Calcium-sensing receptor sequencing in 21 patients with idiopathic or familial parathyroid disorder: pitfalls and characterization of a novel I32 V loss-of-function mutation

Abstract Calcium-sensing receptors (CaSRs) regulate systemic Ca2+ homeostasis. Loss-of-function mutations cause familial benign hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). FHH/NSHPT mutations can reduce trafficking of CaSRs to the plasma membrane. CaSR signaling is potentiated by agonist-driven anterograde CaSR trafficking, leading to a new steady state level of plasma membrane CaSR, which is maintained, with minimal functional desensitization, as long as extracellular Ca2+ is elevated. This requirement for CaSR signaling to drive CaSR trafficking to the plasma membrane led us to reconsider the mechanism(s) contributing to dysregulated trafficking of FHH/NSHPT mutants. We simultaneously monitored dynamic changes in plasma membrane levels of CaSR and intracellular Ca2+, using a chimeric CaSR construct, which allowed explicit tracking of plasma membrane levels of mutant or wild-type CaSRs in the presence of nonchimeric partners. Expression of mutants alone revealed severe defects in plasma membrane targeting and Ca2+ signaling, which were substantially rescued by coexpression with wild-type CaSR. Biasing toward heterodimerization of wild-type and FHH/NSHPT mutants revealed that intracellular Ca2+ oscillations were insufficient to rescue plasma membrane targeting. Coexpression of the nonfunctional mutant E297K with the truncation CaSRΔ868 robustly rescued trafficking and Ca2+ signaling, whereas coexpression of distinct FHH/NSHPT mutants rescued neither trafficking nor signaling. Our study suggests that rescue of FHH/NSHPT mutants requires a steady state intracellular Ca2+ response when extracellular Ca2+ is elevated and argues that Ca2+ signaling by wild-type CaSRs rescues FHH mutant trafficking to the plasma membrane.

Download Full-text

Expression of Calcium-Sensing Receptor and Characterization of Intracellular Signaling in Human Pituitary Adenomas

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.84.8.2848 ◽

1999 ◽

Vol 84 (8) ◽

pp. 2848-2853 ◽

Cited By ~ 14

Author(s):

R. Romoli

Keyword(s):

Pituitary Adenomas ◽

Intracellular Signaling ◽

Calcium Sensing Receptor ◽

Human Pituitary ◽

Calcium Sensing ◽

Human Pituitary Adenomas

Download Full-text

Conditionally immortalized human proximal tubular epithelial cells isolated from the urine of a healthy subject express functional calcium-sensing receptor

AJP Renal Physiology ◽

10.1152/ajprenal.00352.2014 ◽

2015 ◽

Vol 308 (11) ◽

pp. F1200-F1206 ◽

Cited By ~ 11

Author(s):

Annarita Di Mise ◽

Grazia Tamma ◽

Marianna Ranieri ◽

Maria Svelto ◽

Bert van den Heuvel ◽

...

Keyword(s):

Healthy Subject ◽

Cell Line ◽

Cell Lines ◽

Cyclopiazonic Acid ◽

Cytosolic Calcium ◽

Epithelial Cell Line ◽

Loss Of Function ◽

Calcium Sensing Receptor ◽

Calcium Sensing ◽

Proximal Tubular

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor, which plays an essential role in regulating Ca2+ homeostasis. Here we show that conditionally immortalized proximal tubular epithelial cell line (ciPTEC) obtained by immortalizing and subcloning cells exfoliated in the urine of a healthy subject expresses functional endogenous CaSR. Immunolocalization studies of polarized ciPTEC revealed the apical localization of the receptor. By Western blotting of ciPTEC lysates, both monomeric and dimeric forms of CaSR at 130 and ∼250 kDa, respectively, were detected. Functional studies indicated that both external calcium and the positive CaSR allosteric modulator, NPS-R568, induced a significant increase in cytosolic calcium, proving a high sensitivity of the endogenous receptor to its agonists. Calcium depletion from the endoplasmic reticulum using cyclopiazonic acid abolished the increase in cytosolic calcium elicited by NPS-R568, confirming calcium exit from intracellular stores. Activation of CaSR by NPS-R significantly reduced the increase in cAMP elicited by forskolin (FK), a direct activator of adenylate cyclase, further confirming the functional expression of the receptor in this cell line. CaSR expressed in ciPTEC was found to interact with Gq as a downstream effector, which in turn can cause release of calcium from intracellular stores via phospholipase C activation. We conclude that human proximal tubular ciPTEC express functional CaSR and respond to its activation with a release of calcium from intracellular stores. These cell lines represent a valuable tool for research into the disorder associated with gain or loss of function of the CaSR by producing cell lines from patients.

Download Full-text

GENETICS IN ENDOCRINOLOGY: Gain and loss of function mutations of the calcium-sensing receptor and associated proteins: current treatment concepts

Acta Endocrinologica ◽

10.1530/eje-15-1028 ◽

2016 ◽

Vol 174 (5) ◽

pp. R189-R208 ◽

Cited By ~ 32

Author(s):

Bernhard Mayr ◽

Dirk Schnabel ◽

Helmuth-Günther Dörr ◽

Christof Schöfl

Keyword(s):

Calcium Supplementation ◽

Current Treatment ◽

Loss Of Function ◽

Calcium Sensing Receptor ◽

Calcium Sensing ◽

Therapeutic Concepts ◽

Sigma 1 ◽

Associated Proteins ◽

Treatment Concepts

The calcium-sensing receptor (CASR) is the main calcium sensor in the maintenance of calcium metabolism. Mutations of theCASR, the G protein alpha 11 (GNA11) and the adaptor-related protein complex 2 sigma 1 subunit (AP2S1) genes can shift the set point for calcium sensing causing hyper- or hypo-calcemic disorders. Therapeutic concepts for these rare diseases range from general therapies of hyper- and hypo-calcemic conditions to more pathophysiology oriented approaches such as parathyroid hormone (PTH) substitution and allosteric CASR modulators. Cinacalcet is a calcimimetic that enhances receptor function and has gained approval for the treatment of hyperparathyroidism. Calcilytics in turn attenuate CASR activity and are currently under investigation for the treatment of various diseases. We conducted a literature search for reports about treatment of patients harboring inactivating or activating CASR, GNA11 or AP2S1 mutants and aboutin vitroeffects of allosteric CASR modulators on mutated CASR. The therapeutic concepts for patients with familial hypocalciuric hypercalcemia (FHH), neonatal hyperparathyroidism (NHPT), neonatal severe hyperparathyroidism (NSHPT) and autosomal dominant hypocalcemia (ADH) are reviewed. FHH is usually benign, but symptomatic patients benefit from cinacalcet. In NSHPT patients pamidronate effectively lowers serum calcium, but most patients require parathyroidectomy. In some patients cinacalcet can obviate the need for surgery, particularly in heterozygous NHPT. Symptomatic ADH patients respond to vitamin D and calcium supplementation but this may increase calciuria and renal complications. PTH treatment can reduce relative hypercalciuria. None of the currently available therapies for ADH, however, prevent tissue calcifications and complications, which may become possible with calcilytics that correct the underlying pathophysiologic defect.

Download Full-text