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

Valerie N. Babinsky, Fadil M. Hannan, Caroline M. Gorvin, Sarah A. Howles, M. Andrew Nesbit, Nigel Rust, Aylin C. Hanyaloglu, Jianxin Hu, Allen M. Spiegel, Rajesh V. Thakker

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)10876-10885
Number of pages10
JournalJournal of Biological Chemistry
Volume291
Issue number20
DOIs
StatePublished - May 13 2016

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Calcium-Sensing Receptors
GTP-Binding Proteins
Modulation
Mutation
Phosphorylation
Calcium
Flow cytometry
Germ-Line Mutation
HEK293 Cells
Mutant Proteins
Familial Hypercalciuric Hypocalcemia
Immunoassay
Modulators
Flow Cytometry
Carcinogenesis
Proteins
N-(2-hydroxy-3-(2-cyano-3-chlorophenoxy)propyl)-1,1-dimethyl-2-(2-nephthyl)ethylamine

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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Allosteric modulation of the calcium-sensing receptor rectifies signaling abnormalities associated with G-protein α-11 mutations causing hypercalcemic and hypocalcemic disorders. / Babinsky, Valerie N.; Hannan, Fadil M.; Gorvin, Caroline M.; Howles, Sarah A.; Nesbit, M. Andrew; Rust, Nigel; Hanyaloglu, Aylin C.; Hu, Jianxin; Spiegel, Allen M.; Thakker, Rajesh V.

In: Journal of Biological Chemistry, Vol. 291, No. 20, 13.05.2016, p. 10876-10885.

Research output: Contribution to journalArticle

Babinsky, Valerie N. ; Hannan, Fadil M. ; Gorvin, Caroline M. ; Howles, Sarah A. ; Nesbit, M. Andrew ; Rust, Nigel ; Hanyaloglu, Aylin C. ; Hu, Jianxin ; Spiegel, Allen M. ; Thakker, Rajesh V. / Allosteric modulation of the calcium-sensing receptor rectifies signaling abnormalities associated with G-protein α-11 mutations causing hypercalcemic and hypocalcemic disorders. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 20. pp. 10876-10885.
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abstract = "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.",
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AU - Gorvin, Caroline M.

AU - Howles, Sarah A.

AU - Nesbit, M. Andrew

AU - Rust, Nigel

AU - Hanyaloglu, Aylin C.

AU - Hu, Jianxin

AU - Spiegel, Allen M.

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