AP2σ Mutations Impair Calcium-Sensing Receptor Trafficking and Signaling, and Show an Endosomal Pathway to Spatially Direct G-Protein Selectivity

Journal of Molecular Endocrinology

2019

Twenty-five years have elapsed since the calcium-sensing receptor (CaSR) was first identified in bovine parathyroid and the receptor is now recognized as a fundamental contributor to extracellular Ca 2+ (Ca 2+ e ) homeostasis, regulating parathyroid hormone release and urinary calcium excretion. The CaSR is a class C G-protein-coupled receptor (GPCR) that is functionally active as a homodimer and couples to multiple G-protein subtypes to activate intracellular signalling pathways. The importance of the CaSR in the regulation of Ca 2+ e has been highlighted by the identification of >400 different germline loss-and gain-of-function CaSR mutations that give rise to disorders of Ca 2+ e homeostasis. CaSR-inactivating mutations cause neonatal severe hyperparathyroidism, characterised by marked hypercalcaemia, skeletal demineralisation and failure to thrive in early infancy; and familial hypocalciuric hypercalcaemia, an often asymptomatic disorder associated with mild-moderately elevated serum calcium concentrations. Activating mutations are associated with autosomal dominant hypocalcaemia, which is occasionally associated with a Bartter's-like phenotype. Recent elucidation of the CaSR extracellular domain structure enabled the locations of CaSR mutations to be mapped and has revealed clustering in locations important for structural integrity, receptor dimerisation and ligand binding. Moreover, the study of disease-causing mutations has demonstrated that CaSR signals in a biased manner and have revealed specific residues important for receptor activation. This review presents the current understanding of the genetic landscape of CaSR mutations by summarising findings from clinical and functional studies of diseaseassociated mutations. It concludes with reflections on how recently uncovered signalling pathways may expand the understanding of calcium homeostasis disorders.

Section: Future Directionsmentioning

confidence: 99%

Molecular and clinical insights from studies of calcium-sensing receptor mutations

Journal of Molecular Endocrinology

2019

“…The AP2σ protein is ubiquitously expressed, and clathrin-mediated endocytosis is a critical cellular process; however, FHH is a largely benign condition, and the phenotypes observed in FHH3 patients are largely CASR-specific. Thus, by studying the AP2σ mutations identified in FHH3, which have been described in a single residue (Nesbit et al 2013b, Hannan et al 2015, novel insights into the trafficking and signalling mechanisms of CASR have been elucidated, and indicate an important interplay between these two processes (Gorvin et al 2018b).…”

Section: Journal Of Molecular Endocrinologymentioning

confidence: 99%

“…This function of β-arrestin has been recognised for CASR in some studies, providing seemingly contradictory information to that in studies of functional desensitisation. Thus, treatment of cells with dominant-negative forms of β-arrestin1 or β-arrestin2, or with siRNA targeting β-arrestin1 or β-arrestin2, reduces the pERK and membrane ruffling signals downstream of CASR (Bouschet et al 2005, Thomsen et al 2012, Gorvin et al 2018b. Further studies are required to determine whether the discrepancies within these data sets are due to experimental differences, differences in cell type or if both desensitisation and enhanced signalling occur downstream of CASR, but at different spatial or temporal points.…”

Section: Regulation Of Cell Surface Expression By Functional Desensitmentioning

confidence: 99%

Insights into calcium-sensing receptor trafficking and biased signalling by studies of calcium homeostasis

Journal of Molecular Endocrinology

2018

The calcium-sensing receptor (CASR) is a class C G-protein-coupled receptor (GPCR) that detects extracellular calcium concentrations, and modulates parathyroid hormone secretion and urinary calcium excretion to maintain calcium homeostasis. The CASR utilises multiple heterotrimeric G-proteins to mediate signalling effects including activation of intracellular calcium release; mitogen-activated protein kinase (MAPK) pathways; membrane ruffling; and inhibition of cAMP production. By studying germline mutations in the CASR and proteins within its signalling pathway that cause hyper- and hypocalcaemic disorders, novel mechanisms governing GPCR signalling and trafficking have been elucidated. This review focusses on two recently described pathways that provide novel insights into CASR signalling and trafficking mechanisms. The first, identified by studying a CASR gain-of-function mutation that causes autosomal dominant hypocalcaemia (ADH), demonstrated a structural motif located between the third transmembrane domain and the second extracellular loop of the CASR that mediates biased signalling by activating a novel β-arrestin-mediated G-protein-independent pathway. The second, in which the mechanism by which adaptor protein-2 σ-subunit (AP2σ) mutations cause familial hypocalciuric hypercalcaemia (FHH) was investigated, demonstrated that AP2σ mutations impair CASR internalisation and reduce multiple CASR-mediated signalling pathways. Furthermore, these studies showed that the CASR can signal from the cell surface using multiple G-protein pathways, whilst sustained signalling is mediated only by the G pathway. Thus, studies of FHH- and ADH-associated mutations have revealed novel steps by which CASR mediates signalling and compartmental bias, and these pathways could provide new targets for therapies for patients with calcaemic disorders.

“…(28) DNA sequence analysis Variants were validated in G2 mice by Sanger DNA sequencing, using appropriate gene-specific primers (Sigma, Gillingham, UK), followed by dideoxynucleotide sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA) and an automated detection system (ABI3730 capillary sequencer; Applied Biosystems, Carlsbad, CA, USA). (29) DNA samples from G2 and G3 mice were assessed by restriction endonuclease analysis using HindIII and BsrDI restriction endonucleases, as described. (20) Protein sequence alignment and protein prediction Protein sequences were aligned using ClustalW (30) and the effect of mutations predicted using MutationTaster (http://www.…”

Section: Exome Sequence Analysismentioning

confidence: 99%

“…Apoptosis was assessed in whole-kidney lysates using the Caspase-Glo 3/7 luminescence assay (Promega) measuring levels of activecaspase 3/7, as described. (29) Luminescence was measured using a Turner Biosystems luminometer (Promega, Southampton, UK). Experiments were performed in 10 independent kidney samples each, from RCALC2 mutant mice and WT littermates.…”

Section: Apoptosis Assaymentioning

confidence: 99%

An N-Ethyl-N-Nitrosourea (ENU)-Induced Tyr265Stop Mutation of the DNA Polymerase Accessory Subunit Gamma 2 (Polg2) Is Associated With Renal Calcification in Mice

Journal of Bone and Mineral Research

Ahmad

Stechman

et al. 2018

Renal calcification (RCALC) resulting in nephrolithiasis and nephrocalcinosis, which affects ∼10% of adults by 70 years of age, involves environmental and genetic etiologies. Thus, nephrolithiasis and nephrocalcinosis occurs as an inherited disorder in ∼65% of patients, and may be associated with endocrine and metabolic disorders including: primary hyperparathyroidism, hypercalciuria, renal tubular acidosis, cystinuria, and hyperoxaluria. Investigations of families with nephrolithiasis and nephrocalcinosis have identified some causative genes, but further progress is limited as large families are unavailable for genetic studies. We therefore embarked on establishing mouse models for hereditary nephrolithiasis and nephrocalcinosis by performing abdominal X‐rays to identify renal opacities in N‐ethyl‐N‐nitrosourea (ENU)‐mutagenized mice. This identified a mouse with RCALC inherited as an autosomal dominant trait, designated RCALC type 2 (RCALC2). Genomewide mapping located the Rcalc2 locus to a ∼16‐Mbp region on chromosome 11D‐E2 and whole‐exome sequence analysis identified a heterozygous mutation in the DNA polymerase gamma‐2, accessory subunit ( Polg2 ) resulting in a nonsense mutation, Tyr265Stop (Y265X), which co‐segregated with RCALC2. Kidneys of mutant mice ( Polg2 + / Y265X ) had lower POLG2 mRNA and protein expression, compared to wild‐type littermates ( Polg2 +/+ ). The Polg2 +/Y265X and Polg2 + / + mice had similar plasma concentrations of sodium, potassium, calcium, phosphate, chloride, urea, creatinine, glucose, and alkaline phosphatase activity; and similar urinary fractional excretion of calcium, phosphate, oxalate, and protein. Polg2 encodes the minor subunit of the mitochondrial DNA (mtDNA) polymerase and the mtDNA content in Polg2 + / Y265X kidneys was reduced compared to Polg2 +/+ mice, and cDNA expression profiling revealed differential expression of 26 genes involved in several biological processes including mitochondrial DNA function, apoptosis, and ubiquitination, the complement pathway, and inflammatory pathways. In addition, plasma of Polg2 + / Y265X mice, compared to Polg2 + / + littermates had higher levels of reactive oxygen species. Thus, our studies have identified a mutant mouse model for inherited renal calcification associated with a Polg2 nonsense mutation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.