Roles of intraloops‐2 and ‐3 and the proximal C‐terminus in signalling pathway selection from the human calcium‐sensing receptor

Goolam, M.A.; Ward, James H; Avlani, Vimesh Ashvinkumar; Leach, Katie; Christopoulos, Arthur; Conigrave, Arthur D.

doi:10.1016/j.febslet.2014.07.022

Cited by 14 publications

(27 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similarly, although both Ca 2+ o and L‐phenylalanine stimulated Ca 2+ i mobilization in CaS receptor‐transfected HEK293 cells, only Ca 2+ o promoted IP accumulation and diacylglycerol production (Rey et al ., ). Finally, we recently showed that truncation of the CaS receptor after R 866 resulted in a complete inability of the receptor to stimulate Ca 2+ i mobilization, whereas IP accumulation was reduced, but maintained (Goolam et al ., ). In the same study, mutations in intracellular loops 2 and 3 greatly impaired IP accumulation, but had a weaker affect on Ca 2+ i mobilization.…”

Section: Discussionmentioning

confidence: 97%

Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics

Cook

Mistry

Gregory

et al. 2014

British J Pharmacology

View full text Add to dashboard Cite

BACKGROUND AND PURPOSEClinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. EXPERIMENTAL APPROACHWe characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca 2+ i) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. KEY RESULTSPhenylalkylamine calcimimetics were biased towards allosteric modulation of Ca 2+ i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G 670 E). CONCLUSIONS AND IMPLICATIONSThe ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses. Abbreviations

show abstract

Section: Discussionmentioning

confidence: 97%

Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics

Cook

Mistry

Gregory

et al. 2014

British J Pharmacology

View full text Add to dashboard Cite

show abstract

“…(19) Cell surface expression by ELISA Quantification of total and cell surface expression was completed using an ELISA-based assay that has been previously published. (20)…”

Section: Generation Of Cell Linesmentioning

confidence: 99%

Treatment of Autosomal Dominant Hypocalcemia Type 1 With the Calcilytic NPSP795 (SHP635)

Roberts

Gafni

Brillante

et al. 2019

Journal of Bone and Mineral Research

View full text Add to dashboard Cite

Autosomal dominant hypocalcemia type 1 (ADH1) is a rare form of hypoparathyroidism caused by heterozygous, gain‐of‐function mutations of the calcium‐sensing receptor gene (CAR). Individuals are hypocalcemic with inappropriately low parathyroid hormone (PTH) secretion and relative hypercalciuria. Calcilytics are negative allosteric modulators of the extracellular calcium receptor (CaR) and therefore may have therapeutic benefits in ADH1. Five adults with ADH1 due to four distinct CAR mutations received escalating doses of the calcilytic compound NPSP795 (SHP635) on 3 consecutive days. Pharmacokinetics, pharmacodynamics, efficacy, and safety were assessed. Parallel in vitro testing with subject CaR mutations assessed the effects of NPSP795 on cytoplasmic calcium concentrations (Ca2+i), and ERK and p38MAPK phosphorylation. These effects were correlated with clinical responses to administration of NPSP795. NPSP795 increased plasma PTH levels in a concentration‐dependent manner up to 129% above baseline (p = 0.013) at the highest exposure levels. Fractional excretion of calcium (FECa) trended down but not significantly so. Blood ionized calcium levels remained stable during NPSP795 infusion despite fasting, no calcitriol supplementation, and little calcium supplementation. NPSP795 was generally safe and well‐tolerated. There was significant variability in response clinically across genotypes. In vitro, all mutant CaRs were half‐maximally activated (EC50) at lower concentrations of extracellular calcium (Ca2+o) compared to wild‐type (WT) CaR; NPSP795 exposure increased the EC50 for all CaR activity readouts. However, the in vitro responses to NPSP795 did not correlate with any clinical parameters. NPSP795 increased plasma PTH levels in subjects with ADH1 in a dose‐dependent manner, and thus, serves as proof‐of‐concept that calcilytics could be an effective treatment for ADH1. Albeit all mutations appear to be activating at the CaR, in vitro observations were not predictive of the in vivo phenotype or the response to calcilytics, suggesting that other parameters impact the response to the drug. © 2019 American Society for Bone and Mineral Research.

show abstract

“…The human CaSR is encoded by the CASR gene located on chromosome 3q21.1 and consists of: an extracellular domain, which binds Ca 2+ e and mediates receptor dimerization; seven transmembrane domains (TMDs); and an intracellular domain, which is involved in activation of downstream signaling effectors such as G proteins and phospholipase C (PLC) (2, 3). The CaSR stimulates two major signal transduction cascades.…”

Section: Introductionmentioning

confidence: 99%

A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin–biased signaling

et al. 2018

View full text Add to dashboard Cite

The calcium-sensing receptor (CaSR) is a G protein–coupled receptor (GPCR) that signals through Gq/11 and Gi/o to stimulate cytosolic calcium (Ca2+i) and mitogen-activated protein kinase (MAPK) signaling to control extracellular calcium homeostasis. Studies of loss- and gain-of-function CASR mutations, which cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively, have revealed the CaSR to signal in a biased manner. Thus, some mutations associated with FHH1 lead to signaling predominantly through the MAPK pathway, whereas mutations associated with ADH1 preferentially enhance Ca2+i responses. Here, we report a previously unidentified ADH1-associated R680G CaSR mutation, which led to the identification of a CaSR structural motif that mediates biased signaling. Expressing the R680G CaSR mutant in HEK293 cells showed that this mutation increased MAPK signaling without altering Ca2+i responses. Moreover, this gain-of-function in MAPK activity occurred independently of Gq/11 and Gi/o, and was mediated instead by a non-canonical pathway involving β-arrestin scaffolding proteins. Homology modeling and mutagenesis studies showed the R680G CaSR mutation selectively enhanced β-arrestin signaling by disrupting a salt bridge formed between Arg680 and Glu767, which are located in the CaSR transmembrane domain 3 and extracellular loop-2, respectively. Thus, our results demonstrate CaSR signaling through β-arrestin and the importance of the Arg680-Glu767 salt bridge in mediating signaling bias.

show abstract

Roles of intraloops‐2 and ‐3 and the proximal C‐terminus in signalling pathway selection from the human calcium‐sensing receptor

Cited by 14 publications

References 38 publications

Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics

Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics

Treatment of Autosomal Dominant Hypocalcemia Type 1 With the Calcilytic NPSP795 (SHP635)

A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin–biased signaling

Contact Info

Product

Resources

About