The Calcium-Sensing Receptor

CaSR Agonists

The CaSR can be activated by Ca ²⁺ , Mg ²⁺ and certain other polycations (e.g., Gd ³⁺ , polylysine, polyarginine, and neomycin ). These agonists are referred to as type I agonists as they can directly and independently activate the receptor ( Fig. 63.1 ). CaSR activity can also be modulated by other substances or conditions that function by modifying the EC ₅₀ for extracellular Ca ²⁺ (up or down); allosteric activators of the CaSR of this type are referred to as type II allosteric activators ( Fig.63.1 ). Thus, type II activators require the presence of extracellular Ca ²⁺ and function as allosteric modifiers of Ca ²⁺ affinity. Currently identified physiological type II agonists include polyamines (e.g., spermine), L-amino acids (especially aromatic amino acids), extracellular pH, and extracellular ionic strength, primarily changes in NaCl concentration in a physiological context. Increases in polyamine or amino acid concentrations, or isosmotic reductions in ionic strength reduce the EC ₅₀ (increase the affinity) for extracellular Ca ²⁺ . At a constant ionized Ca ²⁺ concentration, these changes in the concentrations of type II agonists will increase activation of the CaSR. As a consequence, CaSR responses to changes in concentrations of divalent minerals or type I agonists must be viewed in the setting of a specific “extracellular environment” (i.e., presence or absence of polyamines and L-amino acids, pH, and ionic strength). Interestingly, the CaSR appears to have initially evolved as a salinity sensor in marine species where these organisms used the effects of ionic strength (salinity) on activation of the receptor by the Ca ²⁺ /Mg ²⁺ present in salt water to regulate tissue responses for salinity adaptation. With the evolution of land-based tetrapods and the loss of the ocean reservoir of Ca ²⁺ /Mg ²⁺ , we see the first appearance of the parathyroid glands and parathyroid hormone (PTH), which are required for divalent mineral regulation of the internal “ocean” represented by the extracellular fluids (ECFs) of terrestrial organisms. Currently identified CaSR-active small molecules (calcimimetics and calcilytics) are used in the treatment of certain disorders of calcium homeostasis. They function as allosteric activators and antagonists, respectively, of the CaSR via the type II mechanism and therefore, require extracellular Ca ²⁺ to act ( Fig. 63.1 ).

Ligand Binding to the CaSR

Class C GPCR receptors have a large (hundreds of residues) N-terminal extracellular domain (ECD) that is joined to the canonical 7-transmembrane (7-TM) domain typical of GPCRs. The ECDs of the class C-related metabotropic glutamate receptors or the distantly related bacterial periplasmic nutrient-binding proteins (e.g., the maltose-binding protein (MBP)) form a bilobed structure that has a ligand binding region within the central cleft between these lobes ( Fig. 63.2A ). Following ligand binding, there is a conformational change in the ECD, which results in the two lobes moving toward one another to enclose the ligand ( Fig. 63.2A , Venus-flytrap model). This molecular motion in the ECD is thought to be translated into conformational changes in the 7-TM domain, leading to G-protein activation.

The ligand-binding sites on class-C receptors, including the CaSR, are located on the ECD and, to a lesser extent, in the 7-TM domains. While an x-ray crystal structure of the ECD of the CaSR has not been obtained, the ECD can be modeled on the basis of the known structure of several metabotropic glutamate receptors. This structural model is shown in Fig. 63.2B with the location of potential Ca ²⁺ -interacting regions composed of negatively charged amino acid residues. In addition, three serine residues (S-147, S-169, and S-170) and proline-823 in the ECD are crucial for maximal responsiveness to extracellular Ca ²⁺ . Receptor activation by Ca ²⁺ is greatly reduced by removal of the ECD, emphasizing the importance of negatively charged acidic (and other) residues in the ECD for the binding of polyvalent cations. Additional acidic residues in the second and third extracellular loops in the 7-TM domain have also been suggested to participate in Ca ²⁺ -binding interactions. In contrast, the response to calcimimetics is retained when the ECD is removed from the CaSR, indicating that these type II activators bind at a different site than does Ca ²⁺ . Observations indicate that the critical sites for interaction of calcimimetics with the CaSR are located in the 7-TM domain, primarily the TM6–TM7 region, with Glu837, at the outer end of TM7, serving as an “anchor” by binding the amino group in the aliphatic linker between the two hydrophobic ends of the molecule. In addition, negative allosteric modulators of the CaSR (calcilytics) have been identified that exhibit a binding site in the 7-TM domain of the CaSR that overlaps with but is not identical to that for the calcimimetics.

The Concept of Superagonism for Agonist Binding to the Class C Receptors

As described previously, type II agonists (including calcimimetics) of the CaSR can act as allosteric enhancers or positive allosteric modulators. It is clear that most GPCRs possess allosteric binding sites that can be recognized by small-molecule ligands. GPCR class-C receptors, including the CaSR, form dimers (or even multimers), and this intermolecular interaction is believed to play an important role in allosteric activation. Type I ligand binding enhances dimerization of the CaSR associated with formation of intermolecular disulfide linkages. Thus, most of the receptors on plasma membranes of cells are in the dimeric (and multimeric) form, since cell surface CaSRs are exposed to millimolar concentrations of extracellular Ca ²⁺ . Although the intermolecular disulfide links between ECDs are not essential for dimerization, they play an important structural role and help to maintain the receptor in its inactive state in the absence of agonist. Initial dimerization takes place in the endoplasmic reticulum, and this interaction may be necessary for the receptor’s transport to the cell surface. Once on the cell surface, intermolecular interactions between CaSR monomers are essential for normal function of this receptor. Specifically, in the presence of agonist, dimerization of the CaSR and other class C receptors appears to enhance downstream cellular signaling, providing “superagonism”. This dimer-driven superagonism, which can also be thought of a positive cooperativity, likely accounts for the ability of type II agonists (e.g., L-amino acids) to activate the receptor by an allosteric mechanism and the remarkable ability of this receptor to detect small changes in ionized Ca ²⁺ from the normal plasma Ca ²⁺ of 1.1-1.3 mM (Ca ²⁺ -Ca ²⁺ allosteric enhancing effect). Given that the Hill coefficient of the dimeric CaSR is 3–4, it is likely that there are at least 2 binding sites for calcium on each of the two monomers, resulting in the substantial degree of positive cooperativity needed for the receptor’s exquisite sensitivity to small changes in Ca ²⁺ . An intact COOH-terminus on the CaSR is also required for cell surface expression.

Complex Signaling: the Receptor is Promiscuous

CaSR coupling to G-protein has been referred to as promiscuous, since type I ligands activate one or more of several G proteins (e.g., Gα _q11 , Gα _i2–i3 ). As with most GPCRs, the COOH-terminal tail of the CaSR and one or more intracellular loops are crucial for signal transduction. The CaSR-generated cytosolic signal is a complex of phospholipase activation (PLC, cPLA ₂ , PLD) and the generation of diverse cellular second messengers (see for reviews), Gα _q⧸⧸11− mediated PLC activation →↑IP ₃ →↑cytosolic Ca ²⁺ concentration (Ca ²⁺ _i ) and Ca ²⁺ _i oscillations as well as →↑DAG→↑PKC; Gα _i -mediated→↓cAMP; ↑intracellular Ca ²⁺ →↑phosphodiesterase (PDE)→↓cAMP; cytosolic (c)PLA ₂ activation→↑arachidonic acid (AA)→↑P ₄₅₀ →↑20-HETE; phosphatidylinositol 3-kinase (PI3K)→ PIP ₃ ; MAP kinases, such as ERK1/2, c-Jun activated N-terminal kinase (JNK), and p38 MAPK; filamin scaffolding of Gα _q →↑Lbc RhoGEF→↑Rho A GTPase leading to activation of a serum response element (SRE) and Gα _12/13 →↑PLD→↑phosphatidic acid (PA). It remains unclear in many cases how this second messenger “soup” and related intracellular signaling pathways integrate to modulate cellular functions, such as PTH secretion or renal responses to extracellular Ca ²⁺ . The ability of the CaSR to be modulated by such a wide variety of agonists and extracellular conditions likely accounts for its multifunctional nature in regulating divalent mineral balance as well as in modulating diverse cellular functions seemingly unrelated to mineral homeostasis. Examples of the latter include CaSR effects on salt and water transport by the kidney and gastrointestinal epithelia. The CaSR also provides proliferation-differentiation-apoptosis signals to certain epithelial cells (e.g., keratinocytes in the skin, mammary gland cells and colonocytes).

CaSR Regulates PTH Secretion

The level and constancy of plasma ionized Ca ²⁺ are set in large part by regulating the release of Ca ²⁺ from bone via the actions of secreted PTH and 1,25(OH) ₂ D ₃ as well as by modulation of the Ca ²⁺ excretion by the kidney, and, indirectly via the action of 1,25(OH) ₂ D ₃ , intestinal calcium absorption, as noted above. Figure 63.3 shows the typical inverse relationship between PTH secretion and plasma Ca ²⁺ . In contrast there is a direct relationship between urinary Ca ²⁺ excretion and plasma Ca ²⁺ ( Fig. 63.4 ). These effects of plasma ionized Ca ²⁺ on PTH secretion and renal Ca ²⁺ excretion are mediated by the CaSR. The role of the CaSR in divalent mineral homeostasis has been established by the identification and characterization of inherited hyper- or hypocalcemic disorders that result from CASR gene mutations (the official designation of the gene for the human CaSR is in all capital letters and italicized) (chromosome 3q13.3–21; Fig. 63.5 ). In vitro studies in mammalian cells expressing normal or mutant CaSR proteins have confirmed that these receptor mutations alter the Ca ²⁺ EC ₅₀ of the CaSR (CaSR refers to the CaSR protein). Loss-of-function mutations in one or both of the CASR alleles result in hypercalcemic disorders due to upward resetting of the CaSR EC ₅₀ for ionized Ca ²⁺ in both the parathyroid gland and kidney. ^48,83,404 Autosomal dominant hypercalcemia, in which one CASR allele has an inactivating mutation, typically present s as a benign hypercalcemic disorder called familial hypocalciuric hypercalcemia (type 1 FHH; OMIM 145980, known as HHC1) or as symptomatic hypercalcemia in neonatal hyperparathyroidism (NHPT). Two phenotypically similar conditions (HHC2, OMIM 145981; and HHC3, OMIM 600740) are linked to the long and short arms of chromosome 19, respectively, but the responsible genes have not yet been identified. Neonatal severe hyperparathyroidism (NSHPT) results from consanguineous unions in FHH families in which both alleles have inactivating mutations or, occasionally, in families in which the two parents have different inactivating mutations (i.e., producing a compound heterozygous infant). Infants with NSHPT exhibit severe hypercalcemia frequently necessitating total parathyroidectomy. The hypercalcemia in these loss-of-function CaSR disorders is usually associated with reduced renal Ca ²⁺ excretion, rather than the increased Ca ²⁺ excretion that is observed in other hypercalcemic states (e.g., 1° HPT and malignant hypercalcemia). The abnormal renal response in FHH demonstrates the important role of the CaSR in the hypercalciuric response to hypercalcemia.

Homozygous CaSR knockout mice exhibit marked parathyroid hyperplasia and hyperparathyroidism and die soon after birth due to severe hypercalcemia ( Fig. 63.5 ). Support for the role of PTH in causing the severe and lethal hypercalcemia came form the observations that the lethal mouse phenotype can be rescued by knocking out the PTH gene or by deletion of the Gcm2 gene, which is the “master gene” needed for development of the parathyroid glands. Studies carried out in the mice with knockout of PTH and/or the CaSR have documented the crucial role of CaSR-regulated PTH secretion as a “floor” preventing hypocalcemia, while the CaSR-mediated upregulation of renal Ca ²⁺ excretion and stimulation of calcitonin secretion are an effective “ceiling” limiting increases in serum calcium in response to dietary or other forms of calcium load. Indeed, the PTH knockout mice defend against hypercalcemia just as well as the wild type mice, even though they lack PTH and the ability to suppress it while hypercalcemic.

Autosomal dominant, gain-of-function (activating) mutations in the CaSR result in an opposite shift in plasma ionized calcium (i.e., hypocalcemia) due to downward resetting of the receptor EC ₅₀ (autosomal dominant hypocalcemia (ADH); OMIM 146200). In some individuals with severe activating mutations, a renal salt wasting disorder with hypercalciuria has been observed that mimics the hyperprostaglandin E ₂ syndrome (type V Bartter syndrome). The latter confirms the important role of the CaSR in regulating renal Ca ²⁺ handling and clearly demonstrates the importance of the CaSR in regulating salt transport in the thick ascending limb (TAL). CASR gene polymorphisms also appear to contribute to the normal variation in steady-state plasma ionized Ca ²⁺ concentration, at least in certain populations. CaSR-activating or -inhibiting autoantibodies can result in autoimmune hypoparathyroidism or an acquired syndrome mimicking FHH, called autoimmune hypocalciuric hypercalcemia, respectively. Mice with an activating mutation in the CaSR exhibit a phenotype similar to that in patients with ADH. All these various lines of evidence convincingly document the key role of the CaSR in mediating the effects of Ca ²⁺ on PTH secretion.

In addition to reducing the secretion of PTH, activation of the CaSR increases the degradation of full length, biologically active PTH1-84 to PTH7-84 and smaller carboxyterminal fragments, thereby decreasing the secretion of intact PTH further still during hypercalcemia and, conversely, increasing it in the setting of hypocalcemia.

CaSR Regulates Expression of the preproPTH Gene

Elevated levels of Ca ²⁺ _o and calcimimetics not only inhibit PTH secretion but also decrease the levels of the mRNA encoding preproPTH; this action of the calcimimetics proves the mediatory role of the CaSR in regulating preproPTH gene expression. The CaSR-mediated alteration in the level of preproPTH mRNA is the result of a change in preproPTH mRNA stability rather than in gene transcription per se. 1,25(OH) ₂ D ₃ , in contrast, acts by a direct inhibitory action on the transcription of this gene. Naveh-Many, Silver and coworkers have clarified the molecular mechanisms by which Ca ²⁺ _o and the CaSR control the stability of preproPTH mRNA. Exposing parathyroid cells to elevated levels of Ca ²⁺ activates the CaSR and, through a pathway that involves stimulation of calmodulin (CaM) and protein phosphatase 2B, post-translationally modifies and reduces the binding of the preproPTH mRNA stabilizing factor, AU-rich factor (AUF-1), to an AU-rich element in the 3’ untranslated region (UTR) of the preproPTH mRNA. The loss of AUF-1 from this binding site permits a second, destablizing protein, K-homology splicing regulator protein (KSRP), to bind to the same site. KSRP subsequently interacts with and is activated by the peptidyl-prolyl isomerase, Pin-1, and, pari passu, recruits the endoribonuclease, PMR1, which is part of the RNA-cleaving exosome. PMR-1 then degrades prepro-PTH mRNA by cleaving it internally.

CaSR Regulates Parathyroid Cellular Proliferation

Studies in humans with NSHPT or in mice homozygous for knock out of the Casr gene (the symbols for mouse genes are italicized with only the first letter capitalized) have proven the CaSR’s importance in regulating parathyroid cellular proliferation. In both cases, marked parathyroid cellular proliferation and glandular enlargement ensue despite severe hypercalcemia, documenting that the CaSR has an essential role in tonically inhibiting parathyroid cellular proliferation. Studies in uremic rat models have illuminated the mechanisms by which high dietary intake of Ca ²⁺ , acting via the CaSR, controls parathyroid proliferation. Induction of the cyclin dependent kinase inhibitor, p21 ^WAF1 , and downregulation of the growth factor, TGF-α, and its receptor, the epidermal growth factor receptor (EGFR), both of which are upregulated in this setting, are key components of this mechanism. 1,25(OH) ₂ D ₃ appears to act in a similar way to inhibit parathyroid cellular proliferation in similar experimental models. An additional mechanism that may participate in stimulation of parathyroid growth during hypocalcemia is an endothelin-1-mediated stimulation of parathyroid cellular growth. Similar studies are difficult to perform in non-uremic animals owing to their much slower rate of parathyroid proliferation, but it seems likely that similar mechanisms participate. Reduced expression of p21 and another cyclin dependent kinase inhibitor, p27, may also participate in the dysregulation of parathyroid growth in both primary (1°) and secondary (2°) hyperparathyroidism (HPT). The second messenger pathway(s) that link the CaSR to the regulation of parathyroid proliferation have not yet been clarified.

Interactions of Vitamin D and the CaSR in the Regulation of Parathyroid Function

A large body of data has stressed the importance of vitamin D in reducing expression of the preproPTH gene and parathyroid proliferation. What is the relative importance of the VDR and CaSR in controlling parathyroid function? The CaSR clearly regulates the secretion of PTH over a time frame from seconds to minutes or longer and is the dominant regulator of acute changes in secretory rate. Over a longer time frame of three weeks, vitamin D deficiency and hypocalcemia both modulate preproPTH mRNA levels in the rat, although hypocalcemia of ~6 mg/dl more powerfully stimulates preproPTH gene expression than does vitamin D deficiency.

Recent studies utilizing mouse knockout models, however, have provided surprising results regarding the relative importance of the VDR and CaSR in controlling parathyroid gland function in vivo . As noted before, homozygous knock out of exon 5 of the CaSR causes striking hyperparathyroidism with marked elevations in both PTH and parathyroid gland size, which clearly cannot be compensated by the remaining VDR gene. This mouse model, if anything, likely underestimates the consequences of loss of the CaSR on parathyroid function, since knock out of exon 5 of the CaSR produces, in some tissues, an alternatively spliced CaSR that lacks exon 5 (which encodes part of the CaSR ECD) and can seemingly still signal. Thus the VDR apparently has limited ability to offset loss of the CaSR in this animal model. In contrast, studies of mice with knockout of the VDR have demonstrated that the CaSR effectively compensates for loss of the vitamin D receptor with regard to the control of parathyroid function. That is, while VDR-/- mice develop strikingly elevated levels of PTH and marked parathyroid enlargement on a standard diet, administering a calcium-rich “rescue” diet normalizes both serum Ca ²⁺ and PTH levels. Thus hypocalcemia per se rather than vitamin D deficiency is seemingly the dominant contributor to the elevated PTH levels. In addition, if the rescue diet is begun early in life, it completely prevents the parathyroid enlargement in the VDR-/- mice, showing that hypocalcemia per se rather than loss of the VDR was also a critical contributor to parathyroid growth in this setting. Subsequently, Meir, et al. created mice with knockout of the VDR only in the parathyroid glands. In this way, the actions of the VDR on the parathyroid could be separated from systemic alterations in mineral ion homeostasis, e.g., owing to loss of the VDR in kidney and intestine. The mice with parathyroid-specific VDR ablation manifest only modest (~30%) elevations in serum PTH but do not exhibit any change in the number of proliferating parathyroid cells and have normal serum calcium concentrations. Thus, while administering exogenous 1,25(OH) ₂ D ₃ clearly suppresses preproPTH gene transcription and parathyroid proliferation in vivo and in vitro , vitamin D seemingly has only a limited role in regulating parathyroid function in vivo under normal physiological conditions. These results should not be taken, however, to mean that 1,25(OH) ₂ D ₃ has no useful therapeutic role, especially in the 2° HPT of renal insufficiency (see below and elsewhere in this volume). Finally, it would be interesting to investigate CaSR signaling efficiency in the parathyroid in mice with deficient VDR signaling to determine to what extent compensatory alterations in the CaSR and/or its downstream signaling components contribute to the phenotypes observed with global or parathyroid-specific knockout of the VDR.