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Calcium (Ca2+) is essential for many biological processes, and plays many intra- and extra-cellular roles [1, 2]. 99% of total body calcium resides in the skeleton as hydroxyapatite; the rest is in extracellular fluids and soft tissues. In serum, approximately half of the calcium is ionized (Ca2+o) and the rest is bound to protein or complexed with citrate and phosphate ions . This tight regulation on Ca2+ is important for the proper function of a variety of bodily functions including nerve and muscles, bone minerialization, blood coagulation, enzymatic activity and the modulation of membrane permeability and excitation[4, 5]. Ca2+ also acts as a second messenger inside cells in a number of signalling cascades. Cytosolic free Ca2+ (Ca2+i) is maintained at a basal level close to 100 nM, however upon cell activation it can attain concentrations as high as 1 Î¼M. Regulation of Ca2+i, and as a result many cellular processes, is dependent on a constant Ca2+ concentration outside cells .
In healthy adults Ca2+o is maintained between 1.1 - 1.3 mM and does not normally deviate by more than 2% on either side of this range [6, 7]. This is accomplished by a highly sensitive regulatory system involving the parathyroid glands, thyroid parafollicular C-cells, kidneys, bone and the gastrointestinal tract (Figure 1.1) [8-10]. Three calciotropic hormones - parathyroid hormone (PTH), calcitonin (CT) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) - are also involved in the endocrine regulation of Ca2+o. PTH and 1,25(OH)2D3 elevate plasma Ca2+ levels, while CT suppresses extracellular Ca2+ levels. These hormones act together on the cells of the kidney, gastrointestinal tract and skeleton to maintain systemic Ca2+ homeostasis (Figure 1.1) .
In humans, the main regulator of Ca2+ homeostasis is PTH, which is secreted from the parathyroid glands. The parathyroid glands sense minute changes in the serum Ca2+ concentrations and, therefore, modulate the secretion of PTH to normalize the serum calcium level .
During hypocalcaemia, or as plasma Ca2+ concentration decreases, parathyroid glands secrete PTH, which then acts on both the kidneys, to enhance renal Ca2+ reabsorption, and bone, to mobilise skeletal Ca2+. Furthermore, during prolonged hypocalcaemia, PTH stimulates renal 1Î±-hydroxylase to convert 25-hydroxyvitamin D3 to the active hormone, 1,25(OH)2D3, resulting in increased intestinal Ca2+ absorption. The net results of these actions are an increase in systemic Ca2+o levels. This rise in Ca2+ levels decreases PTH secretion, providing a negative feedback loop for the control of PTH levels (Figure 1.1).
Figure 1.1: Calcium Homeostasis
The Calcium-Sensing Receptor
The highly sensitive nature of the calcium homeostatic system implies that the cells involved in regulating extracellular Ca2+ are able to recognise and respond to minute changes in the plasma Ca2+ concentration. In accordance with this, certain cells, such as the chief cells of the parathyroid gland, sense small fluctuations in Ca2+o.
Indirect evidence suggested the presence of a Ca2+-sensing mechanism in these cells through a G-protein coupled receptor (GPCR); for example, exposure to high Ca2+ led to the activation of phospholipase C (PLC) leading to an accumulation of inositol 1,4,5-trisphospate (IP3) [13, 14] and the consequent release of intracellular Ca2+ stores . The precise molecular mechanism behind these observations was made clear upon the cloning of an extracellular calcium-sensing receptor (CaSR) from bovine parathyroid cDNA in 1993 .
The cDNA of the human CaSR was subsequently cloned from a library derived from a human adenomatous parathyroid gland . The CaSR has now been cloned from a large number of species including rat , chicken , the salamander Necturus maculosus , and various fish species including the dogfish shark, white winter flounder and Atlantic salmon , goldfish  and Mozambique tilapia .
The 1078-amino acid protein encoded by the human CaSR gene shares 93% homology to the CaSR protein predicted by bovine parathyroid cDNA . The CaSR is a member of the Family C of the GPCR superfamily, which is further sub-divided into four main groups . Group I contains the metabotropic glutamate receptors (mGluRs) 1-8; group III consists of the GABAB receptors, that respond to the neurotransmitter Î³-amino butyric acid (GABA), and three orphan receptors; and group IV which consists of four RAIG1-like orphan receptors . The CaSR belongs to Group II of Family C along with the human basic amino acid receptor GPRC6A [26, 27], the putative odorant and pheromone receptors e.g. goldfish 5.24 receptor , and taste receptors (T1R1, T2R2 and T2R3)  (Figure 1.2).
Figure 1.2: Phylogenetic tree of family C G-protein coupled receptors 
Structure of the CaSR
Hydrophobicity analysis of the amino acid sequence of the CaSR revealed seven hydrophobic stretches of 20 - 25 amino acids, which is characteristic of the seven transmembrane domain motif of GPCRs (Figure 1.3) [17, 31, 32].
Figure 1.3: Domain-based organization of the CaSR . Annotated hydropathy plot (Kyte-Doolittle) is shown to demonstrate the locations of the CaSR's major domains including an N-terminal extracellular Venus Fly Trap domain, a Cysteine-rich domain, a seven-transmembrane domain region and C-terminus. Also shown are the recognized binding sites for amino acids in the VFT domain, phenylalkylamine type-II calcimimetics in the seven-transmembrane region and the cytoskeletal protein filamin in the C-terminus.
Within the GPCR Family C, the CaSR shares only modest sequence similarity to the mGluRs and little sequence similarity with the GABAB receptors, however they all display a similar overall topology comprised of three distinct structural domains: a very large amino-terminal extracellular domain (ECD), a central heptahelical transmembrane region and a carboxyl terminal intracellular tail (Figure 1.4).
The CaSR is expressed mainly as a homodimer on the cell surface (Figure 1.5), although Western blot analysis has also shown that it forms higher order oligomers . Receptor dimerization is independent of the binding of an agonist or other modulator and appears to occur as the newly translated subunits are inserted in the endoplasmic reticulum (ER) membrane . Dimerization is supported at the interface between neighbouring extracellular domains by both covalent and non-covalent interactions [31, 36] and there are two asymmetric intermolecular disulphide bonds between residues C129 and C131 in the dimeric receptors . Furthermore, dimer stability is enhanced by non-covalent interactions involving L112 and L156 .
Dimerization of the receptor promotes trafficking to the plasma membrane, possibly by the mutual masking of ER retention signal peptides in a manner similar to GABAB heterodimers . Dimerization of the CaSR is essential for its activation  and normal function [34, 40].
In addition, the CaSR forms physiologically important heterodimers with other class C GPCRs; including mGluR-1Î± in the bovine brain  and GABAB receptor subunits in growth plate chondrocytes and hippocampal neurons .
Figure 1.5: Graphic Representation of a CaSR dimer on the cell surface . A model of the dimer form of CaSR, with each of the two individual receptor molecules shown in red and blue. The grey circles indicate the nine conserved cysteines residues in the Cys-rich domain, while the yellow triangles show the approximate location of the five putative protein kinase C phosphorylation sites, in each lobe.
Figure 1.4: Schematic Representation of the principal structural features of the predicted CaSR protein . The large N-terminal domain is located extracellularly at the top and the C-terminal tail is located intracellularly at the bottom. Amino acid residues conserved in all mGluRs and the CaSR are shown as filled in red and black filled circles. Symbols are provided in the key.
1.3.1 Extracellular Domain
The CaSR has a large ECD characteristic of all family C GPCRs. The ECD of the human CaSR contains approximately 612 amino acids with eleven potential N-glycosylation sites, which are considered essential for cell surface expression [43, 44].
The N-terminal Venus Fly Trap (VFT) domain extends from residue 20 to 536 and, similar to the N-terminus of metabotropic glutamate receptors, the CaSR's N-terminus is an evolutionary homolog of the nutrient-binding domains in bacterial periplasmic binding proteins [45, 46]. While the CaSR's crystal structure has not been solved, predictions have been made based on solved crystal structures of homologus mGlus including mGlu-1 , mGlu-3 and mGlu-7 . The CaSR's VFT domain is believed to form a bilobed structure with ligand binding sites positioned both within the bilobed cleft and the interprotomeric interface of the homodimers (Figure 1.6). Analogous with the mGlu-1, the CaSR's dimeric VFT domains are expected to adopt four major conformations: open-open, open-closed, closed-open and closed-closed. Binding of ligand is meant to stabilize domain closing, leading to a more favourable energy state which, due to dynamic equilibrium, leads to an increase of the relative population of the "active" conformation . The VFT domain appears to be the primary location of Ca2+ ion binding [50, 51], although additional sites have been identified in the heptahelical transmembrane domains .
Residues required in the VFT for the binding of the Î±-amino and Î±-carboxyl groups of L-amino acid ligands are tightly conserved within mGluRs and other class C GPCRs and indeed, based on the analysis of chimeric receptors  and mutational analyses [54, 55], is also the site of broad spectrum L-amino acid sensing in the CaSR. Two mutations in the VFT, T145A/S170T, selectively impairs L-amino acid sensing, while leaving Ca2+o sensing intact as determined by receptor-dependent activation of Ca2+i mobilization and suppression of intracellular cyclic adenosine monophosphate (cAMP) levels [55, 56].
Figure 1.6: Molecular model of a CaSR protomeric VFT domain . A model of a single subunit, based on the mGlu-1 crystal structure 1EWK . Putative Ca2+ binding sites have been identified using aromatized luminescence analysis of the globular sub-domains .
Comparative in silico modelling of the mGluR-1 and CaSR VFT domains suggests that the ligand binding region in the CaSR is relatively unrestricted and in part explains the relative promiscuity of the receptor for various sub-classes of amino acids . Based on this observation it was predicted that small peptides that contain Î±-amino and Î±-carboxylate functional groups at their N-termini would activate the CaSR. This led to the successful prediction that the CaSR is potently allosterically activated by Î³-glutamyl peptides, including glutathione (Î³-Glu-Cys-Gly) and its analogue S-methylglutathione (SMG) [56, 58, 59]. The double mutant referred to above, T145A/S170T, demonstrated significantly impaired Ca2+i mobilization and cAMP suppression responses to SMG indicating that Î³-glutamyl peptides and L-amino acids activate the CaSR via a common site and/or mechanism .
Furthermore, the VFT domain is also an important site of dimerization . The Ala116 - Pro136 segment of the VFT domain, which appears to contribute to the dimer interface , is specifically sensitive to activating mutations, implicating the importance of this region in the maintenance of the inactive conformation of the CaSR .
It has also been proposed that residues of the CaSR involved in Ca2+-binding are located within the VFT domain, however identification of these residues has been obstructed by the lack of a crystal structure for the receptor, the inability to measure Ca2+ binding directly and the absence of high-affinity agonists for the CaSR . In silico models of the CaSR ECD, based on the x-ray structures of the mGluR1, have led to the identification of multiple Ca2+-binding sites within the ECD (Figure 1.6) [51, 62] and mutational studies have indicated that five amino acids - S147, S170, D190, Y218 and E297 (shown as Site 1 in Figure 1.6) - positioned within a crevice between the two lobes of the VFT are critical for full activation of the CaSR by extracellular Ca2+ [51, 62] . This site also aligns to the conserved L-amino acid binding site of other class C GPCRs, suggesting that Ca2+ and amino acid binding sites are closely associated.
The exact nature of the CaSR's VFT domain's interaction with the immediate adjoining Cysteine-rich domain is currently uncertain, but it is believed that closure of the VFT domain induces turning moments that controls the conformation of the dimeric transmembrane domain and consequently the likelihood of G-protein docking and activation.
1.3.2 Cysteine-rich Domain
The CaSR's VFT domain connects to the transmembrane domain by a 62 residue Cys-rich domain , comprised of nine highly conserved Cys residues , and a 14 residue linker (Figure 1.7) . Molecular analyses indicate that Cys-rich domains are critical for transmitting nutrient or neurotransmitter derived signals from the VFT to the transmembrane domains in class C GPCRs, except for in GABA(B) receptors where the Cys-rich domain is absent . As a result deletion of the entire Cys-rich domain abolished high Ca2+o-dependent activation of phosphatidylinositol-specific phospholipase-C (PI-PLC) without an apparent affect on surface expression . In addition, mutational analysis has indicated that normal receptor function requires the presence of nine Cys residues[37, 63, 66].
While there are no crystal structures for any of the CaSR's major domains, new insights into the relationship between the VFT domain and the transmembrane domain have been made available by the relatively recent solution of the complete extracellular domain of the rat Group II metabotropic glutamate receptor, mGlu-3 . The structure defines roles for all nine conserved Cys residues in the Cys-rich domain.
The mGlu-3 Cys-rich domain contains a rigid structure of 3 Î²-sheets, which are composed of 2 short antiparallel Î²-strands. Four pairs of Cys-residues in the Cys-rich domain (mGlu-3 residues C509 & C528l; C513 & C531; C534 & C564; and C549 & C562) form intra-domain disulphide bridges, which then act to stabilize the interior structure of the domain.. The ninth Cys residue, C527 (aligning to residue 561 in the CaSR) forms an inter-domain disulphide with C240 within the VFT domain (aligning to CaSR residue 236) . This disulphide appears to play an important role by adjusting the angle at which conformational changes induced in the VFT domain by ligand binding are transmitted to the transmembrane domain.
An inter-domain disulphide was predicted within the CaSR, however analysis of proteolytic fragments released from the introduction of an engineered tobacco etch virus cleavage site between the VFT and Cys-rich domains indicated that it was not present , despite conservation of homologous Cys residues in mGlu-2 . These inconsistencies between the mutational work in the CaSR , and the crystal structure findings  and mutation analysis  in members of the homologus mGlus are currently not understood, however, one suggestion has been that the inter-domain disulphide bridge is relatively unstable  and may be affected my ligand binding .
1.3.3 The Transmembrane Domain
All members of the GPCR superfamily display a distinguishing heptahelical (HH) domain which is required for the docking and activation of hetero-trimeric G-proteins. The HH domain consists of seven helices attached by interchanging extracellular loops and intracellular loops, however the residues corresponding to the beginning and ends of the helices and the loops have not yet been definitively identified [69-72].
While the sequence homology between the HH domains of Family C and Family A GPCRs is quite small, they are structurally similar and therefore the crystal structures of class A GPCRs including bovine rhodopsin  and the Î²1-adrenergic  and Î²2-adrenergic [75, 76] receptors have been important in the attempt to understand the mechanisms of GPCR activation using molecular modelling.
Within Family A receptors, the cylindrical arrangement of HH domains forms a binding pocket. Binding of ligand leads to conformation changes in TM3 and TM 4, which releases inhibitory constraints, and allows the binding and activation of G-proteins . Support for this concept has followed from the identification of CaSR mutation A843E in TM7 resulting in a constitutively active receptor . This mutation is proposed to alter the conformation state of the HH domain to promote G-protein coupling .
The exact nature of the interactions between the receptor and G-protein binding, including selectivity and stoichiometry, are currently undefined. While there is a much greater understanding of the activation mechanisms of the homologous mGlu-5 receptors, unlike Group I receptors which specialize in activation of Gq/11, the CaSR is unusually pleiotropic, coupling to Gq/11, Gi/o, G12/13, and even Gs in certain circumstances .
Intracellular loops 2 and 3 appear to be the site of the CaSR's interaction with Gq and G11 as alanine scanning mutagenesis in both intracellular loop-2 and -3 impaired PI-PLC activation . Within intracellular loop-2, residues L704 and F707 were essential for high Ca2+o-mediated coupling to PI-PLC, while in intracellular loop-3, two patches have been identified within close proximity between residues R796 - P799 and N801 - F807  (Figure 1.7).
The analysis described above, for CaSR coupling to Gq/11, has not been applied to other heterotrimeric G-proteins that normally couple to the CaSR, such as Gi/o and G12/13. As mentioned previously, the CaSR's G-protein preference can switch from Gi/o in normal mammary epithelial cells to Gs in two breast cancer cells lines, meaning that CaSR activation promotes cAMP synthesis, instead of inhibiting cAMP, with potential significance for cancer cell growth and/or metastasis . The mechanism that underlies this switching is currently unknown.
Furthermore, the HH domain also plays a role in receptor dimerization through non-covalent interactions [72, 81]. TM5 is believed to be critical for receptor dimerization as a consensus dimerization motif has been identified for non-covalent hydrophobic interactions , and consistent with this, while the A877Stop truncation mutation does not forms dimers, P474fs, a truncation mutant which lacks TM5, TM6 and TM7 does not .
Experiments with chimeric receptors, containing the ECD of rhodopsin fused to the HH domain and C-terminal tail of the CaSR showed that, despite the loss of the ECD, the chimeric receptor was expressed at the cell surface and, in the presence of the calcimimetic compound, NPS R-568, was able to response to Ca2+o . Despite requiring a allosteric modulator to sensitize the receptor to Ca2+o these data suggest that there is at least one site for Ca2+ in the HH domain .
Mutational analysis to determine the site of Ca2+ binding in the HH domain has focused on a number of acidic residues present on the extracellular loop, as they are the only part of the HH domain exposed to the extracellular environment. Individual mutations of three acidic residues to alanines in extracellular loop 2 - D758, E759 and E767 - increased the sensitivity of the CaSR to Ca2+o, while alanine substitution of E837 in extracellular loop 3 impaired the receptor's maximal response to Ca2+o and abolished its response to the phenylalkylamine, NPS R-568 . A salt bridge between the carboxylate side-chain of E837 and the positively charged central amine of R-568 appears to support receptor activation .
Homology modelling of the CaSR's transmembrane domain, based on the crystal structure of rhodopsin, has been useful in identifying potential binding sites of positive and negative allosteric modulators [86, 87] and mutational analysis suggests that calcimimetics and calcilytics interact with an overlapping set of residues in the second and third extracellular loops (Figure 1.7) [87, 88].
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