Calcium-Sensing Receptor (CaSR) Mutations Causing Hyper- and Hypocalcemia Uncouple Intracellular Calcium and Mitogen-Activated Protein Kinase Signalling Responses

Presentation Number: OR05-2
Date of Presentation: April 3rd, 2017

Morten Frost*1, Caroline M Gorvin1, Tomas Malinauskas1, Treena Cranston2, Fadil Hannan3, E. Yvonne Jones1, Christian Siebold1 and Rajesh V Thakker1
1University of Oxford, Oxford, United Kingdom, 2Oxford Molecular Genetics Laboratory, Churchill Hospital, Oxford, United Kingdom, 3University of Liverpool, Liverpool England, UNITED KINGDOM

Abstract

The calcium-sensing receptor (CaSR) is a homodimeric G-protein coupled receptor (GPCR) that signals via intracellular calcium (Ca2+i) and the mitogen-activated protein kinase (MAPK) pathway to regulate parathyroid hormone (PTH) secretion in response to changes in extracellular calcium (Ca2+e). The central importance of the CaSR in Ca2+e homeostasis has been demonstrated by the identification of loss- or gain-of-function CaSR mutations that lead to familial hypocalciuric hypercalcemia (FHH) or autosomal dominant hypocalcemia (ADH), respectively. However, the mechanisms determining whether the CaSR signals via Ca2+i or MAPK have not been established, and we hypothesised that some CaSR residues, which are the sites of both FHH-causing and ADH-causing mutations, may act as intramolecular switches to direct signalling through these pathways. We identified 5 such CaSR residues harbouring 10 FHH- and ADH-causing heterozygous missense mutations respectively comprising: Gln27Pro and Gln27Glu; Asn178Asp and Asn178Tyr; Ser657Tyr and Ser657Cys; Ser820Ala and Ser820Phe; and Thr828Ile and Thr828Asn. We characterised these CaSR switch residue mutations, and studied their effects on CaSR structure and signalling by expressing them in HEK293 cells and using an NFAT-response element containing luciferase reporter to measure Ca2+i–induced gene expression, and a serum-response element (SRE) containing luciferase reporter to measure MAPK-induced gene expression. Three-dimensional homology modelling revealed these CaSR residues to be located in the extracellular (EC) region of the CaSR (Gln27 and Asn178), the transmembrane (TM) domains (Ser657 and Ser820), and the third EC loop (Thr828). All of the FHH-causing mutations resulted in loss-of-function of NFAT and SRE reporter activity, with the exception of Asn178Asp, which did not affect SRE reporter activity, and Ser820Ala, which surprisingly resulted in gain-of-function of SRE activity. All of the ADH-causing mutations resulted in gain-of-function of NFAT and SRE reporter activity, with the exception of Ser657Cys and Ser820Phe, which both resulted in loss-of-function of SRE reporter activity. The Ser657 and Ser820 switch residue mutations are located in TM2 and TM6 domains, which are predicted to interact with downstream effectors such as G-proteins and β-arrestin, and these results indicate that structural alterations to the CaSR TM domains may lead to uncoupling of the Ca2+i and MAPK pathways. The Asn178 residue is located in the EC domain at the interface of the CaSR dimer, thereby illustrating the role of CaSR homodimerisation in possible enhancement of MAPK signalling. Thus, our results reveal critical roles for CaSR residues Ser657 and Ser820 in Ca2+i and MAPK-mediated signal transduction, and for Asn178 in CaSR dimerization, disruption of which causes FHH and ADH.

 

Nothing to Disclose: MF, CMG, TM, TC, FH, EYJ, CS, RVT