Calcium mediates many physiologic processes and hence its tight regulation is critical. Th e calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR) on the surface of parathyroid cells, is sensitive to changes in extracellular ionized calcium and thereby controls parathyroid hormone (PTH) secretion, regulating circulating calcium concentrations. Since its cloning in 1993, the CaSR has been implicated in several disorders of calcium metabolism, including familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalcemia (ADH). Th e more recent discovery of CaSR expression in other tissues, including kidney, bone, intestine, pancreas, thyroid, placenta, central nervous system, heart, epidermis, and breast, suggests widespread and diverse functions for the receptor. This TriPoint: (1) chronicles the basic science investigations that led to identifi cation and characterization of the receptor in parathyroid and non-parathyroid tissues; (2) examines the role of the CaSR in human disease, including how human mutations enlighten our understanding of receptor function; and (3) describes the patient populations that may benefi t, now and in the future, from drugs that activate (calcimimetics) or antagonize (calcilytics) the CaSR.
BASIC RESEARCHER PERSPECTIVE
The Development of the Concept of Extracellular Ca2+-Sensing in Parathyroid (PT) Cells
Sydney Ringer was the first to demonstrate the biological function of ionized calcium (Ca2+) by showing the ability of a trace amount of Ca2+ from tap water to induce contractions of frog hearts in vitro. This unique cation was later shown to be a critical intracellular signaling molecule that mediates diverse functions in literally every cell system. Accompanying these findings was the question of how cells in the most basic unicellular or complex multicellular organisms control intracellular Ca2+ homeostasis in response to fluctuations in the availability of Ca2+ in their immediate environment. Th is question began to be answered when Brown, Shoback, and colleagues demonstrated the ability of a minute change in the concentration of extracellular Ca2+ ([Ca2+]o) to excite several acute (in seconds to minutes) signaling responses in PT cells, including: (1) increases in intracellular [Ca2+]; (2) inhibition of cAMP production via coupling to G-protein subunit GαI; (3) stimulation of mitogen-activated protein kinases; and (4) activation of diacylglycerol/protein kinase C pathways.
In contrast to other endocrine systems where these intracellular signaling events generally stimulate hormone secretion, activation of these signaling responses by Ca2+ actually inhibits the release of PTH in PT cells. These observations led to the hypothesis that a plasma membranedelimited GPCR was responsible for sensing changes in [Ca2+]o, allowing cells to adjust their cellular functions according to extracellular Ca2+ availability. This concept was firmly established by: (1) the cloning of the CaSR from bovine PT glands in 1993; (2) the linkage of large numbers of activating and inactivating mutations in this receptor to the disorders of FHH and ADH; (3) the manifestation of neonatal severe hyperparathyroidism in a mouse model with global deletion of exon 5 of the CaSR gene; and (4) the inability of PT glands lacking CaSR expression to respond to changes in [Ca2+]o in culture.
Extracellular Ca2+-Sensing in Non-Parathyroid (Non-PT) Tissues
Th e cloning of bovine parathyroid CaSR also allowed characterization of its orthologs in other species ranging from elasmobranch fish to humans, demonstrating >85% similarity at the protein level, and permitted identification of the receptor in non-PT cells. The latter cells include those mediating classic calciotropic activities in the kidney, intestine, bone, cartilage, thyroid, placenta, and mammary gland and those not typically involved in maintaining Ca2+ homeostasis, like keratinocytes, neurons, pancreatic β-cells, and smooth and cardiac muscle cells.
To define the biological actions of CaSR in different tissues, a mouse model was generated that enabled the tissue- or cell-specific deletion of the receptor. For example, PT-specific CaSR KO mice, which retain CaSR functions in their kidneys, develop hypercalciuria. In contrast, when the receptor is deleted in all tissues, mice are hypocalciuric, confi rming that renal CaSRs are essential for promoting Ca2+ excretion.
CaSRs play non-redundant roles in mediating the growth, survival, and differentiation of chondrocytes and osteoblasts and in supporting embryonic and postnatal skeletal development. CaSRs expressed in intestinal epithelial cells prevent hyperproliferation, representing a novel target for prevention and/or treatment of colon cancer.
Preliminary studies of mice with CaSR ablated in neurons suggest that this receptor could also modulate general growth, energy metabolism, and skeletal homeostasis by mediating neuroendocrine functions in hypothalamic neurons and thereby the endocrine functions of the pituitary gland. In a transient global ischemia mouse model, CaSR overexpression appeared to be pivotal in the induction of neuronal injury. Studies of other tissue-specific CaSR KO mice are underway and are expected to reveal additional CaSR actions in both physiology and pathology.
Distinct Molecular Actions of CaSR in Pt Vs. Non-PT Tissues
Interestingly, CaSR mutations were often identified in patients with parathyroid disorders who showed none of the skeletal, neuroendocrine, or metabolic disorders seen in the conditional KO mice described above. These observations suggest that the actions of the CaSR in PT tissue are more sensitive to mutations than the CaSRs in non-PT tissues.
Based on cDNA sequences, CaSRs expressed in non-PT tissues are identical to that expressed in PT tissue, so the different CaSR actions in various tissues likely originate from differences in post-translational modifications and/or in its interactions with other accessory proteins and/or signaling molecules. In support of this idea, immunoblotting showed distinct glycosylation patterns of CaSR in nonPT compared to PT tissues.
Moreover, the CaSRs function in the form of multimeric complexes, interacting homomerically with themselves or heteromerically with other GPCR family C members, including type B γ-aminobutyric acid receptor (GABA-B-R) 1 and 2 and metabotropic glutamate receptors (mGluR1 and R5). Different stoichiometric interactions among these receptors, and perhaps with other undefined members of family C GPCRs, could produce receptor complexes with distinct molecular, signaling, and pharmacological characteristics. For example, the CaSR is expressed about 100-fold higher than the GABA-B-R1 in PT glands, favoring CaSR homomeric complexes, whose functions are anticipated to be more susceptible to the mutations in the receptor and manifest dominant negative eff ects as seen in FHH.
In contrast, GABA-B-R1 expression is 10-fold higher than CaSR expression in chondrocytes and neurons, favoring CaSR/GABA-B-R1 heteromeric complexes, which are expected to be less sensitive to CaSR mutations. In addition to changes in their functionalities, these differences in receptor processing and complex formation also provide opportunities for the design of specific compounds to target the CaSR in different tissues.
Though genetic studies in humans and mice demonstrate that the CaSR provides the basis for extracellular Ca2+-sensing by parathyroid cells, it is now clear that the protein plays additional roles in extra-PT tissues. The development and analysis of tissue-specific CaSR knockout models, coupled with biochemical characterization of the CaSR’s processing, partnering, and signaling, will uncover the nature and basis of these novel functions. Such mechanistic insight will enable more rational approaches to the development of therapeutics targeting the CaSR in the parathyroid and other tissues.
CLINICAL RESEARCHER PERSPECTIVE
Extracellular CalciumSensing Receptors and Parathyroid Function in Vivo
Over 30 years ago, pharmacologic and signaling studies clearly documented the capacity of parathyroid and kidney cells to sense and respond to small but physiologically relevant changes in the extracellular concentration of calcium ([Ca2+]e). Clinical investigators meanwhile had described kindreds with hypocalciuric hypercalcemia (FHH), rarely in association with severe hyperparathyroidism (HPT) in infants, and families with autosomal dominant hypocalcemia (ADH). This work supported the hypothesis that a plasma membrane extracellular Ca2+- sensing molecule, akin to members of the G-protein coupled receptor superfamily, existed and might explain the remarkable sensitivity of parathyroid and kidney cells to changes in the extracellular concentration of Ca2+ and Mg2+.
Once the extracellular Ca2+-sensing receptor (CaSR) cDNA was cloned, progress was rapid in identifying human mutations in the CaSR gene. Inactivating mutations were found in families with FHH and neonatal severe primary HPT, while activating mutations explained the hypocalcemia and inappropriately low parathyroid hormone (PTH) levels in many families with ADH.
Inactivating CaSR mutations reduced the capacity of parathyroid and kidney cells to sense and respond to changes in the [Ca2+]e by several mechanisms: (1) Mutations, such as the insertion of a stop codon, disturbed intracellular CaSR biosynthesis and/or folding, and thereby reduced levels of membrane CaSR expression; (2) Point mutations in critical residues blunted the ability of CaSRs to couple to G-proteins and activate downstream signaling pathways mediating inhibition of PTH secretion; (3) Point mutations in the extracellular domain of the CaSR, critical to its ion-sensing function, and in key residues in transmembrane domains, shifted sensitivity of the receptor to changes in the [Ca2+]e; and (4) CaSRs form dimers in the membrane, and certain CaSR mutants act as “dominant-negatives,” suppressing the activation of wildtype CaSRs by high [Ca2+]e.
Conversely, activating mutations of the CaSR enhance coupling to downstream signaling pathways and/ or possess increased sensitivity (i.e., shift to the left) to the [Ca2+]e compared to wild-type CaSRs. Both mechanisms would be predicted to promote the inhibition of PTH secretion at physiologically low [Ca2+]e . Other studies showed that glands from both sporadic primary HPTH (pHPT) and uremic secondary HPT (sHPT) had reduced (by ~50% on average) CaSR mRNA and protein levels. Taken together, this work set the stage for therapeutically targeting CaSRs in disorders of Ca2+-sensing.
Targeting the CaSR to Achieve Therapeutic Efficacy
Shortly after CaSR cloning and expression studies, compounds with the ability to activate the receptor and downstream signaling pathways and modulate PTH secretion were reported. These drugs were designated calcimimetics because they could mimic the actions of Ca2+ on PTH secretion and signaling responses. One such compound cinacalcet has been tested in uremic sHPT and pHPT and is approved to treat both disorders with specifi c indications.
Uremic sHPT is characterized by multiple derangements in serum biochemical parameters (elevated PTH, low 1,25-dihydroxyvitamin D, and high fi broblast growth factor 23 levels, along with hyperphosphatemia and high Ca2+-phosphate product). High PTH levels have long been considered central to morbidity in chronic kidney disease (CKD). Thus, targeting the reduction of PTH levels in patients with stage 5 CKD with cinacalcet was an immediate goal of trials with calcimimetics.
Key studies demonstrated the eff ectiveness of this approach to treat HPT in short-term studies (26 weeks). Higher percentages of patients treated with cinacalcet reached National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF-K/ DOQI) targets for PTH (56%), Ca2+ (49%), phosphate (46%), and Ca2+- phosphate product (65%) compared to placebo-treated patients who met these goals for PTH (10%), Ca2+ (24%), phosphate (33%), and Ca2+-phosphate product (36%). The main side effects of cinacalcet were nausea and hypocalcemia.
The EVOLVE trial examined the efficacy of cinacalcet vs. placebo to reduce composite cardiovascular/peripheral vascular endpoints in patients with stage 5 CKD and moderate to severe sHPT. By intention to treat analysis, the trial was nondefinitive in showing efficacy against cardiovascular/vascular and fracture endpoints. Whether targeting CaSRs has advantages over other approaches in CKD (phosphate binders, diet, vitamin D analogues) remains unproven.
Patients with pHPT due to nonmalignant and malignant parathyroid tumors have been treated with cinacalcet to lower PTH and serum Ca2+ levels. In one study, 78 patients with mild pHPT were randomized to cinacalcet or placebo for 52 weeks. In 80–90% of cinacalcet-treated patients, the serum [Ca2+] was normalized, accompanied by mild reductions in PTH and increases in serum phosphate levels. Treatment with cinacalcet (up to five years) showed no significant effects on bone mineral density.
Patients with intractable pHPT with serum [Ca2+] > 12.5 mg/dL were studied on cinacalcet (dosed up to four times daily) in an open-label trial. Serum [Ca2+] was decreased and quality of life assessments improved vs. baseline. In another trial, patients with severe hypercalcemia and inoperable parathyroid cancer were treated with cinacalcet. This approach produced moderate decrements in serum Ca2+ in the majority of patients. Thus, while skeletal and survival endpoints were not addressed in these small openlabel trials, the potential for improving symptomatic hypercalcemia in such patients with moderate and severe HPT is an important clinical option.
Prescient clinical investigators first suspected that the parathyroid cell harbored a molecule, akin to the G-protein coupled receptors, in the 1970’s when they identified families with apparently benign hypercalcemia and detectable (and even elevated) PTH levels. This clinically generated hypothesis combined with in vitro studies in parathyroid cells demonstrating their remarkable sensitivity to small changes in the ambient [Ca2+] led to the cloning of the CaSR. Knowledge that parathyroid tissues in states of HPT were also insensitive to high [Ca2+] set the stage for targeting CaSR with allosteric modulators to suppress the hypersecretion of PTH in these disorders—a practice that is now common in the clinic.
CLINICAL PRACTITIONER PERSPECTIVE
Evolution of Familial Hypocalciuric Hypercalcemia (FHH)
Prior to the widespread use of automated chemistry analyzers in the 1970’s, hyperparathyroidism was generally considered an uncommon, symptomatic condition with familial cases being quite rare. Once the frequency of hypercalcemia was more apparent, asymptomatic hyperparathyroidism and familial hyperparathyroidism became increasingly recognized.
Starting in the 1960’s, families with “hereditary hyperparathyroidism” began to be described with relatively few clinical consequences from the disease, and they were also found to have persistent hypercalcemia despite sub-total parathyroidectomy. It was later realized that these families had low urinary calcium excretion, and that this could be used to distinguish families who might not benefit from parathyroid exploration. This new entity was called familial benign hypercalcemia or familial hypocalciuric hypercalcemia (FHH).
Discovery of the CalciumSensing Receptor and Its Connection to Human Disease
In the earliest descriptions of FHH, there was speculation about an “abnormality of the receptor mechanism for calcium,” though no such receptor had been discovered for an ion to that point. After various lines of suggestive evidence throughout the 1980’s, the calcium-sensing receptor (CaSR) was cloned and characterized in 1993. Simultaneously, the same group described inactivating mutations of the receptor as the most common cause of FHH. The following year, they described activating mutations as a cause of autosomal dominant hypocalcemia (ADH). These findings revolutionized our understanding of calcium homeostasis and led to various therapeutic advances for human disease.
Once the concept of the CaSR was solidified, it was not long before compounds were developed that modified the action of the receptor. Calcimimetics are compounds that activate, or sensitize the receptor to extracellular calcium. By doing so in the parathyroid chief cells, parathyroid hormone (PTH) secretion is suppressed with subsequent lowering of serum calcium levels. One agent in this class, cinacalcet, has been approved since 2004 for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease on hemodialysis as well as in patients with hypercalcemia from parathyroid carcinoma. There are reports of effective use in rare cases of FHH with more severe hypercalcemia, though given the generally benign nature of this uncommon disease, treatment would not typically be indicated for the majority of cases.
Based on cinacalcet’s mechanism of action, there is a natural impulse to use the medication in the management of primary hyperparathyroidism. However, the drug was only approved by the FDA in 2011 for patients with primary hyperparathyroidism and severe hypercalcemia who are unable to undergo parathyroidectomy. The rationale for this has been practical, as surgery is usually an excellent option for a definitive cure. Additionally, while cinacalcet lowers serum calcium and PTH levels in patients with primary hyperparathyroidism, there is no improvement in bone mineral density (BMD) after five years of therapy. An improvement in BMD is typically seen after surgical cure of primary hyperparathyroidism, so it is unclear that cinacalcet will off er the same long-term benefits.
For these reasons, cinacalcet should not be considered an equivalent alternative to parathyroidectomy for patients in whom surgery would be indicated. Conversely, if surgery is not indicated, typically due to lack of symptoms and a low risk of complications from the hyperparathyroidism, cinacalcet would not likely off er benefit. Hence, cinacalcet would only be recommended for use in less common situations. As an example, I have helped manage a 65-year-old woman with primary hyperparathyroidism, corrected serum calcium levels as high as 11.7 mg/dL (normal <10.1 mg/dL), and progressive renal failure. No autonomous parathyroid tissue was localized despite various imaging modalities. Attempts at surgical exploration were postponed due to repeated bouts with decompensated congestive heart failure. Due to progressive hypercalcemia, she was treated with cinacalcet. The starting dose of 30 mg twice daily actually caused modest, asymptomatic hypocalcemia (with similarly elevated PTH levels). She has since responded well to 600 mg of calcium carbonate twice daily and 50,000 international units of ergocalciferol weekly while continuing the same dose of cinacalcet.
Calcilytics are compounds that antagonize the CaSR, thereby stimulating endogenous PTH secretion. Since brief, daily exposure to exogenous PTH or its analogs is known to stimulate anabolic bone growth with a reduction in fracture risk, agents in this class are being studied as a potential anabolic therapy for osteoporosis. The first generation of these agents was shown to cause a dramatic increase in bone turnover in ovariectomized rats, though there was no change in BMD. This was felt to be due to a longer duration of action leading to a more prolonged elevation of PTH (>4 hours). More recently, a shorter-acting calcilytic, ronacaleret, has been shown to increase trabecular BMD in postmenopausal women, though there were small decreases in cortical BMD, still implying excess PTH exposure. Therefore, while it is not clear where these agents will take us, they do off er the promise of an orally administered, anabolic therapy for osteoporosis.
The discovery of the CaSR has revolutionized our understanding of calcium homeostasis. Not only has it allowed us to define the etiology of certain disorders of calcium metabolism, but it has also provided a target at which to aim therapeutics for a wide array of pathologies.