A New Multisystem Disorder Caused by the Gαs Mutation p.F376V

Abstract

Context: The α subunit of the stimulatory G protein (Gαs) links numerous receptors to adenylyl cyclase. Gαs, encoded by GNAS, is expressed predominantly from the maternal allele in certain tissues. Thus, maternal heterozygous loss-of-function mutations cause hormonal resistance, as in pseudohypoparathyroidism type Ia, whereas somatic gain-of-function mutations cause hormone-independent endocrine stimulation, as in McCune-Albright syndrome.

Objective: We report two unrelated boys presenting with a new combination of clinical findings that suggest both gain and loss of Gαs function.

Design and setting: Clinical features were studied and sequencing of GNAS was performed. Signaling capacities of wild-type and mutant Gαs were determined in the presence of different G protein-coupled receptors (GPCRs) under basal and agonist-stimulated conditions.

Results: Both unrelated patients presented with unexplained hyponatremia in infancy, followed by severe early onset gonadotrophin-independent precocious puberty and skeletal abnormalities. An identical heterozygous de novo variant (c.1136T>G; p.F376V) was found on the maternal GNAS allele in both patients; this resulted in a clinical phenotype that differed from known Gαs-related diseases and suggested gain of function at the vasopressin 2 receptor (V2R) and lutropin/choriogonadotropin receptor (LHCGR), yet increased serum PTH concentrations indicative of impaired proximal tubular PTH1 receptor (PTH1R) function. In vitro studies demonstrated that Gαs-F376V enhanced ligand-independent signaling at the PTH1R, LHCGR, and V2R and, at the same time, blunted ligand-dependent responses. Structural homology modeling suggested mutation-induced modifications at the C-terminal α5 helix of Gαs that are relevant for interaction with GPCRs and signal transduction.

Conclusions: The Gαs p.F376V mutation causes a previously unrecognized multisystem disorder.

Figure 1.

Radiographic studies of skeletal features in the patients. Radiographs of patient 1 showing the skeletal abnormalities such as antecurvatura of the left distal tibia, bowing of both upper legs, and enlargement of the metaphyses. (A) Radiographic study of patient 1 at 1 year 8 months and 7 years 6 months. (B) Radiographic study of patient 2 at 1 year 3 months.

Figure 2.

(A–E) Functional characterization of wt-Gαs and variants coexpressed with several different GPCRs. 2B2 cells were cotransfected with GPCRs and either wt or mutant Gαs, as indicated. After 2 days, cAMP accumulation was determined in the absence or presence of maximal concentration of indicated ligands by α screen technology. Results of four independent experiments performed in triplicated are shown as mean ± SEM. Statistical analysis was performed with one-way ANOVA with Kruskal-Wallis test, and indicated receptors + Gαs were tested against receptors in the presence of different Gαs variants. *P ≤ 0.05. **P ≤ 0.01. ***P ≤ 0.001. ****P ≤ 0.0001.

Figure 3.

(A–E) Functional characterization of wt Gαs and variants coexpressed with different GPCRs. 2B2 cells were cotransfected with GPCRs and wt or mutant Gαs, as indicated. After 2 days, cAMP accumulation was determined in the presence of decadic increasing concentration of indicated ligands by α screen technology. Results of four independent experiments performed in triplicates are shown as mean ± SEM. hPTH, human parathyroid hormone; hTSH, human TSH; α-MSH, alpha-melanocyte stimulating hormone.

Figure 4.

Phenylalanine 376 in different Gαs conformations and activity states. (A–A1) In the inactive state, conformation of the heterotrimeric Gαs amino acid F376 is cagelike embedded by several hydrophobic and/or aromatic amino acids (shown as green sticks). F376 is not directly located at the GDP/GTP (shown as orange spheres) binding pocket, unlike known pathogenic Gαs mutations, for example, residue A366, R201, or Q227 (7) (magenta sticks). (A2) Mutational variants such as p.F376V, p.F376Y, and p.F376M lead to specific changes in the tight hydrophobic/aromatic environment at position 376. Methionine is aliphatic in contrast to phenylalanine but is hydrophobic and interacts with aromatic ring systems. Valine is characterized by a shorter and more branched side chain as compared to phenylalanine. The patient mutant F376V therefore most likely leads to a loss of the tight interactions, which finally can cause structural modifications in the relative spatial α5 helix orientation compared with wt. (B) From the crystal structure Gαs/β-2AR complex [PDB entry 3SN6 (17)], it is known that the Gαs protein undergoes structural reorganization during interaction with the receptor, for example, a global movement of the helical domain (arrow), enabled by ligand-induced conformational changes in the receptor. (B1) The superimposition between the active and the inactive Gαs crystal structure shows that F376 changes the spatial localization during G protein activation related to local movements of the C-terminal α5 helix.