Calcium Sensing Receptor (CASR)
The calcium-sensing receptor (CaSR) is a heterotrimeric G-protein-coupled receptor (GPCR), one of the largest family of cell-surface receptors, containing 14 other members including the metabotropic glutamate receptors (mGluR) and ɣ-aminobutyric acid (GABA) B receptors (Zhang et al. 2016a). CaSR was first cloned by Dr. Edward M. Brown in 1993 from the bovine parathyroid gland (Brown et al. 1993). The major function of CaSR is to maintain calcium homeostasis by balancing the ingestion and absorption of calcium in the gastrointestinal tract, excretion of calcium through the urinary system, and the breakdown and formation of bone. CaSR regulates parathyroid hormone (PTH) secretion from parathyroid glands and calcitonin secretion from thyroidal C-cells upon sensing the alteration of the extracellular calcium concentration (Zhang et al. 2016a). This receptor has been reported to be present in different tissues and organs for systemic Ca2+ homeostasis such as parathyroid, thyroid, gastrointestinal tract, kidney, and bone (osteoclasts, osteoblasts, and osteocytes). Additionally, CaSR is also expressed in renal tubule, liver, lung, breast, placenta, vasculature, chondrocytes, lens epithelial cells, pancreas, neurons and glia in the CNS, exocrine cells in the pituitary, peripheral perivascular sensory nerves, keratinocytes, and prostate (Hendy and Canaff 2016). Furthermore, the detection of CaSR in multiple cardiovascular cells and tissue types, such as vascular smooth muscle cells (VSMCs), human aortic smooth muscle cells (HAoSMC), cardiac myocytes, myocardial microvasculature, cardiac fibroblasts, the atria and ventricle of the rat heart, human aortic endothelial cells (HAECs), etc., implicates the importance of CaSR function in cardiovascular system (Smajilovic et al. 2011).
In addition to the diverse expression of CaSR in many tissues and organs, CaSR activation and mechanism are also complicated with numerous types of agonists and coagonists that are able to cooperatively mediate CaSR signaling. Furthermore, the sensitivity of CaSR to extracellular [Ca2+] can vary in different cells and tissues. For example, in bone, the local extracellular [Ca2+] can reach 40 mM, but in skin, the extracellular [Ca2+] ranges from 0.03 mM to >0.5 mM (Breitwieser 2014). Moreover, numerous mutations related to CaSR diseases and CaSR-associated cancer along with diversified expressions of CaSR with altered signalings are prevalent. In this review, we will focus on CaSR activators, its varied tissue- and species-specific genetic information, and its disease-related mutations in order to understand the versatile function of CaSR in broad microenvironments at genetic and molecular levels.
Activation and Regulation of CaSR
The CaSR consists of a 612 amino-acid extracellular domain (ECD) that is critical for cotranslational processing, receptor dimerization, binding ligands, and transmitting activation signals through the seven transmembrane domains (TMDs), which comprises residues 613 to 862, and through the intracellular domain (ICD), which comprises residues 863 to 1078 (Zhang et al. 2016a). The principal agonist of CaSR is known to be the extracellular Ca2+. The functional diversity of CaSR results from its ability to activate multiple downstream signaling pathways through Gq/11, Gi/o, G12/13, and Gs proteins when a ligand binds to the CaSR ECD (Conigrave and Ward 2013). In addition to Ca2+, CaSR is also regulated through several orthosteric agonists such as various divalent and trivalent cations, polyamines including Mg2+, Al3+, Sr+, Mn2+, Ni2+, Gd3+, and Ba2+, cationic polypeptides, and endogenous allosteric modulators such as the extracellular pH and ionic strength (Saidak et al. 2009). The potency of these agonists can vary in various cells types. For example, in parathyroid, Mg2+ and Ca2+ has similar potency, but in sheep parafollicular cells of thyroid, the potency is ranked as Gd3+>Ba2+>Ca2+>>Mg2+ (McGehee et al. 1997). Furthermore, aromatic and aliphatic L-amino acids such as L-Phe and L-Trp is known to increase the sensitivity of CaSR for Ca2+ and are considered as positive allosteric modulators of the receptor (Saidak et al. 2009). Synthetic positive allosteric modulators such as calcimimetics are known to left-shift, whereas the negative allosteric modulators known as calcilytics right-shift the concentration-response curve of Ca2+ (Brown et al. 1993). Recent work by Zhang et al. has reported a novel Trp derivative as a coagnoists for CaSR’s activation (Zhang et al. 2016b).
Ligand- and tissue-specific modulation of signaling pathways through the activation of CaSR known as biased signaling has been widely studied. Brauner-Osborne has explored the effect of 12 orthosteric CaSR agonists on inositol (1,4,5)-triphosphate (IP3) accumulation, cAMP inhibition, and ERK1/2 phosphorylation in HEK293 cells transfected with CaSR (Colella et al. 2016). Similarly, Ziegelstein et al. showed that the induction of Ca2+ release and nitric oxide production is possible with spermine alone and not with Ca2+, Gd3+, or neomycin. Also, Smajilovic et al. demonstrated a concentration-dependent vasodilation in rat aorta when treated with cinacalcet but not with neomycin or Gd3+. To add to the complicated mechanism of CaSR function, the amount of CaSR expressed at the cell membrane can be dynamically regulated through agonist-dependent trafficking of intracellular receptors. Furthermore, allosteric modulators of CaSR function as pharmacoperones have been extensively studied. NPS R-568 rescues the signaling and expression of loss-of-function mutant CaSR, NPS 2143 promotes trafficking of CaSR mutants to cell membrane while causing a decrease in CaSR signaling, and cinacalcet rescues both the trafficking and signaling of CaSR mutants while causing a loss of cell surface expression (Breitwieser 2014; Leach et al. 2014).
Evolution of CaSR
Of the 1059 amino acid residues of the matured human CaSR, 44% sites are completely conservative during the evolution including the nine cysteine residues in cysteine rich (CR)-domain and the predicted calcium-binding site 1 (S147, S170, D190, Y218, E297) (Huang et al. 2009) (Fig. 1). Most of these conserved sites are buried inside the protein in 3D structure, which might be necessary for the maintenance of protein structure and function (Fig. 1). The calcium binding site 1 overlap with the L-Trp binding site determined in CaSR crystal structure which are located in the cleft between the two lobes of VF (Geng et al. 2016). The other metal binding sites determined by CaSR crystallization or predicted by computational approach are relatively conserved but with slight heterogeneity, which might be related to the alteration of habitat during evolution.
CASR is expressed in multiple tissues and organs at various levels. The variation of expression of CASR gene as multiple alternative transcripts in multiple tissues/organs hints at the ability to diversify its functions and regulations. In addition to the wild-type variant II CASR, low abundant variant I exists which contains additional 10 amino acid inserted after amino acid 536 in the CaSR (GenBank #U20760). This variant I is known to function similarly to variant II (Hendy and Canaff 2016). While, in human kidney, both the 5.4-kb transcript and the 10-kb transcript are similarly predominant (Hendy and Canaff 2016). The use and presence of the two promoters in CaSR gene allows for variation in transcription of alternative 5′ UTR exons (1A and 1B) (Hendy et al. 2009). The real-time PCR (qPCR) analysis has shown that exon 1B-transcripts in human parathyroid cells were significantly more expressed than exon 1A-containing transcripts. The 5.4 and 10-kb transcripts in human parathyroid adenomas and normal glands use exon 1A exclusively as shown by northern blot analysis, while the 4.2-kb transcripts are either derived from 1A or 1B (Hendy and Canaff 2016). 5.4 and 4.2-kb sized transcripts are derived from the use of the two alternative polyadenylation sites in the 3′ UTR tract (Hendy and Canaff 2016).
An exon 3-deleted CASR transcript has been reported in thyroid TT cells, placental cytotrophoblast, and in parathyroid, thyroid, and kidney (Hendy and Canaff 2016). This protein is poorly expressed and does not traffic to the cell surface. Another alternative transcript of CaSR exists in human keratinocytes where exon 5 is deleted (with a 77-amino acid inframe deletion in the exodomain). The relative amounts of full-length is lowered during keratinocyte differentiation and the alternatively spliced variant is shown to cause the full-length protein to be less responsive to Ca2+. This variant of CaSR is also known to be upregulated in skin and kidney in the knockout mice, and is shown to compensate for the absence of the full-length CaSR in bone and cartilage (Hendy et al. 2009).
CaSR Mutations and Disease
Disorder of Ca2+ Homeostasis and CaSR
Over 225 mutations in the Ca2+-sensing receptor result in hypercalcemic or hypocalcemic disorders, such as familial hypocalciuric hypercalcemia, neonatal severe primary hyperparathyroidism, and autosomal dominant hypocalcemic hypercalciuria. Abnormal function of CaSR is associated with Ca2+ homeostatic disorders. Loss-of-function mutations in CaSR lead to potentially fatal neonatal severe primary hyperparathyroidism, while gain-of-function mutations cause autosomal dominant hypocalcemia (Hendy et al. 2009; Ward et al. 2012). However, not all the mutations detected from patients are disease related, and some nucleotide diversity can be a rare allele of single nucleotide polymorphism. For example, a CaSR variant (Glu250Lys) identified in FHH and ADHH probands was demonstrated to be a functionally neutral polymorphism (Hannan et al. 2012).
Cancer and CaSR
CaSR plays a vital role in the regulation of cancer expression. CaSR expression and function are highly dubious in various cancer cells. Several studies indicated that CaSR gene in cancer is regulated epigenetically. For example, in colon cancer, 69% CaSR gene showed methylation and showed an inverse correlation with the degree of differentiation. The methylation was significantly correlated with reduced mRNA and protein expression of CaSR. The study showed a significant role of epigenetic silencing of CASR in colorectal carcinogenesis (Hizaki et al. 2011). In addition, meta-analysis studies investigating the association of CASR polymorphisms with cancer risks have presented many inconclusive results. The role of CASR polymorphisms as a tumor suppressor or an oncogene is known to differ by cancer site and environmental condition. High calcium intake is shown to reduce the risk of colorectal cancer development as E-cadherin stimulated by CaSR is known to interact with β-catenin, an important proto-oncogen (Jeong et al. 2016). The increased expression of CaSR by high calcium levels is known to promote MCF-7 breast cancer cells and PC-3 and C4-2B prostate cancer cells that are known to metastasize to the bone. These cancer cell proliferation process is linked to extracellular signal-regulated kinases 1 and 2 (ERK 1/2) phosphorylation (Jeong et al. 2016).
There are many single-nucleotide polymorphisms (SNPs) in CaSR that are associated with cancer. Three common nonsynonymous SNPs (rs1801725, rs1042636, and rs1801726) have been the primary research targets for cancer risk, but inconsistent results have been reported in MEDLINE, EMBASE, Web of Science, Scopus, and the HuGE databases (Jeong et al. 2016). rs1801725 (A986S, 2956G.T) is where T allele is associated with higher levels of serum calcium, e.g., in colorectal adenoma, whereas rs1042636 (R990G, 2968A.G) mostly present in Asians induces a gain-of-function mutation associated with primary hyperparathyroidism and calcium stone formation (Jeong et al. 2016). It is shown to be lower in colorectal adenoma. On the other hand, rs1801726 (Q1101E, 3403C.G) is a common polymorphism in African ethnicity, and is less carried in prostate cancer patients. Patients with this SNP has increased risk of colorectal adenoma. Another polymorphism called rs17251221 (1378–1412A.G) is an SNP in introns. rs1801725 induces a gain-of-function mutation associated with total serum calcium concentration and stone multiplicity in patients with nephrolithiasis, noticeable association with prostate and breast cancer increased risk (Jeong et al. 2016).
CaSR is a versatile receptor that allows control of serum Ca2+ concentration at microscopic level. The recent advancement in its study such as the crystal structure obtained in the past year has expanded our knowledge on the function of CaSR at a molecular level to some degree, highlighting its modulation through divalent cations, such as Ca2+ and Mg2+, and Trp-derivative ligand as coagonist. However, the agility of this receptor as a multifunctional receptor comes from many factors such as its diversity in transcripts and expression levels in various tissues, organs, and species, along with various modes of regulation such as multitude of agonists and coagonists, and disease-mutations that affect its signaling and expression. An in-depth understanding of molecular and genetic modulation of CaSR expression and function in broad microenvironments and its effect to the downstream pathways is necessary in order to help us design and develop organ- or tissue-specific therapeutics and targeting methods against CaSR-related maladies.
This work was supported in part by National Institutes of Health Grants GM081749 and EB007268 and by the American Heart Association Grant 16GRNT31210016 (to J. J. Y.), and a Center for Diagnostics and Therapeutics fellowship (to R.G.) from Georgia State University.
- Colella M, Gerbino A, Hofer AM, Curci S. Recent advances in understanding the extracellular calcium-sensing receptor. F1000Res. 2016;5:1–12.Google Scholar
- Geng Y, Mosyak L, Kurinov I, Zuo H, Sturchler E, Cheng TC, et al. Structural mechanism of ligand activation in human calcium-sensing receptor. elife. 2016;5:1–25.Google Scholar
- Hannan FM, Nesbit MA, Zhang C, Cranston T, Curley AJ, Harding B, et al. Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients: evidence for clustering of extracellular domain mutations at calcium-binding sites. Hum Mol Genet. 2012;21(12):2768–78.CrossRefPubMedGoogle Scholar