Excessive Signal Transduction of Gain-of-Function Variants of the Calcium-Sensing Receptor (CaSR) Are Associated with Increased ER to Cytosol Calcium Gradient

Marianna Ranieri,Grazia Tamma,Giuseppe Vezzoli,Annarita Di Mise,Giovanna Valenti,Maria Svelto,Laura Soldati,Nanette H. Bishopric

doi:10.1371/journal.pone.0079113

Abstract

In humans, gain-of-function mutations of the calcium-sensing receptor (CASR) gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA) and reducing expression of Plasma Membrane Calcium-ATPase (PMCA). Wild-type CaSR (hCaSR-wt) and its gain-of-function (hCaSR-R990G; hCaSR-N124K) variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate) receptor inputs to cell function.

Highlights

The extracellular calcium-sensing GPCR (CaSR) belongs to the C family of the G-protein-coupled receptors GPCR, expressed primarily, but not exclusively, in parathyroid glands and kidney [1,2]
We demonstrated for the first time that functional expression of gain-of-function variants of the GPCR CaSR results in parallel modulation of the expression level and activity of an integrated set of regulatory proteins contributing to an excessive signal transduction of mutant receptors
The main findings reported in this work are summarized as follows: (i) both the hCaSR-R990G and the hCaSR-N124K mutant receptors had significant reduced EC50 values when compared with hCaSR-wt, (ii) basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its activating variants compared to mock or loss-offunction hCaSR-D121 expressing cells, (iii) FRET experiments with the D1ER probe, which detects [Ca2+]endoplasmic reticulum (ER) directly, demonstrated a significantly higher calcium accumulation in the ER in cells expressing the activating CaSR variants compared to hCaSRwt expressing cells, (iv) SarcoEndoplasmic Reticulum Calcium-ATPase (SERCA) expression and activity increased significantly in cells expressing activating CaSR variants compared to hCaSR-wt expressing cells

Summary

Introduction

The extracellular calcium-sensing GPCR (CaSR) belongs to the C family of the G-protein-coupled receptors GPCR, expressed primarily, but not exclusively, in parathyroid glands and kidney [1,2]. The CaSR senses changes in extracellular calcium concentrations and regulates parathyroid hormone (PTH) secretion and renal tubular calcium reabsorption to maintain serum calcium levels within the normal range [3,4,5,6,7]. Loss-of-function CaSR mutations result in familial (benign) hypocalciuric hypercalcemia (FBHH), and neonatal severe primary hyperparathyroidism (NSHPT), characterized by resistance to the normal inhibition of PTH secretion by the hormone agonist, extracellular calcium [10,11,12]

Methods

Results

Conclusion