Heterogeneity of G protein activation by the calcium-sensing receptor.

Hasnat Ali Abid,Asuka Inoue,Caroline M Gorvin

doi:10.1530/jme-21-0058

Hasnat Ali Abid, Asuka Inoue + Show 1 more

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https://doi.org/10.1530/jme-21-0058

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Abstract

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 (Gαq/11, Gαi/o, Gα12/13, Gαs), 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 Gαq/11 family and several Gαi/o family proteins, although Gαz was not activated. CaSR stimulated dissociation of Gα12/13 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, Gαz, Gαi1 and Gα13 genes were highly expressed in parathyroid tissue, indicating CaSR most likely activates Gα11 and Gαi1 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.

Highlights

The calcium-sensing receptor (CaSR) is a class C homodimeric G protein-coupled receptor (GPCR) that plays a fundamental role in extracellular calcium (Ca2+e) homeostasis by regulating parathyroid hormone (PTH) release and urinary calcium excretion
Our studies confirm that the CaSR can activate multiple G proteins, and this may account for the increasingly varied functions that have been described for the receptor in a wide range of tissues (Hofer et al 2000, Rossol et al 2012, Yarova et al 2015, Zietek & Daniel 2015)
Other G proteins that are highly expressed in parathyroid tissue, such as Gαz, were not activated by CaSR in the NanoBiT dissociation assays, indicating it is unlikely to be important in calcium-mediated responses

Summary

Introduction

The calcium-sensing receptor (CaSR) is a class C homodimeric G protein-coupled receptor (GPCR) that plays a fundamental role in extracellular calcium (Ca2+e) homeostasis by regulating parathyroid hormone (PTH) release and urinary calcium excretion. Consistent with its critical role in maintaining serum calcium concentrations, the CaSR is highly expressed on the surface of parathyroid and kidney cells (Riccardi & Brown 2010). Inactivating mutations cause familial hypocalciuric hypercalcaemia type-1 (FHH1), characterised by lifelong elevated serum calcium, moderate-to-high PTH concentrations, and low renal calcium excretion, and rarely cause neonatal severe hyperparathyroidism, which can be fatal if untreated (Pollak et al 1993). Activating CaSR mutations cause autosomal dominant hypocalcaemia type-1 (ADH1), characterised by mild-to-moderate hypocalcaemia, with inappropriately low-to-normal serum PTH (Pearce et al 1996). Inactivating mutations in the G protein-α11 (Gα11), by which the CaSR signals and the adaptor protein-2 σ-subunit (AP2σ), which regulates endocytosis, cause FHH2 and FHH3, respectively; whereas activating Gα11 mutations cause ADH2 (Mannstadt et al 2013, Nesbit et al 2013a,b)

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