Ogo I. Egbuna, M.D., M.Sc., and Edward M. Brown, M.D.

Dr. Egbuna is a research fellow in the Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women's Hospital, a staff physician in the Department of Medicine, Division of Nephrology, Beth Israel Deaconess Medical Center, and Instructor in Medicine, Harvard Medical School; Dr. Brown is a staff physician in the Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women's Hospital, and Professor of Medicine, Harvard Medical School.

Within the past 12 months, Dr. Brown has been on the Speakers Bureau for Athena Diagnostics and receives royalties from NPS Pharmaceuticals, Inc. Dr. Egbuna reports no commercial conflicts of interest.

Release Date: 07/15/2008
Termination Date: 07/15/2011

Estimated time to complete: 1 hour(s).

Albert Einstein College of Medicine designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.

• Describe Ca²⁺_o homeostasis, calcium-sensing receptors (CaSRs) and the role of the prototypical CaSR in Ca²⁺_o homeostasis
• Discuss recent advances in our understanding of the structure-function relationships and ligand binding sites of the CaSR
• List the spectrum of mutations involving the CaSR that cause human disease and describe disorders arising from anti-CaSR autoantibodies
• Discuss the actions of the CaSR in CaSR-expressing tissues in defending against both hypo- and hypercalcemia
• Discuss the current and potential utility of CaSR-based therapeutics
• Outline future directions in CaSR physiology and research.

The calcium-sensing receptor (CaSR) plays key roles in the maintenance of a narrow range (1.1-1.3 mM) of extracellular ionized calcium concentration (Ca²⁺_o), primarily by modulating the function of chief cells of the parathyroid gland. The CaSR regulates the synthesis and secretion of parathyroid hormone (PTH), as well as parathyroid cellular proliferation, inhibiting all three processes when Ca²⁺_o is high and stimulating them when Ca²⁺_o is low.(1) It serves as a "calciostat," informing the parathyroid glands and other tissues where it is expressed of the precise level of Ca²⁺_o.

The CaSR (also known as CaSR1 or GPRC2A) was identified using the expression-cloning technique in Xenopus laevis (the African clawed frog) oocytes.(2) It is a member of family C of the superfamily of 7-transmembrane, G protein-coupled receptors (GPCRs). Other members of this family are the so-called metabotropic receptors for glutamate (mGluRs), receptors for gamma-aminobutyric acid (GABA), as well as GPCRs for sensing pheromones, taste and odorants (in fish). Recently, another member of family C, GPRC6A, has been found to share several pharmacological properties with the CaSR.(3),(4) Like the CaSR, GPRC6A is sensitive towards certain L-amino acids but is most responsive to basic amino acids and less so to calcium;(3),(5) nevertheless, it is possible that GPRC6A is a second calcium-sensing receptor (CaSR2).

The physiological relevance of the CaSR in humans has been proven by experiments-of-nature in inherited human disorders caused by mutations in the receptor leading to either loss- or gain-of-function.(6) Heterozygous inactivating mutations of the CaSR cause a disorder of calcium metabolism, familial hypocalciuric hypercalcemia (FHH), that manifests as asymptomatic hypercalcemia with relative or absolute hypocalciuria. Homozygous mutations, on the other hand, cause a severe, sometimes lethal form of hyperparathyroidism, with marked hypercalcemia and hyperparathyroidism if left untreated. Activating mutations, in contrast, cause an autosomal dominant form of hypocalcemia (ADH) associated with low normal or low PTH levels, despite hypercalcemia and relative or absolute hypercalciuria.

CaSR expression is greatest in the parathyroid glands, calcitonin-secreting C-cells of the thyroid gland and kidney. The CaSR is also found in the two other key organs that participate in calcium homeostasis: gut and bone.(7) Available data have demonstrated that the CaSR is expressed not only in the organs that secrete calcium-regulating hormones (e.g., the parathyroid glands and C-cells of the thyroid glands) but also in target tissues for these hormones and in tissues not known to be involved in Ca²⁺_o homeostasis (see Figure 1).(8) These latter tissues regulate Ca²⁺_o by translocating calcium ions into or out of the bodily fluids and include the kidney, which expresses the CaSR at robust levels in certain nephron segments, as well as bone and intestine, which express the receptor at lower levels. By acting on both hormone-secreting and hormone-responsive, calcium-transporting tissues through its own cell surface receptor, Ca²⁺_o acts, in effect, as another Ca²⁺_o-regulating "hormone" (in this case Ca²⁺_o-lowering) or "first messenger."

Current Understanding of Ligand Binding Sites of the CaSR

The human CaSR has a large extracellular domain (ECD) (612 residues) -- a transmembrane domain (TMD) of 250 amino acids containing the 7-membrane spanning helices characteristic of the GPCRs and an intracellular C-terminal domain (ICD) of 216 amino acids. The receptor exhibits substantial N-linked glycosylation, which is important for normal cell membrane expression of the receptor but does not appear to modify the function of the receptor per se.(9) The functional cell surface form of the CaSR is a dimer and the two monomers within the dimeric CaSR are linked by disulfide bonds involving cysteine residues 129 and 131 within each monomer (see Figure 2A).(10) The ECD of each CaSR monomer consists of a bilobed, venus fly trap motif (VFTM), with a crevice between the lobes likely participating in ligand binding, and a cysteine-rich region just proximal to the first transmembrane domain.

Extracellular calcium is the prototypical ligand that activates the CaSR, although several other polyvalent cations and polycationic amine ligands have been identified that activate, inactivate or allosterically modulate the receptor.(7) The ECD of the CaSR likely contains more than one binding site for Ca²⁺_o because the Hill coefficient (a measure of cooperativity in binding of a ligand to its receptor) for the activation of the receptor by Ca²⁺_o is 3-4, consistent with the presence of positive cooperativity among at least this number of binding sites within the dimeric CaSR.(11),(12) The TMD is also apparently involved in Ca²⁺_o-sensing, since a mutant CaSR lacking the ECD also responds to Ca²⁺_o and other polyvalent cations.(13)

A major barrier to advancing our understanding of the role of Ca²⁺_o in the regulation of the CaSR is our incomplete understanding of its Ca²⁺_o binding sites, which is largely hindered by the lack of a solved three-dimensional structure and rapid association-dissociation binding rates for Ca²⁺_o as a result of low binding affinities. Delineation of the Ca²⁺ binding pocket within the conserved VFTM of the human CaSR has been attempted using molecular modeling, as well as mutational and functional analyses, and is thought to reside in a crevice between lobes 1 and 2 of the VFTM(14) that appears to be conserved within several other class III GPCRs that are also responsive to Ca²⁺_o. This information provides the foundation for an eventual molecular understanding of the effects of Ca²⁺ on these receptors.

Further insight into the three predicted binding sites [located in lobes 1 and 2 and in the crevice between the two lobes (Figure 2B-C)] has been derived from recent studies utilizing site-directed mutagenesis, nuclear magnetic resonance spectroscopy, fluorescent energy transfer analysis and insertion of peptides containing putative Ca²⁺_o-binding sites into a scaffold protein to identify Ca²⁺-binding sites in the ECD of the CaSR.(15) The importance of these sites for binding to Ca²⁺ was confirmed by directed mutagenesis of amino acids present in these sites and the observation of significant potentiating and inhibiting effects on the EC₅₀ (half maximal effective concentration) for Ca²⁺_o-induced alterations in the function of the receptor. For further insights, the reader is referred to reviews of structure-function relationships of the CaSR from mutations and allosteric modifiers, as well as clinical conditions resulting from mutations of the CaSR.(16),(17)

A. Location of the putative binding sites of Ca²⁺ in the lobes of the CaSR monomer B. C. Ultrastructural details of putative binding sites of Ca²⁺ in the lobes of the CaSR monomer.

Printed with permission of the authors.(15)