title: Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein α-11 Mutations Causing Hypercalcemic and Hypocalcemic Disorders.
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.
authors.affiliation: From the Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom.
authors.forename: Valerie N
authors.initials: VN
authors.lastname: Babinsky
authors.affiliation: 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.
authors.forename: Fadil M
authors.initials: FM
authors.lastname: Hannan
authors.affiliation: From the Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom.
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Gorvin
authors.affiliation: From the Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Howles
authors.affiliation: 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.
authors.forename: M Andrew
authors.initials: MA
authors.lastname: Nesbit
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
authors.forename: Nigel
authors.initials: N
authors.lastname: Rust
authors.affiliation: Department of Surgery and Cancer, Institute of Reproductive Biology and Development, Imperial College London, London W12 0NN, United Kingdom.
authors.forename: Aylin C
authors.initials: AC
authors.lastname: Hanyaloglu
authors.affiliation: Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Hu
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: Allen M
authors.initials: AM
authors.lastname: Spiegel
authors.affiliation: From the Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom, rajesh.thakker@ndm.ox.ac.uk.
authors.forename: Rajesh V
authors.initials: RV
authors.lastname: Thakker
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects / Q000235:genetics; D019943:Amino Acid Substitution; D000069449:Cinacalcet / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006934:Hypercalcemia / Q000235:genetics / Q000378:metabolism*; D006996:Hypocalcemia / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D009281:Naphthalenes / Q000494:pharmacology; D044169:Receptors, Calcium-Sensing / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C095550:CASR protein, human; C436740:N-(2-hydroxy-3-(2-cyano-3-chlorophenoxy)propyl)-1,1-dimethyl-2-(2-nephthyl)ethylamine; D009281:Naphthalenes; D044169:Receptors, Calcium-Sensing; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000069449:Cinacalcet
keywords: G protein; GNA11; autosomal dominant hypocalcemia; calcium; drug action; familial hypocalciuric hypercalcemia; genetic disease; parathyroid hormone; uveal melanoma