Aim: 

Hyperparathyroidism (and the related hypercalcemia) is the third most frequent endocrine disorder. In some cases, hyperparathyroidism is a genetic-related disease, as for instance familial hypocalciuric hypercalcemia (FHH). Parathyroid chief cells are the Ca2+-sensing cells in the body. This is accomplished by the G protein-coupled Ca2+-Sensing Receptor (CaSR), which is expressed on the cell surface and is activated by extracellular Ca2+. Shortly after its cloning, different mutations of CaSR were shown to be the cause of FHH. Such polymorphic variants could also be associated with other complications of the disease (for example, adenomas, fractures, chondrocalcinosis and pancreatitis) although the mechanisms underlying parathyroid dysfunction associated with altered CaSR properties are not yet entirely understood.

Methods and Results: 

Patients followed at the Endocrinology and Metabolism Unit of the University Hospital of Pisa underwent the standard clinical evaluation for the diagnosis of hypercalcemia. Biochemical data and genetic analysis were also obtained in the available family members. In particular, the entire coding sequence and splice junctions of the CASR gene were sequenced, and different unreported CASR mutations were found. In this respect, the clinical features of the probands and affected family members were consistent with the diagnosis of FHH. COS-7 cells were then transfected with CaSR gene coding for wild-type or carrying unreported polymorphic variants (loss-of-function mutations). The evaluation of CaSR expression and its spatial distribution within the cells was performed by means of immunofluorescence confocal microscopy. Functionally, CaSR was activated by treating cells with increasing concentrations of Ca2+. CaSR coupling to inositol trisphosphate release was evaluated by radiochemical approaches. In addition, we studied the CaSR-induced increase of intracellular Ca2+ concentration by means of spectrofluorimetry-based assays. The pharmacological parameters of responses (pEC50 and Emax) were determined by non-linear regression curve analysis. Finally, CaSR coupling to mitogen-activated protein kinase pathway was evaluated by Western blot experiments.

Conclusion: 

Data obtained with different polymorphic variants of CaSR were compared in order to elucidate the pharmacological and the functional features of distinct loss-of-function mutations. Taken toghether, our data provided an explanation for the clinical phenotype in the patients and suggest that conformational changes account for altered CASR activity. Moreover, a reduced complement of normal CASRs in heterozygous patients, may contribute to defective Ca2+-sensing in vivo. From a practical point of view, CaSR potentially represents an important therapeutic target for disorders in which the receptor is inappropriately underactive. In this line, the development of allosteric activators ("calcimimetics") which may specifically and efficiently target the mutated CaSRs might offer a new therapeutic perspective in CaSR-based therapy.