Abstract by Wei Du

The calcium-sensing receptor (CaSR) is a member of the class C G protein-coupled receptor (GPCR) superfamily, and the receptor plays a crucial role in human calcium homeostasis through its regulation of the release of parathyroid hormone and calcitonin. As a result of this, genetic mutations (variants) in CaSR and its partner proteins are associated with calcium homeostasis disorders such as hypercalcemia and hypocalcemia. Moreover, positive or negative allosteric modulators (PAMs or NAMs) of CaSR that increase or inhibit CaSR function, respectively, can be applied in the treatment of these diseases. Unfortunately, the CaSR PAMs presently used in the clinic are often characterized by significant side effects, suggesting the need for better CaSR-targeted therapies.

The present PhD thesis is based on the results from three different studies. In the first study, we characterized the effects of five CaSR variants (Y63C, I81T, Q459R, W818stop, R955stop) associated with familial hypocalciuric hypercalcemia type 1 (FHH1) or autosomal dominant hypocalcemia type 1 (ADH1) on the expression, functional properties and pharmacology of CaSR. Via expression of wild-type (WT) CaSR and these variants in human embryonic kidney 293T (HEK293T) cells, we found that the variants induce significant changes in protein expression levels and the functional properties of CaSR, and these changes correlated with the clinical phenotypes caused by the variants. Interestingly, mechanistically different PAMs were found to mediate distinct degrees of functional rescue of the reduced functionality exhibited by the FHH1-associated CaSR variants.

In the second study, we characterized the effects of a novel variant (T347A) in the G-protein α-subunit G_α11 linked to hypocalciuric hypercalcemia type 2 (FHH2) on CaSR-mediated signaling through this G-protein in HEK293T cells. While the T347A variant did not alter G_α11 expression levels, it caused a modest but significant reduction in the responsiveness of CaSR to calcium, which correlates to the clinical phenotype caused by the variant.

In the third study, we performed a detailed in vitro functional characterization of four distinct nanobodies that target and act as PAMs at CaSR: Nb4, Nb5, Nb10, and Nb45. Each of the nanobodies displayed very similar PAM properties at CaSR in five different functional assays, with Nb4 and Nb10 being fairly potent CaSR PAMs. Interestingly, the activity of Nb45 was found to be completely dependent on epitope-tagging of the receptor, since this nanobody did not exhibit significant PAM activity at the untagged WT CaSR. 

In conclusion, the findings outlined in this thesis offer in-depth insights into the molecular phenotypes caused by five CaSR variants and a novel G_α11 variant and detail the functional properties of four novel nanobodies acting as CaSR PAMs. Thus, the results disclose the functional consequences of several variants underlying calcium homeostatic disorders and reveal the potential of nanobodies as a novel type of CaSR PAMs. Collectively, the findings in this work thus advance our understanding of the molecular basis underlying these disorders and the putative treatment of these with new generations of CaSR modulators.