Stephen J. Quinn

Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells

Several proteins implicated in the pathogenesis of polycystic kidney disease (PKD) localize to cilia. Furthermore, cilia are malformed in mice with PKD with mutations in TgN737Rpw (encoding polaris). It is not known, however, whether ciliary dysfunction occurs or is relevant to cyst formation in PKD. Here, we show that polycystin-1 (PC1) and polycystin-2 (PC2), proteins respectively encoded by Pkd1 and Pkd2, mouse orthologs of genes mutated in human autosomal dominant PKD, co-distribute in the primary cilia of kidney epithelium. Cells isolated from transgenic mice that lack functional PC1 formed cilia but did not increase Ca2+ influx in response to physiological fluid flow. Blocking antibodies directed against PC2 similarly abolished the flow response in wild-type cells as did inhibitors of the ryanodine receptor, whereas inhibitors of G-proteins, phospholipase C and InsP3 receptors had no effect. These data suggest that PC1 and PC2 contribute to fluid-flow sensation by the primary cilium in renal epithelium and that they both function in the same mechanotransduction pathway. Loss or dysfunction of PC1 or PC2 may therefore lead to PKD owing to the inability of cells to sense mechanical cues that normally regulate tissue morphogenesis.

The Ca2+-sensing receptor: a target for polyamines

The Ca2+-sensing receptor (CaR) is activated at physiological levels of external Ca2+(Cao) but is expressed in a number of tissues that do not have well-established roles in the control of Cao, including several regions of the brain and the intestine. Polyamines are endogenous polyvalent cations that can act as agonists for the CaR, as shown by our current studies of human embryonic kidney (HEK-293) cells transfected with the human CaR. Cellular parameters altered by polyamines included cytosolic free Ca2+(Cai), inositol phosphate production, and the activity of a nonselective cation channel. Spermine stimulated Cai transients in CaR-transfected HEK cells, with a concentration producing a half-maximal response (EC50) of ∼500 μM in the presence of 0.5 mM Ca2+, whereas sustained increases in Cai had an EC50 of ∼200 μM. The order of potency was spermine > spermidine >> putrescine. Elevation of Cao shifted the EC50 for spermine sharply to the left, with substantial stimulation below 100 μM. Addition of subthreshold concentrations of spermine increased the sensitivity of CaR-expressing HEK cells to Cao. Parathyroid hormone secretion from bovine parathyroid cells was inhibited by 50% in the presence of 200 μM spermine, a response similar to that elicited by 2.0 mM Cao. These data suggest that polyamines could be effective agonists for the CaR, and several tissues, including the brain, may use the CaR as a target for the actions of spermine and other endogenous polycationic agonists.

Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that plays a key role in extracellular calcium ion homeostasis. We have engineered 11 CaR mutants that have been described in the disorders familial benign hypercalcemia (FBH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcaemia (ADH), and studied their function by characterizing intracellular calcium [Ca2+]i transients in response to varying concentrations of extracellular calcium [Ca2+]o or gadolinium [Gd3+]o. The wild type receptor had an EC50 for calcium (EC50[Ca2+]o) (the value of [Ca2+]o producing half of the maximal increase in [Ca2+]i) of 4.0 mM (+/- 0.1 SEM). However, five missense mutations associated with FBH or NSHPT, (P55L, N178D, P221S, R227L, and V817I) had significantly higher EC50[Ca2+]os of between 5.5 and 9.3 mM (all P < 0.01). Another FBH mutation, Y218S, had an EC50[Ca2+]o of > 50 mM but had only a mildly attenuated response to gadolinium, while the FBH mutations, R680C and P747fs, were unresponsive to either calcium or gadolinium. In contrast, three mutations associated with ADH, (F128L, T151M, and E191K), showed significantly reduced EC50[Ca2+]os of between 2.2 and 2.8 mM (all P < 0.01). These findings provide insights into the functional domains of the CaR and demonstrate that mutations which enhance or reduce the responsiveness of the CaR to [Ca2+]o cause the disorders ADH, FBH, and NSHPT, respectively.

The Extracellular Calcium-sensing Receptor Dimerizes through Multiple Types of Intermolecular Interactions

Recent studies have shown that the G protein-coupled, extracellular calcium ([Ca2+] o )-sensing receptor (CaR) forms disulfide-linked dimers through cysteine residues within its extracellular domain and that dimerization of the CaR has functional implications. In this study, we have investigated which of these disulfide linkages are essential for dimerization of the CaR and whether they are required for these functional interactions. Our results confirm the key roles of Cys129 and Cys131 in CaR dimerization. However, utilizing cross-linking of the CaR or immunoprecipitation of a non-FLAG-tagged CaR with a FLAG-tagged CaR using anti-FLAG antibody, we demonstrate that CaRs with or without these two cysteines form dimers on the cell surface to a similar extent. In addition, reconstitution of CaR-mediated signaling by cotransfection of two individually inactive mutant CaRs is nearly identical in the presence or absence of both Cys129 and Cys131, showing that covalent linkage of CaR dimers is not needed for functional interactions between CaR monomers. These findings suggest that the CaR has at least two distinct types of motifs mediating dimerization and functional interactions, i.e. covalent interactions involving intermolecular disulfide bonds and noncovalent, possibly hydrophobic, interactions.

Sodium and Ionic Strength Sensing by the Calcium Receptor

The calcium-sensing receptor (CaR) is activated by small changes in extracellular calcium [Ca2+] o ) in the physiological range, allowing the parathyroid gland to regulate serum [Ca2+] o ; however, the CaR is also distributed in a number of other tissues where it may sense other endogenous agonists and modulators. CaR agonists are polycationic molecules, and charged residues in the extracellular domain of the CaR appear critical for receptor activation through electrostatic interactions, suggesting that ionic strength could modulate CaR activation by polycationic agonists. Changes in the concentration of external NaCl potently altered the activation of the CaR by external Ca2+ and spermine. Ionic strength had an inverse effect on the sensitivity of CaR to its agonists, with lowering of ionic strength rendering the receptor more sensitive to activation by [Ca2+] o and raising of ionic strength producing the converse effect. Effects of osmolality could not account for the modulation seen with changes in NaCl. Other salts, which differed in the cationic or anionic species, showed shifts in the activation of the CaR by [Ca2+] o similar to that elicited by NaCl. Parathyroid cells were potently modulated by ionic strength, with addition of 40 mm NaCl shifting the EC50 for [Ca2+] o inhibition of parathyroid hormone by at least 0.5 mm. Several CaR-expressing tissues, including regions of the brain such as the subfornical organ and hypothalamus, could potentially use the CaR as a sensor for ionic strength and NaCl. The Journal guidelines state that the summary should be no longer than 200 words.

pH sensing by the calcium-sensing receptor

The calcium-sensing receptor (CaR) is activated by small changes in the ionic extracellular calcium concentration (Cao) within the physiological range, allowing the parathyroid gland to regulate serum Cao; however, the CaR is also distributed in a number of other tissues where it may sense other endogenous agonists and modulators. CaR agonists are polycationic molecules, and our previous studies suggest that charged residues in the extracellular domain of the CaR are critical for receptor activation through electrostatic interactions. Therefore, pH could also potentially modulate CaR activation by its polycationic agonists. Changes in the concentration of extracellular H+ substantially altered the activation of the CaR by Cao and other CaR agonists. The effects of external pH on the CaRs sensitivity to its agonists were observed for both acidic and basic deviations from physiological pH of 7.4, with increases in pH rendering the receptor more sensitive to activation by Cao and decreases in pH producing the converse effect. At pH values more acidic than 5.5, CaR sensitivity to its agonists showed some recovery. Changes in the intracellular pH could not account for the effects of external pH on CaR sensitivity to its agonists. Other G-protein-coupled receptors, which are endogenously expressed in human embryonic kidney 293 cells, showed little change in activity with alterations in external pH or effects opposite those found for the CaR. Extracellular pH directly alters the CaR in the case of Cao and Mgo activation; however, the charges on many organic and inorganic agonists are pH-dependent. Activating CaR mutations show reduced pHo modulation, suggesting a molecular mechanism for increased CaR activity at physiological pHo. Several CaR-expressing tissues, including regions of the stomach, the kidney, bone, and the brain, could potentially use the CaR as a sensor for pH and acid-base status.

L-Amino acid sensing by the calcium-sensing receptor: a general mechanism for coupling protein and calcium metabolism?

Cellular sensing of L-amino acids is widespread and controls diverse cellular responses regulating, for example, rates of hormone secretion, amino acid uptake, protein synthesis and protein degradation (autophagy). However, the nature of the sensing mechanisms involved has been elusive. One important sensing mechanism is selective for branched chain amino acids, acts via mTOR (mammalian target of rapamycin) and regulates the rates of insulin and IGF-1 secretion as well as hepatic, and possibly muscle, autophagy. A second sensing mechanism is selective for aromatic L-amino acids and regulates the rate of gastric acid secretion and other responses in the gastro-intestinal tract. Interactions between calcium and protein metabolism, including accelerated urinary calcium excretion in subjects consuming high-protein diets and secondary hyperparathyroidism in subjects consuming low-protein diets, suggest an additional amino acid sensing mechanism linked to the control of urinary calcium excretion and parathyroid hormone (PTH) release. New data demonstrating L-amino acid-dependent activation of the calcium-sensing receptor (CaR), which regulates PTH secretion and urinary calcium excretion, suggests an unexpected explanation for these links between calcium and protein metabolism. Furthermore, expression of the CaR in gastrin-secreting G-cells and acid-secreting parietal cells, together with data indicating that the CaR exhibits selectivity for aromatic amino acids, would appear to provide a molecular explanation for amino acid sensing in the gastrointestinal tract. This review examines what is known about the CaR as a gene, a receptor, a physiological regulator and, now, as an amino acid sensor. Possible new roles for the CaR are also considered.

G-protein-coupled, extracellular ca2+-sensing receptor : A versatile regulator of diverse cellular functions

Publisher Summary This chapter examines the importance of G-protein-coupled, extracellular Ca 2+ -sensing receptor as a versatile regulator of diverse cellular functions. In the extracellular space, calcium is a cofactor for clotting factors, adhesion molecules, and other proteins. It also regulates neuronal excitability and is an essential component of the mineral phase of the skeleton. Bone provides both a structural framework protecting crucial bodily structures and enabling locomotion and a large reservoir of mineral ions that can be mobilized in times of need. The maintenance of near constancy of Ca 2+ in tetrapods necessitates that specific cells of the mineral ion homeostatic system detect and respond in a homeostatically appropriate manner to the changes in plasma calcium concentration of the same order as its normal variability. Elevated levels of Ca 2+ also stimulate several aspects of osteoblast function in vitro that could promote increased bone formation in vivo and, therefore, reductions in Ca 2+ . The evidence for the presence of a G-protein-coupled CaR came from studies in bovine parathyroid cells examining the actions of Ca 2+ , the physiological agonist for the putative CaR, on intracellular second messengers. A variety of polyvalent cations mimic the actions of Ca 2+ on the osteoclast, but their pharmacological profile in this cell type differs distinctly from that in parathyroid and other CaR-expressing cells.

Deficiency of the Calcium-Sensing Receptor in the Kidney Causes Parathyroid Hormone–Independent Hypocalciuria

Rare loss-of-function mutations in the calcium-sensing receptor (Casr) gene lead to decreased urinary calcium excretion in the context of parathyroid hormone (PTH)-dependent hypercalcemia, but the role of Casr in the kidney is unknown. Using animals expressing Cre recombinase driven by the Six2 promoter, we generated mice that appeared grossly normal but had undetectable levels of Casr mRNA and protein in the kidney. Baseline serum calcium, phosphorus, magnesium, and PTH levels were similar to control mice. When challenged with dietary calcium supplementation, however, these mice had significantly lower urinary calcium excretion than controls (urinary calcium to creatinine, 0.31±0.03 versus 0.63±0.14; P=0.001). Western blot analysis on whole-kidney lysates suggested an approximately four-fold increase in activated Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). In addition, experimental animals exhibited significant downregulation of Claudin14, a negative regulator of paracellular cation permeability in the thick ascending limb, and small but significant upregulation of Claudin16, a positive regulator of paracellular cation permeability. Taken together, these data suggest that renal Casr regulates calcium reabsorption in the thick ascending limb, independent of any change in PTH, by increasing the lumen-positive driving force for paracellular Ca(2+) transport.

Interactions between calcium and phosphorus in the regulation of the production of fibroblast growth factor 23 in vivo

Calcium and phosphorus homeostasis are highly interrelated and share common regulatory hormones, including FGF23. However, little is known about calciums role in the regulation of FGF23. We sought to investigate the regulatory roles of calcium and phosphorus in FGF23 production using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR; PTH-CaSR DKO). In wild-type, PTH KO, and PTH-CaSR DKO mice, elevation of either serum calcium or phosphorus by intraperitoneal injection increased serum FGF23 levels. In PTH KO and PTH-CaSR DKO mice, however, increases in serum phosphorus by dietary manipulation were accompanied by severe hypocalcemia, which appeared to blunt stimulation of FGF23 release. Increases in dietary phosphorus in PTH-CaSR DKO mice markedly decreased serum 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] despite no change in FGF23, suggesting direct regulation of 1,25(OH)(2)D(3) synthesis by serum phosphorus. Calcium-mediated increases in serum FGF23 required a threshold level of serum phosphorus of about 5 mg/dl. Analogously, phosphorus-elicited increases in FGF23 were markedly blunted if serum calcium was less than 8 mg/dl. The best correlation between calcium and phosphorus and serum FGF23 was found between FGF23 and the calcium × phosphorus product. Since calcium stimulated FGF23 production in the PTH-CaSR DKO mice, this effect cannot be mediated by the full-length CaSR. Thus the regulation of FGF23 by both calcium and phosphorus appears to be fundamentally important in coordinating the serum levels of both mineral ions and ensuring that the calcium × phosphorus product remains within a physiological range.