Reduced affinity of calcium sensing-receptor heterodimers and reduced mutant homodimer trafficking combine to impair function in a mouse model of Familial Hypocalciuric Hypercalcemia Type 1

Heterozygous loss-of-function mutation of the calcium sensing-receptor (CaSR), causes familial hypocalciuric hypercalcemia type 1 (FHH1), a typically benign condition characterized by mild hypercalcemia. In contrast, homozygous mutation of this dimer-forming G-protein coupled receptor manifests as the lethal neonatal severe hyperparathyroidism (NSHPT). To investigate the mechanisms by which CaSR mutations lead to these distinct disease states, we engineered wild-type (WT) and an exon 5-deficient disease-causing mutation, and transfected expression constructs into human embryonic kidney (HEK) cells. WT protein was mainly membrane-expressed whereas the mutant CaSR protein (mCaSR) was confined to the cytoplasm. Co-expression of WT CaSR directed mCaSR to the cell membrane. In assays of CaSR function, increases in extracellular [Ca 2+ ] ([Ca 2+ ] o ) increased intracellular [Ca 2+ ] ([Ca 2+ ] i ) in cells expressing WT CaSR while the response was reduced in cells coexpressingmutant and WT receptor. Untransfected cells or those expressing mCaSR alone, showed minimal, equivalent responses to increased [Ca 2+ ]o. Immunoprecipitation experiments confirmed an association between mutant and wild-type CaSR. The affinity of the WT CaSR for calcium was three times greater than that of the heterodimer. The maximal functional response to [Ca] o was dependent on localization of CaSR to the membrane level and independent of homo- or heterodimerizations. In summary, these results suggest that heterodimerization of WT and mCaSR receptors, rescues the trafficking defect of the mutant receptors and also reduces the affinity of the WT-mutant heterodimer for [Ca] o . In contrast, the homozygous mutants do not produce functional receptors on cell membrane. These data indicate how substantial differences between signaling of hetero- and homodimeric mutants lead to profound differences in the severity of disease in heterozygous and homozygous carriers of these mutations.