Metabolism
Physiological significance of l-amino acid sensing
by extracellular Ca2+-sensing receptors
A.D. Conigrave1 , H.-C. Mun and S.C. Brennan
School of Molecular and Microbial Biosciences, G08, University of Sydney, Sydney, NSW 2006, Australia
Abstract
The calcium-sensing receptor is a multimodal, multimetabolic sensor that mediates the feedback-dependent
control of whole body calcium metabolism. Remarkably, in addition to its role in Ca2+ o (extracellular Ca2+ )
sensing, the CaR (Ca2+ -sensing receptor) also responds to l-amino acids. l-amino acids appear to activate,
predominantly, a signalling pathway coupled with intracellular Ca2+ mobilization, require a threshold
concentration of Ca2+ o for efficacy and sensitize the receptor to activation by Ca2+ o . Here, we review the
evidence that the CaR, like other closely related members of the class 3 GPCR (G-protein-coupled receptor)
family including GPRC6A, is a broad-spectrum amino acid-sensing receptor, consider the nature of the
signalling response to amino acids and discuss its physiological significance.
Introduction
The calcium-sensing receptor is a class 3 GPCR (G-proteincoupled receptor) that mediates the effects of multiple
nutrients and metabolic signals including Ca2+ o (extracellular
Ca2+ ) and extracellular Mg2+ , organic multivalent cations
including the polyamine spermine, ionic strength, and
α-amino acids and related oligopeptides (for reviews, see
[1,2]). In this respect, the CaR (Ca2+ -sensing receptor)
shares pluripotency along with other class 3 GPCRs that
mediate taste (for a review, see [3]). With various signals to
process and numerous signalling pathways to access, the
CaR acts as a multichannel switch box, providing ligandselective control of physiological responses. The physiological response to changes in the concentrations of
specific nutrients depends on context. Cellular context,
for example, determines which signalling adaptors and
enzymes are expressed and, thus, which pathways are
available for receptor-dependent activation. Cellular context
also determines, of course, which physiological effector
responses can be accessed. Compartment-specific effects
arise from variations in ionic and nutrient composition.
What are the normal concentration ranges for the receptor’s
activators in the compartment? Are they relatively stable or
subject to substantial fluctuations? Excursions in nutrient
concentration may have a substantial impact on receptor
response. In the present paper, we review the evidence
suggesting that the CaR plays a key role in sensing variations
in the concentrations of L-amino acids and explore its
physiological significance. We show that the CaR’s response
to amino acids is dependent not only on variations in amino
Key words: amino acid-sensing receptor, calcium-sensing receptor, extracellular Ca2+
concentration, ligand-selective signalling, l-phenylalanine, l-tryptophan.
Abbreviations used: Ca2+ o , extracellular Ca2+ ; CaR, Ca2+ -sensing receptor; ERK1/2, extracellularsignal-regulated kinase 1/2; GI tract, gastrointestinal tract; GPCR, G-protein-coupled receptor;
HEK, human embryonic kidney; PTH, parathyroid hormone; VFT domain, Venus Fly Trap domain.
1
To whom correspondence should be addressed (email [email protected]).
acid concentration to which the receptor is exposed but also
on the prevailing Ca2+ o concentration with different amino
acids exhibiting differential Ca2+ o concentration thresholds
for receptor activation. The response is also dependent on the
extent to which amino acid binding induces the activation of
signalling pathways, the membrane location of the receptor
and the functions of the cells in which the receptor itself is
expressed.
Activation of the CaR by l-amino acids
As a member of the class 3 GPCR subgroup, the CaR is
related structurally to various receptors for amino acids
including the metabotropic glutamate receptors, which are
specific for the acidic amino acid glutamate, and several taste
receptors that respond in some cases to D-amino acids and,
in others, to short peptides such as aspartame (Asp-D-Phe).
The class 3 subgroup also includes at least two receptors with
broad-spectrum amino acid-sensing properties: GPRC6A,
which recognizes basic and various aliphatic amino acids,
its apparent orthologue, the fish 5.24 receptor, and a specific
taste receptor heterodimer (T1R1–T1R3), which responds to
aliphatic and polar amino acids (for a review, see [4]).
L-Amino acid activation of the CaR induces intracellular
Ca2+ mobilization, which, in single HEK-293 cells
(human embryonic kidney cells) that stably express the CaR,
otherwise known as HEK-CaR cells, and human parathyroid
cells, takes the form of various patterns of response
including simple stepwise increases in cytoplasmic free-Ca2+
concentration, particularly at low Ca2+ o concentrations and
above a certain threshold Ca2+ o concentration, characteristic
slow wave oscillations with a frequency of approx. 1–
2 · min−1 . The highest potency activators are the aromatic
amino acids L-phenylalanine and L-tryptophan but potency
is critically dependent on Ca2+ o [5].
C The
C 2007 Biochemical Society
Authors Journal compilation 1195
1196
Biochemical Society Transactions (2007) Volume 35, part 5
Figure 1 Hypothetical model of interacting actions of amino
acids and Ca2+ on the calcium-sensing receptor
The model shows initially the dimeric CaR in its resting state with both VFT
domains open. Ca2+ binding at low concentrations in the transmembrane
region primes the receptor for activation by amino acids. Amino acid
binding in turn sensitizes the receptor to Ca2+ . In the absence of amino
acids, higher concentrations of Ca2+ are required for activation. The
model suggests that distinct G-proteins may, at least in part, mediate
the actions of amino acids and Ca2+ . kf, rate constant of the forward
reaction.
Dependence of amino acid activation
on Ca2+ o concentration
Ca2+ o is absolutely required for receptor activation by amino
acids. In this sense, amino acids are allosteric activators and
Ca2+ ions prime the receptor for activation by amino acids
(Figure 1). One possible explanation is that Ca2+ binding in
the receptor’s VFT domain (Venus Fly Trap domain) leads to
adoption of a conformation that supports amino acid binding
[6]. Alternatively, Ca2+ binding in the seven-transmembrane
domain region may couple the amino acid-bound VFT domain with the recruitment of G-proteins and activation of
other receptor-associated proteins. The demonstration that
the CaR responds to Ca2+ in truncated receptors that lack the
VFT domain [7,8] is consistent with this idea.
Regardless of its origin, a defined Ca2+ o concentration
threshold can be identified for the effects of amino acid
activators of the CaR. In our initial studies on intracellular
Ca2+ mobilization in cell populations, we identified a Ca2+
threshold concentration of approx. 0.5–1.0 mM for L-phenylalanine and L-tryptophan in HEK-CaR cells [5]. More recent
C The
C 2007 Biochemical Society
Authors Journal compilation studies support this conclusion but demonstrate that
intracellular Ca2+ oscillations require higher Ca2+ o threshold
concentrations, e.g. approx. 1.0–1.5 mM for L-phenylalanine.
Activation of intracellular Ca2+ oscillations by Ca2+ o in
the absence of amino acids, however, requires much higher
concentrations, approx. 2.0–2.5 mM. In this sense, amino
acids sensitize the receptor to Ca2+ . Investigation of the
interactions between Ca2+ and amino acids for intracellular
Ca2+ mobilization indicates that there are some surprising
differences between the Ca2+ o thresholds required for
activation by different amino acids. In particular, although
L-alanine is a much less potent activator of the CaR, its Ca2+ o
threshold concentration for intracellular Ca2+ mobilization
is significantly lower. In populations of human parathyroid
cells, for example, the threshold for L-alanine lies around
0.5 mM. The threshold for L-phenylalanine, on the other
hand, lies around 0.75 mM. This implies that different classes
of amino acids have differential effects on the receptor’s
response dependent on the prevailing Ca2+ o concentration
and suggests, surprisingly, that the response to amino acids
with larger side chains requires higher Ca2+ concentrations.
For parathyroid cells, the results suggest that amino
acids have no effect on the receptor’s response at grossly
subphysiological Ca2+ o concentrations (<0.5 mM), exert
only a small effect at Ca2+ o concentrations at the low end of
the physiological normal range (approx. 1.0 mM) but exhibit
progressively higher levels of receptor activation as the Ca2+ o
concentration increases between 1.0 and 1.5 mM, i.e. encompassing the normal physiological range for Ca2+ o concentration [9]. What is the potential physiological significance of
this phenomenon for parathyroid cells? One of the predicted
consequences is a tighter range over which changes in Ca2+ o
concentration titrate the intracellular signalling response and
the cellular rate of PTH (parathyroid hormone) secretion
(Figure 2). In this way, physiological mixtures of amino
acids appear to narrow the effective Ca2+ o response range,
assisting a receptor that responds to Ca2+ o concentrations in
the millimolar range to orchestrate a concentration response
relationship in which Ca2+ has a minimal effect on PTH secretion at 1.0 mM and a maximal suppressive effect at 1.4 mM.
Differential activation of distinct signalling
pathways by amino acids when compared
with Ca2+ o
The initial studies on amino acid activation of the CaR
demonstrated that amino acids induced intracellular Ca2+
mobilization, required threshold levels of Ca2+ o for activity
and enhanced the sensitivity of the response to Ca2+ o [5].
Subsequent analyses have demonstrated that amino acids are
much more effective activators of intracellular Ca2+ mobilization than various other signalling responses including inositol
phosphate turnover [10] and ERK1/2 (extracellular-signalregulated kinase 1/2) activation [11]. More recent analysis
using a novel quantitative assay of ERK1/2 indicates that
amino acids do, in fact, enhance Ca2+ o -induced ERK1/2 responses but that the enhanced sensitivity to Ca2+ o is modest,
Metabolism
Figure 2 Effect of increasing the total amino acid concentration
Ca2+
on the co-operativity of receptor activation by
Increasing fold-concentrations of plasma-like amino acid mixtures lower
Ca2+ -dependent
the threshold for
activation, elevate the maximum
response and increases co-operativity, i.e. narrows the effective Ca2+
concentration range for activation. 䊊, control; 䉭, ×0.2; 䊐, ×0.5; 䊉,
×1; 䉱, ×2; 䊏, ×5. *Change in fluorescence ratio with respect to the
c 2004 American Society
baseline. Modified from [9] with permission. for Biochemistry and Molecular Biology.
i.e. the EC50 for Ca2+ o falls by approx. 0.2–0.3 mM [12]. Since
the Ca2+ o concentration is tightly regulated between 1.1 and
1.3 mM, a fall in EC50 for Ca2+ o of this order can have a major
impact on the physiological response. Thus the concept that
the effects of amino acids are restricted solely to the regulation
of intracellular Ca2+ mobilization seems unreliable and it
seems more likely that amino acids activate multiple CaRregulated signalling pathways that differ in the gain of the
amino acid-dependent fine-tuning control mechanism.
It is interesting to contemplate whether there are
circumstances in which amino acids are not just modulators
of Ca2+ o -dependent responses but the primary physiological
regulators of the CaR. The key requirements for amino
acid-dependent control of the CaR are, first, that the Ca2+ o
concentration should be stable and above the threshold
required for amino acid activation of the receptor and,
secondly, that the amino acid concentration should be subject
to excursions that are capable of activating the receptor. The
lumen of the GI tract (gastrointestinal tract) and hepatocytes
exposed to food and portal blood respectively are two
obvious sites that are susceptible to marked increases in amino
acid concentration in the millimolar range. The systemic
blood also exhibits significant variations in amino acid
concentrations following the ingestion of dietary protein [3].
Expression of the CaR by epithelial cells
and evidence for amino acid regulation of
physiological function
The CaR is expressed widely in mammalian tissues and is
expressed at high levels in endocrine cells such as parathyroid
chief cells, thyroid C cells and anterior pituitary cells [13]. In
Figure 3 Model of amino acid- and Ca2+ -activated gastric acid
secretion
The model incorporates the amino acid (AA) and Ca2+ o -activated CaR
along with histamine H2 and muscarinic receptors as one of the
basolateral membrane receptors that promote gastric acid secretion [16].
A possible role for the L-type amino acid transporter in the control of
gastric acid secretion is also demonstrated [23]. ACh, acetylcholine; ECL,
enterochromaffin-like; MLCK, myosin light-chain kinase. Modified from
c 2006 The American Physiological Society.
[3] with permission. the brain, the CaR is expressed in the ionic strength-sensing
subfornical organ that provides inputs to hypothalamic
centres that control antidiuretic hormone secretion, various
other organs including the hippocampus and more diffusely
on myelin-producing oligodendrocytes. Otherwise, it is
widely expressed in epithelial tissues of the gut and kidney.
Since the parathyroid and thyroid arise embryologically
from the pharyngeal pouches and retain some epithelial
organization, the localization of the CaR in these sites is also
usefully considered from an epithelial perspective. In the
parathyroid, it localizes primarily to the apical membranes,
which are also the primary sites for the exocytosis of
PTH-containing secretory vesicles.
In the GI tract, the CaR is expressed at high levels on
the basolateral membranes of gastric parietal cells [14]. It
provides an explanation for the recognized phenomena of
C The
C 2007 Biochemical Society
Authors Journal compilation 1197
1198
Biochemical Society Transactions (2007) Volume 35, part 5
Ca2+ -dependent and amino acid-dependent activation of acid
secretion [15,16], and the CaR’s selectivity for aromatic
amino acids provides an explanation for the recognized
selectivity of gastric acid secretion for aromatic amino acids
[17] (Figure 3). The CaR is also expressed in epithelial cells
and enteroendocrine cells of the small intestine [18,19]. It
seems plausible that the CaR mediates the known effects of
aromatic amino acids, on cholecystokinin release and thus bile
flow and pancreatic enzyme secretion (for a review, see [3]).
In the kidney, the CaR is expressed at highest density on the
basolateral membranes of the cortical thick ascending limb
of Henle’s loop and the apical membranes of the collecting
ducts [20]. Consistent with these locations, elevated Ca2+
concentrations interfere with Ca2+ reabsorption in the cortical thick ascending limb and promote free water clearance
in the collecting tubules by interfering with antidiuretic
hormone-dependent water reabsorption [21]. Previous
experiments on Ca2+ and water excretion demonstrate
that amino acids as well as other type II calcimimetics
promote calcium and water excretion consistent with the
idea that amino acids activate CaRs in several renal tubular
segments [22].
References
1 Conigrave, A.D., Quinn, S.J. and Brown, E.M. (2000) Trends Pharm. Sci.
21, 401–407
2 Breitwieser, G.E., Miedlich, S.U. and Zhang, M. (2004) Cell Calcium 35,
209–216
3 Conigrave, A.D. and Brown, E.M. (2006) Am. J. Physiol. Gastrointest.
Liver Physiol. 291, G753–G761
4 Conigrave, A.D. and Hampson, D.R. (2006) Trends Endocrinol. Metab. 17,
398–407
C The
C 2007 Biochemical Society
Authors Journal compilation 5 Conigrave, A.D., Quinn, S.J. and Brown, E.M. (2000) Proc. Natl.
Acad. Sci. U.S.A. 97, 4814–4819
6 Silve, C., Petrel, C., Leroy, C., Bruel, H., Mallet, E., Rognan, D. and Ruat, M.
(2005) J. Biol. Chem. 280, 37917–37923
7 Ray, K. and Northup, J. (2002) J. Biol. Chem. 277, 18908–18913
8 Mun, H.C., Franks, A.H., Culverston, E.L., Krapcho, K., Nemeth, E.F. and
Conigrave, A.D. (2004) J. Biol. Chem. 279, 51739–51744
9 Conigrave, A.D., Mun, H.-C., Delbridge, L., Quinn, S.J., Wilkinson, M. and
Brown, E.M. (2004) J. Biol. Chem. 279, 38151–38159
10 Rey, O., Young, S.H., Yuan, J., Slice, L. and Rozengurt, E. (2005)
J. Biol. Chem. 280, 22875–22882
11 Davies, S.L., Gibbons, C.E., Vizard, T. and Ward, D.T. (2006) Am. J. Physiol.
Cell Physiol. 290, 1543–1551
12 Lee, H., Mun, H.-C., Lewis, N.C., Crouch, M.F., Culverston, E.L., Mason, R.S.
and Conigrave, A.D. (2007) Biochem. J. 404, 141–149
13 Brown, E.M. and MacLeod, R.J. (2001) Physiol. Rev. 81, 239–297
14 Cheng, I., Qureshi, I., Chattopadhyay, N., Qureshi, A., Butters, R.R., Hall,
A.E., Cima, R.R., Rogers, K.V., Hebert, S.C., Geibel, J.P. et al. (1999)
Gastroenterology 116, 118–126
15 Geibel, J.P., Wagner, C.A., Caroppo, R., Qureshi, I., Gloeckner, J.,
Manuelidis, L., Kirchhoff, P. and Radebold, K. (2001) J. Biol. Chem. 276,
39549–39552
16 Busque, S.M., Kerstetter, J.E., Geibel, J.P. and Insogna, K. (2005) Am. J.
Physiol. Gastrointest. Liver Physiol. 289, G664–G669
17 Strunz, U.T., Walsh, J.H. and Grossman, M.I. (1978) Proc. Soc.
Exp. Biol. Med. 157, 440–441
18 Chattopadhyay, N., Cheng, I., Rogers, K., Riccardi, D., Hall, A., Diaz, R.,
Hebert, S.C., Soybel, D.I. and Brown, E.M. (1998) Am. J. Physiol.
Gastrointest. Liver Physiol. 274, G122–G130
19 Ray, J., Squires, P., Curtis, S., Meloche, M. and Buchan, A. (1997)
J. Clin. Invest. 99, 2328–2333
20 Riccardi, D., Hall, A.E., Chattopadhyay, N., Xu, J.Z., Brown, E.M. and
Hebert, S.C. (1998) Am. J. Physiol. 274, F611–F622
21 Brown, E.M. and Hebert, S.C. (1997) Bone 20, 303–309
22 Conigrave, A.D. and Lok, H. (2004) Clin. Exp. Pharmacol. Physiol. 31,
368–371
23 Kirchhoff, P., Dave, M.H., Remy, C., Kosiek, O., Busque, S.M., Dufner, M.,
Geibel, J.P., Verrey, F. and Wagner, C.A. (2006) Pflügers Arch. 451,
738–748
Received 18 June 2007
doi:10.1042/BST0351195