: In conclusion, our study demonstrated that the Gα_s-Lys58Gln variant is associated with constitutive activation of GNAS signalling, similar to Arg201 mutations previously reported in adrenocortical adenomas, potentially representing a new pathogenic mechanism in a subset of patients with adrenal Cushing syndrome. This variant may also affect cell proliferation and requires further study.

Keywords: ACTH receptor; G protein; cAMP/PKA signalling; cortisol-producing adenomas; somatic mutation.

Figure 1

Predicted effects of the Lys58Gln variant on the Gα_s protein. (A) Multiple protein sequence alignment of Gα_s 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 Gα_s. (B) Predicted three-dimensional structure of Gα_s 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 Gα_s in the active conformation (PDB: 1AZS, pink) overlaid over the structure of inactive Gα_s (green) showing the consequences of activation on the positioning of the amino acid residue at position 58 (blue/light blue: inactive, red: active) on both the (D) wild-type Lys58 residue and (E) Gln58 variant. The Arg201 residue that is frequently mutated in adrenocortical tumours is shown in orange.

Figure 2

Establishment of cell lines stably expressing Gα_s 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 Gα_s/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-Gα_s plasmids in the WT and Lys58Gln cell lines. The parental HEK Gα_s/l knockout cells were used as a negative control. HEK293 cells were used to illustrate absence of the SNAP-Gα_s and endogenous expression of Gα_s/l. HEK293 transiently transfected with the SNAP-Gα_s were used to illustrate the presence of the transfected SNAP-Gα_s and endogenous Gα_s/l. Calnexin was used as a loading control.

Figure 3

The Gα_s-Lys58Gln variant enhances constitutive activity of the ACTH receptor. (A) ACTH-mediated cAMP responses measured by GloSensor in Gα_s wild-type and Gα_s-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 Gα_s transfected with the ACTH receptor. n = 5 biological replicates. (D) ACTH-mediated phospho-CREB (pCREB) responses measured by AlphaLISA in Gα_s wild-type and Gα_s-Lys58Gln variant cell lines transiently expressing MC2R and MRAP1, with (E) pEC₅₀. n = 4 biological replicates. (F) ACTH-mediated CRE luciferase activity in Gα_s wild-type and Gα_s-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 Gα_s variant enhances cell growth and apoptosis. (A) Cell viability in Gα_s wild-type and Gα_s-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.