An activating CaSR variant with biased signaling reveals a critical residue for Gα11 coupling

Autosomal dominant hypocalcemia (ADH) is due to enhanced calcium-dependent signaling caused by heterozygous gain-of-function (GOF) variants in the CASR gene (ADH1) or in the GNA11 gene, encoding Gα11 (ADH2). Both ADH1 and ADH2 are associated with hypocalcemia and normal or inappropriately low levels of circulating PTH. ADH1 patients typically manifest hypercalciuria, while ADH2 is associated with short stature in approximately 42% of cases. We evaluated a 10-year-old boy with hypoparathyroidism and short stature. Biochemical analyses revealed hypocalcemia, hyperphosphatemia and inconsistent hypercalciuria. Genetic analyses revealed a de novo heterozygous p.Leu723Arg variant in CASR. We characterized the expression of recombinant wild type and Leu723Arg CaSR proteins in HEK293 cells and assessed G protein activation in vitro by CaSR using Bioluminescence Resonance Energy Transfer (BRET). Transient expression studies showed the Leu723Arg variant was normally expressed but resulted in a significantly lower EC50 for extracellular calcium activation of G11 but not other G proteins (i.e. Gi, Gq, Gs). The Leu723Arg substitution has a novel GOF phenotype that leads to biased CaSR activation of G11 signaling, suggesting that residue 723 specifies activation of G11 but not other G proteins. Similar studies of a previously described CaSR variant associated with hypoparathyroidism and short stature, Leu616Val, showed no changes in any G protein pathways, indicating it is likely to be a benign variant. Given the preferential activation of G11 by the Leu723Arg CaSR variant, we propose that the patient's short stature shares a similar basis to that in patients with ADH2 due to GOF variants in GNA11.