Heterogeneity of G protein activation by the calcium-sensing receptor

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that plays a fundamental role in extracellular calcium (Ca²⁺ _e) homeostasis by regulating parathyroid hormone release and urinary calcium excretion. The CaSR has been described to activate all four G protein subfamilies (Gα_q/11, Gα_i/o, Gα_12/13, Gα_s), and mutations in the receptor that cause hyper/hypocalcaemia, have been described to bias receptor signalling. However, many of these studies are based on measurements of second messengers or gene transcription that occurs many steps downstream of receptor activation and can represent convergence points of several signalling pathways. Therefore, to assess CaSR-mediated G protein activation directly, we took advantage of a recently described NanoBiT G protein dissociation assay system. Our studies, performed in HEK293 cells stably expressing CaSR, demonstrate that Ca²⁺ _e stimulation activates all Gα_q/11 family and several Gα_i/o family proteins, although Gα_z was not activated. CaSR stimulated dissociation of Gα_12/13 and Gαs from Gβ-subunits, but this occurred at a slower rate than that of other Gα-subunits. Investigation of cDNA expression of G proteins in three tissues abundantly expressing CaSR, the parathyroids, kidneys and pancreas, showed Gα₁₁, Gα_z, Gα_i1 and Gα₁₃ genes were highly expressed in parathyroid tissue, indicating CaSR most likely activates Gα₁₁ and Gα_i1 in parathyroids. In kidney and pancreas, the majority of G proteins were similarly expressed, suggesting CaSR may activate multiple G proteins in these cells. Thus, these studies validate a single assay system that can be used to robustly assess CaSR variants and biased signalling and could be utilised in the development of new pharmacological compounds targeting CaSR.