Mandibulofacial dysostosis with alopecia results from ETAR gain-of-function mutations via allosteric effects on ligand binding
Yukiko Kurihara, … , Jeanne Amiel, Hiroki Kurihara
Yukiko Kurihara, … , Jeanne Amiel, Hiroki Kurihara
Published January 13, 2023
Citation Information: J Clin Invest. 2023;133(4):e151536. https://doi.org/10.1172/JCI151536.
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Research Article Development Genetics

Mandibulofacial dysostosis with alopecia results from ETAR gain-of-function mutations via allosteric effects on ligand binding

Abstract

Mutations of G protein–coupled receptors (GPCRs) cause various human diseases, but the mechanistic details are limited. Here, we establish p.E303K in the gene encoding the endothelin receptor type A (ETAR/EDNRA) as a recurrent mutation causing mandibulofacial dysostosis with alopecia (MFDA), with craniofacial changes similar to those caused by p.Y129F. Mouse models carrying either of these missense mutations exhibited a partial maxillary-to-mandibular transformation, which was rescued by deleting the ligand endothelin 3 (ET3/EDN3). Pharmacological experiments confirmed the causative ETAR mutations as gain of function, dependent on ET3. To elucidate how an amino acid substitution far from the ligand binding site can increase ligand affinity, we used molecular dynamics (MD) simulations. E303 is located at the intracellular end of transmembrane domain 6, and its replacement by a lysine increased flexibility of this portion of the helix, thus favoring G protein binding and leading to G protein–mediated enhancement of agonist affinity. The Y129F mutation located under the ligand binding pocket reduced the sodium-water network, thereby affecting the extracellular portion of helices in favor of ET3 binding. These findings provide insight into the pathogenesis of MFDA and into allosteric mechanisms regulating GPCR function, which may provide the basis for drug design targeting GPCRs.

Authors

Yukiko Kurihara, Toru Ekimoto, Christopher T. Gordon, Yasunobu Uchijima, Ryo Sugiyama, Taro Kitazawa, Akiyasu Iwase, Risa Kotani, Rieko Asai, Véronique Pingault, Mitsunori Ikeguchi, Jeanne Amiel, Hiroki Kurihara

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Figure 2

Craniofacial phenotypes of Ednra mutant mice are rescued by Edn3 deletion.

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Craniofacial phenotypes of Ednra mutant mice are rescued by Edn3 deletio...
(A–D) Facial appearance and skeletal morphology of WT and heterozygous mutant (EdnraY129F/+and EdnraE303K/+) mice. Mutant adult mice show over-folded pinnae and skin erosion around the eyelid (A). E18.5 bone and cartilage stains show transformation of the upper jaw components into a rod-shaped bone in the mutants (arrows in B and C) and deformity of the incus (arrowheads in D). The mandible is almost normal, except the angular process, which is slightly reduced (arrowhead in B). (E) Facial abnormalities in the EdnraY129F/+ and EdnraE303K/+ mutants are variably rescued by heterozygous deletion of the Edn3 gene. (F and G) Transformation of the upper jaw components in the mutants is rescued by heterozygous Edn3 deletion (arrows). (H) deformity of the incus (arrowheads) is not rescued by heterozygous Edn3 deletion. (I) The incus of the mutants is normalized by homozygous loss of Edn3 (arrowheads). (J) Schematic diagrams indicating the skeletal components affected in the Ednra mutants (green). (K) Comparison of Edn1 and Edn3 mRNA levels at E9.5. 4 pairs of RNA samples were extracted from pools of mandibular or maxillary arches, in each of which 7 to 14 WT littermate samples were combined and were subjected to q-RT-PCR. *P < 0.05 by Mann-Whitney U test (n = 4). (L) Schematic diagrams summarizing the effect of ET signals in normal and pathogenic states. Red lines indicate ET3-dependent activation of mutant receptors. (M) Dlx5 and Dlx6 expression at E9.5 is restricted to the mandibular region of PA1 in WT embryos, whereas the expression of both genes extends into the maxillary region of PA1 in EdnraY129F/+and EdnraE303K/+ embryos (arrowheads). n = 3–6. agp, angular process; dnt, dentary; gn, gonial; in, incus; jg, jugal; ma, malleus; Mc, Meckel’s cartilage; mx, maxilla; sq, squamosal. Scale bars: 1 mm (B, C), 500 µm (D), and 100 µm (M)