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School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Sydney, Australia
* To whom correspondence should be addressed. E-mail: a.conigrave{at}mmb.usyd.edu.au.
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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-amino butyric acid; and several receptors that act as broad-spectrum amino acid sensors. The CaR is one of these broad-spectrum amino acid sensors that, along with several other members of the subgroup, also responds to extracellular Ca2+. In this mini-review, we consider evidence that the CaR is a sensor of aromatic amino acids, that it has broad-spectrum amino acid sensing properties, that it provides an amino acid binding site in its extracellular N-terminal Venus Fly Trap domain, and that amino acids have a physiological impact on systems in which the CaR is expressed.
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L-Phe, L-Tyr, L-Trp, and L-His are activators of the CaR expressed heterologously in HEK293 or CHO-K1 cells (4,6) or expressed endogenously in human parathyroid cells (5) and their potencies depend on the extracellular Ca2+ concentration. At a subthreshold Ca2+ concentration (< 
0.5 mmol/L), these amino acids are ineffective. However, at Ca2+ concentrations above threshold, amino acids activate the receptor and enhance its sensitivity to Ca2+. The effects are stereoselective; D-amino acids are much less effective than L-amino acids. In addition, at a Ca2+ concentration of 2.5 mmol/L in CaR-expressing HEK293 cells, aromatic amino acids were clearly more effective than other amino acids. Thus, the order of effectiveness was aromatics > aliphatics, polars > acidic > basic, branch-chain amino acids (4). Furthermore, in human parathyroid cells that express the CaR endogenously, the aromatic amino acids L-Phe and L-Trp exhibited higher potencies than other amino acids (5). At an extracellular Ca2+ concentration of 1.5 mmol/L, for example, the activator concentration inducing 50% efficacy values for L-Phe and L-Trp were 0.12 and 0.14 mmol/L, respectively, lower than for any of the other amino acids tested (Table 1).
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Investigation of the effects of the standard 20 genetically encoded amino acids revealed that almost all, with the exception of basic and branch-chain amino acids, were effective activators of the CaR (4). We considered their potencies and fasting plasma concentrations and found many of these amino acids were equivalent activators (5). These amino acids include L-Trp, L-Phe, L-Tyr, L-His, L-Ala, L-Ser, and L-Thr. The results suggest the conclusion that CaR, like other class 3 G-protein coupled receptors (GPCR) including goldfish 5.24 receptors, GPRC6A, and taste receptor T1R1/T1R3 heterodimers, are broad-spectrum amino acid sensors. Furthermore, increasing fold-concentrations of a fasting amino acid mixture also enhanced receptor activity (Fig. 2). These findings implicate the CaR as a sensor of changes in protein metabolism and also indicate that the CaR may provide a molecular link between protein and calcium metabolism.
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The CaR extracellular, N-terminal Venus Fly Trap (VFT) domain is related to nutrient sensing, bacterial periplasmic binding proteins (7), and, in the mammalian genome, to the VFT domains of other class 3 GPCR as well as several ionotropic glutamate receptors [iGlus; (6)]. However, type-II calcimimetics of the phenylalkylamine class (e.g. cinacalcet), which like amino acids are positive allosteric modulators of the CaR, bind in the receptor's transmembrane domain region (8). Chimeric receptor analysis was used to resolve whether the amino acid binding site is located in the VFT or transmembrane domains (9). Consistent with previous analyses, the amino acid L-Phe and the phenylalkylamine type-II calcimimetic NPS R467 both markedly enhanced the sensitivity of the wild-type CaR to extracellular Ca2+ (Fig. 3). R467 failed, however, to activate a chimeric receptor in which the CaR transmembrane domain was replaced by the rat mGlu-1 transmembrane domain (Fig. 4A), whereas the effect of L-Phe on these CaR/Glu/Glu chimeric receptors was retained (Fig. 4B). However, R467, but not L-Phe, activated an N-terminally truncated CaR (not shown) (9). Thus, unlike phenylalkylamine activators of the CaR, which bind in the transmembrane domain, L-amino acids bind in the CaR VFT domain and mutations of residues in the VFT domain were subsequently used to develop a mutant receptor that retains full sensitivity to Ca2+ ions but is completely insensitive to amino acids (10).
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The finding that amino acids activate cultured cells that express the CaR heterologously led to the prediction that amino acids are also likely to regulate cells that express the CaR endogenously. To test this hypothesis, we prepared cells from small samples of normal human parathyroid cells and investigated their sensitivity to amino acids in 2 key assays: extracellular Ca2+-induced Ca2+ mobilization in fura2-loaded cells; extracellular Ca2+-regulated PTH secretion.
We found amino acids had a considerable impact on both assays (5). First, in fura2-loaded human parathyroid cells, L-Phe and various other CaR-active amino acids, including L-Trp, L-His, and L-Ala, markedly stimulated intracellular Ca2+ mobilization. Second, perifused parathyroid cells exhibited enhanced Ca2+-dependent suppression of intact PTH secretion in response to all these amino acids. CaR-inactive amino acids, however (including D-isomers of the active amino acids as well as the basic amino acid, L-Arg, and branch chain amino acid, L-Leu), were much less effective. It is interesting that amino acids, which are required for the synthesis of peptide hormones such as PTH, should also suppress its secretion.
High dietary protein intake stimulates urinary calcium excretion in humans (11,12) and other species including the laboratory rat (13). Because the CaR is a key regulator of renal tubular calcium reabsorption and, thus, urinary calcium excretion, and amino acids stimulate the CaR, it seems feasible that protein-induced hypercalciuria arises from amino acid-dependent activation of CaR in the kidney and in endocrine cells that contribute to the control of urinary calcium excretion. To test this hypothesis, individual CaR-active amino acids were infused intravenously into female Wistar rats to generate serum levels between 0.5 and 1.0 mmol/L (similar to the change in total serum amino acid concentration that occurs acutely following the ingestion of a protein-rich meal). Interestingly, both L-Phe and L-Ala stimulated urinary calcium excretion (14).
In addition to sensing changes in the extracellular Ca2+ concentration, the CaR also senses aromatic amino acids, which act as allosteric activators. Although aromatic amino acids are the most potent activators of the CaR, the CaR also responds sensitively to multiple amino acids and, based on their potencies and fasting concentrations, as many as 6–8 different amino acids (including L-Trp, L-Phe, L-Tyr, L-His, L-Ala, L-Ser, and L-Thr) are equivalent activators. There are striking positive interactions between extracellular Ca2+ and amino acids so that amino acids enhance the sensitivities of the CaR intracellular signaling pathways to extracellular Ca2+ and the extracellular Ca2+ concentration sets the receptor's level of amino acid responsiveness. A threshold extracellular Ca2+ concentration is required for amino acid activation of the CaR; in parathyroid cells that express the CaR endogenously, it is 0.5 mmol/L. Amino acids and amino acid mixtures have predictable effects in tissues that express the CaR. In the parathyroid gland, elevations in the concentrations of CaR-active amino acids markedly sensitize intracellular Ca2+ mobilization to the extracellular Ca2+ concentration and suppress PTH secretion. In addition, amino acids activate gastric acid secretion at least in part via CaR on the basolateral membranes of gastric parietal cells (15) and infusions of CaR-active amino acids activate renal calcium excretion. It remains to be determined whether amino acids have a physiological role as modulators of CaR activity in the brain (e.g. in the control of myelination by oligodendrocytes) or whether toxic elevations of aromatic amino acids (e.g. in phenylketonuria or tyrosinemia) induce some of their effects by inappropriate activation and/or downregulation of CaR in key sites.
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FOOTNOTES |
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2 Author disclosures: H-C. Munn, no relationships to disclose, H-C. Lok, no relationships to disclose; A. D. Conigrave's travel expenses to attend meeting paid by Ajinomoto Company, Inc.
3 Funding arrangements: The authors' research is funded by the NHMRC of Australia.
4 Abbreviations used: CaR, calcium-sensing receptor; GPCR, G-protein coupled receptor; PTH, parathyroid hormone; VFT, Venus Fly Trap domain.
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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1. Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev. 2001;81:239–97.
2. Ho C, Conner DA, Pollak MR, Ladd DJ, Kifor O, Warren HB, Brown EM, Seidman JG, Seidman CE. A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat Genet. 1995;11:389–94.[Medline]
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5. Conigrave AD, Mun H-C, Delbridge L, Quinn SJ, Wilkinson M, Brown EM. L-Amino acids regulate parathyroid hormone secretion. J Biol Chem. 2004;279:38151–9.
6. Conigrave AD, Quinn SJ, Brown EM. Cooperative multi-modal sensing and therapeutic implications of the extracellular Ca2+-sensing receptor. Trends Pharmacol Sci. 2000;21:401–7.[Medline]
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8. Hauache OM, Hu J, Ray K, Xie R, Jacobson KA, Spiegel AM. Effects of a calcimimetic compound and naturally activating mutations on the human Ca2+ receptor and on Ca2+ receptor/metabotropic glutamate chimeric receptors. Endocrinology. 2000;141:4156–63.
9. Mun HC, Franks AH, Culverston EL, Krapcho K, Nemeth EF, Conigrave AD. The Venus Fly Trap domain of the extracellular Ca2+-sensing receptor is required for L-amino acid sensing. J Biol Chem. 2004;279:51739–44.
10. Mun H, Culverston E, Franks A, Collyer C, Clifton-Bligh R, Conigrave A. A double mutation in the extracellular Ca2+-sensing receptor's Venus fly trap domain that selectively disables L-amino acid sensing. J Biol Chem. 2005;280:29067–72.
11. Johnson NE, Alcantara EN, Linkswiler H. Effect of level of protein intake on urinary and fecal calcium and calcium retention of young adult males. J Nutr. 1970;100:1425–30.
12. Allen LH, Oddoye EA, Margen S. Protein-induced hypercalciuria: a longer term study. Am J Clin Nutr. 1979;32:741–9.
13. Amanzadeh J, Gitomer WL, Zerwekh JE, Preisig PA, Moe OW, Pak CYC, Levi M. Effect of high protein diet on stone-forming propensity and bone loss in rats. Kidney Int. 2003;64:2142–9.[Medline]
14. Conigrave AD, Lok H. Activation of renal calcium and water excretion by novel physiological and pharmacological activators of the calcium-sensing receptor. Clin Exp Pharmacol Physiol. 2004;31:368–71.[Medline]
15. Busque SM, Kerstetter JE, Geibel JP, Insogna K. L-type amino acids stimulate gastric acid secretion by activation of the calcium sensing receptor in parietal cells. Am J Physiol. 2005;289:G664–9.
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from baseline) and were derived from 4 separate experiments. The figure has been reproduced from reference (
