SAT-036 Extracellular Domain Calcium-Sensing Receptor (CaSR) Mutations Leading to Hypercalcemic and Hypocalcemic Disorders Cluster at the Homodimeric Interface

The calcium-sensing receptor (CaSR) is a homodimeric G-protein coupled receptor that plays a pivotal role in extracellular Ca(2+) (Ca(2+)(e)) homeostasis, and loss- and gain-of-function mutations of the CaSR cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively. Crystal structure analysis has shown that the CaSR has a large extracellular domain (ECD) comprising lobes 1 and 2, and a cysteine-rich domain (CRD), which connects the ECD to the transmembrane domain. The CaSR ECD binds Ca(2+)(e) at multiple sites, which induces conformational changes at the extracellular dimer interface that lead to intracellular signaling via the inositol trisphosphate (IP(3)) and mitogen-activated protein kinase (MAPK) pathways. To further elucidate the structure-function relationships of the ECD, we examined the location of all CaSR ECD mutations reported to-date in FHH1 and ADH1 probands using available CaSR crystal structures. These studies identified that 127 FHH1 and 66 ADH1 mutations affected ECD residues, with >50% of these mutations being located at the dimer interface. Mutations predicted to disrupt key CaSR dimer-dimer interactions included: a lobe 1 Tyr161Cys mutation, which impaired an interprotomer interaction with the lobe 1 Pro55 residue; a Ser171Asn mutation predicted to disrupt a lobe 2 interprotomer salt bridge, which forms upon agonist binding; and a Gly553Arg mutation, which altered interprotomer hydrophobic interactions within the CRD. We investigated the effect of the Tyr161Cys, Ser171Asn and Gly553Arg mutations on receptor dimerization using western blotting and showed that these mutant proteins all form dimers similar to the wild-type (WT) CaSR. Furthermore, we evaluated the effect of these mutations on CaSR function in HEK293 cells following stimulation with Ca(2+)(e), by measuring accumulation of inositol monophosphate (IP(1)), which is an IP(3) metabolite, and also by measuring serum response element (SRE)-containing luciferase reporter fold-change responses, which is an indicator of MAPK activation. The Tyr161Cys, Ser171Asn and Gly553Arg dimer interface mutations were shown to significantly impair CaSR-mediated signaling. Indeed, cells expressing the Tyr161Cys, Ser171Asn or Gly553Arg mutant CaSRs all showed a >30% reduction in IP(1) accumulation and >50% reduction in SRE-reporter responses, respectively, compared to WT-expressing cells (p<0.05, results are from 3 independent experiments). Thus, these studies demonstrate that the majority of FHH1- and ADH1-causing CaSR ECD mutations are located at the dimer interface, but likely do not disrupt dimer formation. Instead, these mutations may potentially influence conformational changes that occur at the dimer interface upon CaSR activation.