Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein α-11 Mutations Causing Hypercalcemic and Hypocalcemic Disorders

Research output: Contribution to journalArticlepeer-review

Authors

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

Colleges, School and Institutes

External organisations

  • From the Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom.
  • From the Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom, Department of Musculoskeletal Biology, University of Liverpool, Liverpool L69 3GA, United Kingdom.
  • From the Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom, Biomedical Sciences Research Institute, Ulster University, Coleraine BT52 1SA, United Kingdom.
  • Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
  • Imperial College London
  • Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and.
  • Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461;
  • From the Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom, rajesh.thakker@ndm.ox.ac.uk.

Abstract

Germline loss- and gain-of-function mutations of G-protein α-11 (Gα11), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca(2+) 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-regulating MAPK signaling. 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 downstream Gα11 protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca(2+) i alterations associated with FHH2- and ADH2-causing Gα11 mutations, and evaluated the influence of germline gain-of-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 Ca(2+) i and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca(2+) (Ca(2+) o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca(2+) i responses of FHH2- and ADH2-associated Gα11 loss- and gain-of-function mutations, respectively. ADH2-causing Gα11 mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca(2+) 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 somatic Gα11 mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα11 mutations induce non-constitutive alterations in MAPK signaling.

Details

Original languageEnglish
Pages (from-to)10876-85
Number of pages10
JournalJournal of Biological Chemistry
Volume291
Issue number20
Early online date18 Mar 2016
Publication statusPublished - 13 May 2016

Keywords

  • Allosteric Regulation, Amino Acid Substitution, Cinacalcet Hydrochloride, GTP-Binding Protein alpha Subunits, Gq-G11, HEK293 Cells, Humans, Hypercalcemia, Hypocalcemia, Mutation, Missense, Naphthalenes, Receptors, Calcium-Sensing, Signal Transduction, Journal Article, Research Support, Non-U.S. Gov't