Identification of a novel constitutively active G[alpha]<SUB>s</SUB> variant associated with cortisol-producing adrenocortical adenoma

Aqfan Jamaluddin; Rachael Wyatt; Andreea Pasaliu; Oliver Ruggles; Davide Calebiro; Caroline Gorvin; Cristina L. Ronchi

doi:10.1530/endoabs.94.P157

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Endocrine Abstracts (2023) 94 P157 | DOI: 10.1530/endoabs.94.P157

SFEBES2023 Poster Presentations Adrenal and Cardiovascular (78 abstracts)

Identification of a novel constitutively active Gα_s variant associated with cortisol-producing adrenocortical adenoma

Aqfan Jamaluddin ^1,2 , Rachael Wyatt ^1,2 , Andreea Pasaliu ¹ , Oliver Ruggles ¹ , Davide Calebiro ^1,2 , Caroline Gorvin ^1,2 & Cristina L. Ronchi ^1,3

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¹Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, United Kingdom. ²Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Birmingham, United Kingdom. ³Centre for Endocrinology, Diabetes and Metabolism, University Hospitals Birmingham NHS Foundation Trust, Birmingham Health Partners, Birmingham, United Kingdom

Adrenocortical adenomas are among the most commonly identified human neoplasias, with a prevalence of 2-3% in the population. In some cases they are associated with autonomous cortisol excess that leads to increased morbidity and mortality. Altered cAMP/protein kinase A (PKA) signalling is common in sporadic cortisol-producing adenomas (CPA), mostly caused by somatic mutations in the genes coding for the catalytic subunit α of PKA (PRKACA) or the stimulatory G-protein α subunit, Gαs (GNAS), CPA-associated mutations in GNAS commonly affect the Arg201 residue, located within the GTPase binding domain, and are associated with constitutive cAMP activity. In a previous study, we identified a private Lys58Gln GNAS somatic variant in a patient (female, 44 yrs) with a 5.3 cm adenoma and overt Cushing’s syndrome (Ronchi et al 2016). This variant was predicted likely pathogenic, and structural modelling showed Lys58 is located near to the critical Arg201 residue, suggesting Lys58Gln may affect Gα_s function. To clarify whether this Lys58Gln variant had a functional effect we assessed signalling by the melanocortin-2-receptor (MC2R), an adrenocorticotropic hormone (ACTH) receptor. We utilised HEK293 cells depleted of GNAS to establish GNAS-WT and GNAS-Lys58Gln stable cell-lines and investigated ACTH-induced MC2R receptor activity using cAMP Glosensor kinetic assays. This showed the Lys58Gln Gα_s variant had a significantly higher basal cAMP concentration and a significantly greater response to ACTH between 0-10nM (n=6, P<0.001), when compared to wild-type Gα_s. The Lys58Gln Gαs variant diminished the receptor’s maximal response to ACTH (P<0.001), although it did not affect the ligand’s potency. In conclusion, we demonstrated that Lys58Gln is a likely pathogenic Gα_s variant associated with constitutive MC2R signalling, similarly to the previously described Arg201 mutations. Our findings identify this variant as a new potential pathogenic mechanism that could be observed in a small subset of patients with adrenal Cushing syndrome.