SFEBES2012 Oral Communications Young Endocrinologists prize session (8 abstracts)
Structure-function analysis of calcium-sensing receptor (CaSR) mutations reveal clustering at calcium binding sites of the extracellular bilobed venus flytrap domain
Fadil Hannan 1 , M Andrew Nesbit 1 , Chen Zhang 2 , Treena Cranston 3 , Paul Christie 1 , Carl Fratter 3 , Edward Brown 4 , Jenny Yang 2 & Rajesh Thakker 1
1Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom; 2Department of Chemistry, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA; 3Oxford Molecular Genetics Laboratory, Churchill Hospital, Oxford, United Kingdom; 4Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.
The calcium-sensing receptor (CaSR) is a G protein-coupled receptor with an extracellular bilobed venus flytrap domain (VFTD) predicted to contain five calcium-binding sites. The major site for calcium-binding is comprised of amino acid residues that line the cleft between the two lobes of the VFTD. We investigated the structure-function relationships of VFTD CaSR mutations identified in patients with familial hypocalciuric hypercalcaemia (FHH), neonatal severe primary hyperparathyroidism (NSHPT), or autosomal dominant hypocalcaemic hypercalciuria (ADHH). DNA sequence analysis was undertaken in 295 unrelated probands with FHH, NSHPT or ADHH and CaSR mutations affecting key VFTD residues were analysed by in vitro expression studies. Three-dimensional CaSR modelling, based on the structure of the rat metabotropic glutamate receptor, was performed to predict the locations of mutations in the VFTD. A total of 85 CaSR mutations were identified, 49 in FHH, 10 in NSHPT and 26 in ADHH patients. Thirty-four CaSR missense mutations were located in the VFTD, and 18 mutations were within 10 Angstroms of one or more of the calcium-binding sites, and were situated mainly within the VFTD cleft region. CaSR mutations at residues 173 (Leu173Phe or Leu173Pro) and 221 (Pro221Leu or Pro221Gln) were found in both FHH and in ADHH patients and were demonstrated to have inactivating and activating effects, respectively, on CaSR function. Three-dimensional modelling of residues 173 and 221 revealed that these are both located at the entrance to the VFTD cleft, and mutations of these residues were predicted to influence the entry and binding of calcium within the cleft region. Thus, our findings, which help to further elucidate the structure-function relationships of the CaSR extracellular domain, demonstrate that VFTD mutations cluster at calcium-binding sites and provide support for the VFTD cleft having a major role in the binding of extracellular calcium and regulation of CaSR function.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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