Allosteric modulation of the calcium-sensing receptor rectifies signaling abnormalities associated with G-protein α-11 mutations causing hypercalcemic and hypocalcemic disorders

Germline loss- and gain-of-function mutations of G-protein α-11 (Gα₁₁), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca²⁺i) signaling, lead to familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2), respectively, whereas somatic Gα₁₁ mutations mediate uveal melanoma development by constitutively up-regulatingMAPKsignaling. Cinacalcet and NPS-2143 are allosteric CaSR activators and inactivators, respectively, that ameliorate signaling disturbances associated with CaSR mutations, but their potential to modulate abnormalities of the downstreamGα₁₁ protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca²⁺i alterations associated with FHH2- and ADH2-causing Gα₁₁ mutations, and evaluated the influence of germline gainof- function Gα₁₁ mutations on MAPK signaling by measuring ERK phosphorylation, and assessed the effect of NPS-2143 on a uveal melanoma Gα₁₁ mutant. WT and mutant Gα₁₁ proteins causing FHH2, ADH2 or uveal melanoma were transfected in CaSR-expressing HEK293 cells, and Ca²⁺i and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca²⁺ (Ca²⁺ o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca²⁺i responses of FHH2- and ADH2-associated Gα₁₁ lossand gain-of-function mutations, respectively. ADH2-causing Gα₁₁ mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca²⁺o stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somaticGα₁₁ mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα₁₁ mutations induce non-constitutive alterations in MAPK signaling.