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A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
Published on: February 20, 2018
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Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein
Valerie N Babinsky1, Fadil M Hannan2, Caroline M Gorvin1
1From the Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom.
The Journal of Biological Chemistry
|March 20, 2016
Summary
This study shows that CaSR-targeted drugs can correct signaling issues in Gα11 mutations linked to calcium disorders and uveal melanoma. These compounds effectively normalize intracellular calcium levels and MAPK signaling in affected cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Germline Gα11 mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2).
- Somatic Gα11 mutations are implicated in uveal melanoma development through MAPK signaling.
- Allosteric CaSR modulators (cinacalcet, NPS-2143) affect CaSR signaling but their impact on downstream Gα11 is unclear.
Purpose of the Study:
- Investigate if cinacalcet and NPS-2143 can correct intracellular calcium (Ca(2+)i) alterations in FHH2 and ADH2.
- Evaluate the effect of Gα11 mutations on MAPK signaling (ERK phosphorylation).
- Assess NPS-2143's efficacy on a uveal melanoma Gα11 mutant.
Main Methods:
- Transfection of wild-type (WT) and mutant Gα11 proteins (FHH2, ADH2, uveal melanoma) into HEK293 cells expressing CaSR.
- Measurement of Ca(2+)i and ERK phosphorylation using flow cytometry and Alphascreen immunoassay.
- Exposure to extracellular calcium (Ca(2+)o) and allosteric modulators.
Main Results:
- Cinacalcet and NPS-2143 corrected Ca(2+)i responses in FHH2 and ADH2 Gα11 mutants.
- ADH2-associated Gα11 mutations did not show constitutive activation, inducing ERK phosphorylation only upon Ca(2+)o stimulation.
- NPS-2143 rectified increased ERK phosphorylation in ADH2 and uveal melanoma Gα11 mutants.
Conclusions:
- CaSR-targeted compounds can rectify signaling disturbances from germline and somatic Gα11 mutations.
- These compounds show potential for treating calcium disorders and uveal melanoma.
- ADH2-causing Gα11 mutations involve non-constitutive alterations in MAPK signaling.
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