Cell Calcium 35 (2004) 197–207
Structure–function relationship of the extracellular
calcium-sensing receptor
Mei Bai∗
Department of Medicine, Division of Endocrinology, Diabetes, and Hypertension,
Brigham and Women’s Hospital, 221 Longwood Ave., Boston, MA 02115, USA
Received 20 October 2003; accepted 27 October 2003
Abstract
The extracellular calcium-sensing receptor (CaR) originally cloned from bovine parathyroid gland is a G protein-coupled receptor. The
physiological relevance of the cloned CaR for sensing and regulating the extracellular calcium concentration has been established by
identifying hyper-and hypocalcemic disorders resulting from inactivating and activating mutations, respectively, in the CaR. The cloned
CaR has been stably or transiently expressed in human embryonic kidney cells and significant progress has been made in elucidating
its regulation and activation process using physiological, biochemical and molecular biological methods. A large collection of naturally
occurring CaR mutations offers a valuable resource for studies aimed at understanding the structure-function relationships of the receptor,
including functional importance of CaR dimerization. In turn, characterization of these naturally occurring mutations has clarified the
pathogenesis of clinical conditions involving abnormalities in the CaR, such as familial hypocalciuric hypercalcemia and neonatal severe
hyperparathyroidism.
© 2003 Elsevier Ltd. All rights reserved.
Keywords: Extracellular calcium-sensing receptor; Posttranslational modification; Functional dimerization; Activation by calcium; Allosteric modulation;
Activation of G proteins and downstream signaling; Negative regulation by protein kinase C
1. Introduction
The extracellular calcium (Ca2+
o )-sensing receptor
(CaR), originally cloned from bovine parathyroid, is a G
protein-coupled receptor (GPCR) [1]. The CaR is well
conserved across different species [2–7]. For instance, the
amino acid sequences of CaRs from human, rat, and rabbit are more than 90% identical to that of bovine CaR.
The human homologue of the CaR consists of 1078 amino
acid residues. As shown in Fig. 1, the CaR from humans
has three major structural domains [3]: a large, extracellular amino (N)-terminal domain (ECD) of 612 amino
acid residues; a central core of 250 amino acid residues
containing a 7-transmembrane domain (TMD); and a hydrophilic, 216-amino-acid-containing carboxy (C)-terminus
(C-tail) [3].
Protein sequence determination of the isolated ECD of
the receptor has revealed that the putative signal peptide at
the N-terminus of the CaR has been cleaved [8]. Thus, the
∗ Tel.:
+1-617-732-5661; fax: +1-617-732-5764.
E-mail address: [email protected] (M. Bai).
0143-4160/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ceca.2003.10.018
first residue encountered is the tyrosine predicted at amino
acid position 20 of the human CaR cDNA. Sequence analysis predicts that the receptor has 11 putative N-linked glycosylation sites in the ECD, five phosphorylation sites of
protein kinase C (PKC), and two sites of protein kinase A
(PKA) within its intracellular loops and C-tail.
The CaR belongs to a unique subfamily of GPCRs called
family C, which includes five groups of receptors: the
metabotropic glutamate receptors, mGluRs 1–8 [9–11]; the
metabotropic GABAB receptors [12–15]; the CaR; a subgroup of putative pheromone receptors [16–19]; and the
taste receptors [20]. All of these receptors possess unusually
large (500–600-residue) ECDs having sequence similarity
to bacterial periplasmic binding proteins that have a structural feature of a biolobed Venus-flytrap (VFT) [21–23].
The three-dimensional structures of the ECD from mGluR1
were recently determined [24].
In several localized regions of the CaR and mGluRs,
amino acid residues are strikingly conserved. For instance,
the relative positions of 20 cysteines (17 in the N-terminal
ECD, one each in the first and second predicted extracellular loops and one in the fifth transmembrane span) are
conserved in the CaR and mGluRs. A Cys-rich region with
198
M. Bai / Cell Calcium 35 (2004) 197–207
Fig. 1. Schematic representation of the principal structural features of the predicted CaR protein. The large N-terminal domain is located extracellularly
at the top, and the C-terminal domain is located intracellularly at the bottom of diagram. Symbols are provided in the key. Amino acid residues that are
conserved in all mGluRs and the CaR are shown as black and red filled circles.
M. Bai / Cell Calcium 35 (2004) 197–207
nine highly conserved cysteines in a closely spaced sequence
(about 60 amino acids long) is present between the VFT domain and the TMD in the ECDs of all of the members of
family 3 except GABAB R1. The VFT and Cys-rich domains
are not linked by disulfide bonds [25]. In addition, a stretch
of non-charged residues (between amino acid positions 141
and 171) in the ECD of the CaR is quite similar (48% identity and 70% similarity) to the so-called A segment in the
equivalent region of the ECDs of the mGluRs [26]. These
conserved elements may provide a structural framework for
correct protein folding of both the CaR and mGluRs.
Besides the regions in ECDs described above, the putative first and third intracellular loops (i1 and i3) are significantly conserved in the CaR and mGluRs (49 and 69%
identity, respectively). It has been reported for the mGluRs
that i2 and a part of the C-tail determine the specificity
for G-protein coupling [e.g. Gq/11 and Gi , which activate
phospholipase C (PLC) and inhibit adenyl cyclase (AC),
respectively] [27–29]. The specificity of mGluR3 could be
converted from coupling to Gi to Gq/11 by replacing its i2
and C-tail with those of mGluR1. These two determinants
for coupling to a specific class of G-proteins are not well
conserved in the CaR, which may permit the CaR to couple
to both activation of PLC and inhibition of AC.
2. N-Glycosylation with complex carbohydrates is
important for cell-surface expression of the CaR
CaR proteins isolated from CaR-transfected human embryonic kidney (HEK293) cells have expression patterns
similar to those of CaR proteins isolated from parathyroid
cells [30]. The detection of the CaR isolated from these cells
using the antibody to the receptor showed three CaR-specific
immunoreactive bands between 120 and 200 kDa and additional bands of higher molecular mass (∼350 kDa). The
minor species at 120 kDa corresponds to the nonglycosylated form of the receptor; the two major species at 140 and
160 kDa are N-glycosylated with high mannose and complex carbohydrates, respectively [30,31]. Only a very small
fraction of the mature receptor is expressed on the cell surface [32]. Site-directed mutagenesis revealed that 8 of 11
predicted N-glycosylation sites are efficiently glycosylated
and that disruption of four to five of these sites reduced
cell-surface expression by 50–90% [31]. Glycosylation of
at least three sites is critical for cell-surface expression but
does not appear to be critical for signal transduction, as assessed by high Ca2+
o -induced increases in the accumulation
of inositol phosphates (IPs) [31].
3. The CaR dimerizes via both covalent and
non-covalent interactions
As mentioned above, Western analysis using reducing
agents revealed variable amounts of immunoreactivity in
199
bands above 200 kDa, in addition to the monomeric forms
of the CaR [30]. These are not simply artifacts produced by
aggregation of the receptor during its isolation from cells
nor subsequent PAGE in a SDS-containing running buffer.
Using three different approaches, we demonstrated that the
CaR resides on the cell surface of transfected HEK293 cells,
mostly in a dimeric form [32]. Moreover, no monomeric
form is detectable either on the cell surface or inside the
cells. Dimeric forms of the CaR have also been found in
detergent extracts prepared from the inner medulla of the rat
kidney [33]. These CaR dimers are disulfide-linked, as the
inclusion of reducing agents, such as dithiothreitol, converts
the dimer to the monomer on the SDS–PAGE gel [32].
As mentioned above, the CaR shares with the mGluRs the
same relative positions of 20 cysteines. Biochemical characterization of mutant receptors with substitutions of serines
for various cysteines revealed that Cys-129 and Cys-131
mediate the intermolecular disulfide linkages [22,34]. Substitutions of both cysteines are required for disruption of the
covalent intermolecular interaction. Further study showed
that the CaR dimerizes even in the absence of covalent
disulfide linkage [34]. Therefore, other dimerization motifs in addition to the formation of intermolecular disulfide
bonds are present in the CaR.
Our molecular modeling of the ECD using crystal structures of mGluR1a as templates suggested that the conserved
leucines at the dimer interface of the mGluR1a could be important for hydrophobic interactions of these two receptors
(Fig. 2). Our unpublished studies have shown that Leu-112
and Leu-156 at the putative dimer interface were essential
for the dimerization of the CaR through noncovalent hydrophobic interactions and functional reconstitution. However, the additional hydrophobic interprotomer interaction
sites are also present in the last six transmembrane spans,
even though they are not as strong as the site in the ECD.
One of these sites may be located in its fifth transmembrane
span (TM5), through a consensus sequence mediating the
dimerization of the ␤2 -adrenergic receptor [35].
4. Dimerization of the CaR is functionally important
Even though Cys-129 and Cys-131 are not essential for
dimerization of the CaR, the CaR with these cysteines replaced by serines is substantially more sensitive to Ca2+
o
than is the wild-type receptor, suggesting that these intermolecular disulfide bonds could participate in constraining
the receptor in its inactive conformations [22,34].
Furthermore, we demonstrated that heterodimerization of
mutant CaRs can lead to reconstitution of Ca2+
o -inducible
intracellular signaling and that intermolecular interactions
within the heterodimeric receptor are functionally important for receptor-mediated signaling [34,36]. For instance,
coexpression of two CaRs carrying mutations within the
ECD of one and within the C-tail of the other resulted in
substantial recovery of intracellular signaling through the
200
M. Bai / Cell Calcium 35 (2004) 197–207
Fig. 2. Molecular modeling of the dimeric ECD of the CaR. Using the free form I of the dimeric ECD of mGluR1 as a template (1EWT), we generated
the model of the CaR ECD by SWISS-MODEL, a fully automated protein structure homology-modeling server. The ECD is predicted to consist of two
lobes, LB1 and LB2. In LB1s, the cyteines at 129 and 131 forming interprotomer disulfide linkages are shown in yellow spheres and the leucines at 112
and 156 at the non-covalent dimer interface are shown in cyan spheres. The serine that is important for the potentiating effect of an allosteric amino acid
modulator, phenylalanine, is shown in gray spheres at the interface of LB1 and LB2. The C-terminal residue of the ECD is shown in purple spheres.
CaR-dependent PLC pathway, even though these individual mutant CaRs were completely inactive or severely attenuated in their abilities to activate intracellular signaling
when transfected alone. Co-immunoprecipitation of cotransfected receptors indicated that the heterodimer is the principal form of the receptor contributing to the reconstituted
Ca2+
o -sensing and signal transduction in these cotransfected
cells. Neither Cys-129 nor Cys-131 is required for functional
reconstitution or dimerization.
Our studies [36] suggest that the CaR may consist of
at least two separable functional domains, one comprising
the ECD and the other including the i3 and C-tail of the
CaR. The possession of at least one complete set of normal functional domains is necessary for reconstitution of
receptor-dependent signaling in cells cotransfected with inactive mutant receptors. For instance, coexpression of two
mutant CaRs, each with a different mutation in its ECD (e.g.
G143E and E297K), or of a CaR with a mutation in the i3
and another with a truncated C-tail did not lead to a substantial recovery of function. In contrast, coexpression of a
CaR containing a mutation in its ECD with another CaR
bearing mutations in the i3 and/or C-tail usually resulted
in an apparent gain of function. In other words, for heterodimers containing either the truncated mutant or the i3
mutant, intracellular signaling must occur through the nor-
mal i3/C-tail domain of the coexpressed CaR containing a
mutation in its ECD. One possible mechanism for this type
of functional complementation is domain swapping, as proposed by Gouldson and Reynolds [37].
However, a truncation mutant possessing an intact
amino-terminal extracellular and first transmembrane domain is not sufficient to complement ECD mutants [38],
even though the truncated receptor forms heterodimers with
the ECD mutants on the cell surface. Furthermore, the mutation in the i3 of one monomeric receptor significantly reduces the maximal response of the resultant heterodimer in
comparison to the heterodimers with two wild-type TMDs
but lacking one cytoplasmic tail. This result suggests that
receptor–receptor interaction between TMDs or receptor–G
protein interaction through both TMDs within the context
of a heterodimer is important for CaR signaling.
In addition, we found that a normal domain of the CaR
can be negatively affected by the presence of an abnormal
domain in the heterodimeric receptor [36]. For instance,
abnormal ECDs affect the apparent ligand affinities and
cooperativities of their respective heterodimeric receptors. The apparent affinities of various heterodimers are
quite different, even though the same C-tail-truncated receptor was used for cotransfection with various mutant
CaRs harboring mutations in their ECDs. For instance,
M. Bai / Cell Calcium 35 (2004) 197–207
the EC50 [Ca2+
o ] of G143E/A877Stop is about two-fold
higher than that of R185Q/A877Stop. In addition, the apparent cooperativities (as reflected in the Hill coefficients)
of putative heterodimers, such as G143E/A877Stop and
E297K/A877Stop, were not only much less than that of the
wild-type CaR but also much less than that of another heterodimer, R185Q/A877Stop. Therefore, the heterodimeric
ECDs likely act as a unit (i.e. as a disulfide-linked dimeric
ECD), producing EC50 values determined in part by the
particular ECD mutant used in the cotransfection.
It appears that the functional consequences of the intermolecular interactions between CaR monomers documented
[36] in the heterodimeric complex are considerably more
extensive than those observed with other GPCRs, such as
the V2 vasopressin [39] and muscarinic receptors [40,41].
For example, several inactive vasopressin receptors carrying
single mutations in the i3 or TM6 could be rescued by cotransfection with a C-terminal V2 receptor peptide spanning
the sequence within which the various mutations occurred.
In most cases, the sensitivities of the reconstituted receptors
to the agonist arginine vasopressin were similar to that of
the wild-type receptor. Therefore, these mutated segments
appear to have little impact on the ligand-binding properties
of the reconstituted receptors. This difference in functional
consequences of receptor–receptor interactions observed
with heterodimeric mutant CaRs and heterodimeric mutant
V2 vasopressin receptors may originate from the fundamental differences in their structural features, in particular, the
sizes of their ECDs, and the domains involved in agonist
binding. As discussed earlier, the ECD of the CaR is much
larger (consisting of more than 600 amino acid residues)
[1,3] than the ECD of the V2 vasopressin receptor (containing about 40 amino acid residues) [39]. The Ca2+
o -binding
sites reside within the large ECD of the CaR, as discussed
below [42–44], whereas agonist binding to the V2 vasopressin receptor most probably involves residues near the
extracellular ends of several TMs and within extracellular
loops [45].
Since abnormal functional domains within a heterodimeric CaR complex adversely affect the function of normal
domains via intermolecular interactions, heterodimerization likely contributes to a more severe expression of disease phenotype in some clinical conditions involving the
CaR. For instance, a form of neonatal severe hyperparathyroidism (NSHPT) could be resulted from heterozygous
mutations [30,46] in the CaR. Furthermore, heterodimerization may play a role in Ca2+
o -induced differentiation of
keratinocytes [47]. It has been shown that an alternatively
spliced form of the CaR lacking exon 5 that encodes a
portion of the extracellular domain, is expressed along with
the full-length receptor in the keratinocytes. Moreover,
the ratio of the splice variant to the full-length form increases during keratinocyte differentiation. It is possible that
the inactive, alternatively spliced receptor interferes with
the function of the full-length CaR through formation of
heterodimers.
201
5. The CaR interacts with its principal physiological
agonist, Cao2+ , through its ECD
The large size of the ECD of the human homologue of
the CaR (612 amino acid residues) is a unique feature found
among the subfamily C of GPCRs. The first direct evidence
that Ca2+
o binds to the ECD of the CaR was provided by
studies using chimeric receptors in which the ECD of either the CaR or a mGluR was fused to the TMD/C-tail of
the other receptor [42,43]. The chimeric receptors are expressed in Xenopus laevis oocytes. A chimeric receptor containing the ECD of the CaR and the TMD/C-tail of mGluR1a
was activated by high Ca2+
o but not by mGluR agonists.
Conversely, a chimeric receptor comprising the ECD of the
mGluR and the TMD/C-tail of the CaR was activated by glutamate but not by high Ca2+
o . In addition, polyamines, such
as neomycin, also were shown to interact with the CaR via
its ECD [43]. In contrast, trivalent ions, such as Gd3+
o , bind
not only to the ECD of the receptor but also to the TMD of
the receptor [43].
Brauner-Osborne et al. [44] used transfected HEK293
cells to confirm that Ca2+
o acts on the CaR by binding to its
ECD. They took a similar approach that used chimeric receptors and showed that a chimeric receptor comprising the
ECD of the CaR and the TMD/C-tail of the mGluR1a was
2+
2+
activated by Ca2+
o , Mgo , and Ba o with EC50 values very
similar to that of the wild-type CaR. Therefore, divalent ions
2+
2+
other than Ca2+
o , such as Mgo and Ba o , also activate to
the ECD of the receptor. In addition, Brauner-Osborne et al.
found that the serine residues in positions homologous to
those in mGluR1a, which bind to glutamate, also are important for Ca2+
o -elicited responses via the CaR. Therefore,
they concluded that the binding of Ca2+
o by the CaR involves
these serines at positions 147 and 170.
There are presently no assays for the binding of Ca2+
o
to the CaR. Therefore, identification of the determinants
within the ECD, which are important for Ca2+
o -binding, has
relied on the measurement of high Ca2+
-evoked
increases
o
in Ca2+
or
PLC
activity
(e.g.
as
assessed
by
accumulation
i
of IPs). Clearly, interfering with the binding of Ca2+
o to a
specific amino acid residue is only one of many possible
ways to alter the function of the CaR. However, secondarily
perturbing agonist binding and/or subsequent steps in the
activation of intracellular signal transduction is a relatively
common mechanism for producing inactivating mutations
in the ECD, such as R62M, G143E, and R185Q [30,48].
Thus, direct structural studies (e.g. using X-ray crystallography) will be necessary to establish definitively whether
residues with functional importance, such as Ser-147,
Thr-138, Ser-170, and Glu-297 within the ECD of the CaR,
participate directly or indirectly in Ca2+
o binding.
Based on the Hill coefficient of 3 for CaR activation by
high Ca2+
o [30], there are probably at least three binding
sites for Ca2+
o within the ECD of the CaR. It is possible that
dimerization of the CaR also contributes to this apparent
positive cooperativity of this receptor in its binding of Ca2+
o .
202
M. Bai / Cell Calcium 35 (2004) 197–207
6. Calcimimetics and l-amino acids allosterically
modulate the CaR through distinct sites
The CaR can be activated allosterically by phenylalkylamine calcimimetics in the presence of Ca2+
in the
o
millimolar concentration range [49]. These effects were
stereoselective and the R enantiomers were 10–100-fold
more potent than the S enantiomers. Recently, the mutation of E837A in extracellular loop 3 (e3) was shown to
drastically reduce the sensitivity of the CaR to NPS R-568
even though the mutation did not alter Ca2+ sensitivity of
the CaR [50]. Mutation of any of three in the extracellular loop 2 (e2), Asp-758, Glu-759, and Glu-767, increased
the sensitivity of the CaR to activation by Ca2+ . These
studies strongly suggest that the key interaction site of the
calcimimetic is in the TMD and that the binding of the calcimimetics primarily reduce the charge repulsion between
negatively charged amino acid residues in e2.
The CaR can also be activated allosterically by l-amino
acids in the presence of Ca2+
o in the millimolar concentration range (0.1–10 mM) with a preference for aromatic and
small aliphatic l-amino acids [51]. Moreover, these amino
acids stereoselectively enhance the sensitivity of the CaR to
its agonists. It has been documented that these two homeostatic systems are intimately related. For instance, a reduction in protein intake below the normal level results in
secondary hyperparathyroidism in the context of normocalcemia [52], and high dietary protein intake induces elevated
urinary calcium excretion [53,54]. The CaR may serve as
a fundamental link between protein and Ca2+
o metabolism.
Our recent study has shown that Ser-170, corresponding to
Thr-188 in the ECD of mGluR1a important for the binding of glutamate to mGluRs, appears to be essential for
the modulating actions of phenylalanine on the CaR [55].
A single mutation, S170A, significantly blocked the potentiating effect of l-phenylalanine and, moreover, mutating
two other serines at 169 and 171 together with Ser-170
yielded a more complete block of the phenylalanine modulation than did the single mutation. These studies suggest
that the binding site of l-amino acids is in the ECD of
the CaR.
Recently, we demonstrated that many mutations had differential effects on the functional modulation of the CaR by
these two allosteric activators, supporting the idea that these
modulators act through distinct sites [55]. Ten millimolar l-phenylalanine and 1 ␮M NPS R-467—submaximal
doses of the two agents—each elicited similar modulation
of R185Q. There are different relative potencies for these
two modulators, however, with some receptors being more
responsive to l-phenylalanine and others being more responsive to NPS R-467. Responsiveness of the CaR to
Ca2+
o appears to be essential for the potentiating action of
l-phenylalanine, but not that of NPS R-467, on the receptor.
NPS R-467 reduces the Hill coefficients of the wild-type
as well as mutant receptors, suggesting that engagement of
all Ca2+ -binding sites is not required when the receptor is
activated by NPS R-467. In contrast, l-phenylalanine has
little effect on the Hill coefficients of mutant receptors.
Consistent with earlier findings that these two allosteric
modulators act through distinct sites of the CaR, they exert
a synergistic effect on mutant CaRs that are less active than
the wild-type receptor but responsive to both modulators.
7. Amino acid residues in i2 and i3 of the CaR are
important for receptor activation
Receptors belonging to the rhodopsin/␤-adrenergic family are characterized by the presence of a large i3. Amino
acid residues within these microdomains play a key role in
determining selective coupling to heteroterimeric G proteins
[56,57]. Unlike these GPCRs, the CaR and other members
of GPCRs in the family C have a much smaller i3 that is well
conserved among the members of the family, despite their
different coupling properties [10,27,28,58]. Conversely, i2
is little conserved among mGluRs and other members of the
family C. The analysis of chimeric receptors derived from
the Gi -coupled mGluR3 bearing different portions of the intracellular domains of the Gq -coupled mGluR1a has shown
that i2 is necessary, but not sufficient, for the specific activation of PLC and that both i3 and the C-tail of the receptor
also appear to be necessary for efficient coupling to this pathway [10,27,28]. Substitutions within i2 of mGluR1a alter
selectivity of the receptor for G proteins, while substitutions
within i3 of mGluR1a inactivate both PLC and cAMP pathways [58]. For instance, within i2, Thr-695, Lys-697, and
Ser-702 were found to be involved selectively in the interaction with Gq class ␣ subunits, whereas mutation of Pro-698
and the deletion of both Cys-694 and Thr-695 affected only
Gs coupling. Furthermore, the mutation K690A profoundly
altered mGluR1a-signaling properties and conferred to the
receptor the ability to couple to the inhibitory cAMP pathway. Two residues within i3, Arg-775 and Phe-781, are
important for mGluR1-mediated activation of both PLC
and AC.
The corresponding residues in i2 (except Thr-695), which
are critical for selectivity of the mGluR1a, are not conserved
in the CaR. In addition, the region containing the corresponding residues (positions 712–720) in the i2 of the CaR
is not important for CaR-mediated PLC activation [59]. Instead, two residues at the N-terminus of the i2 (Phe-706 and,
to a lesser extent, Leu-703) are important for the activation
of PLC. Substitution of Tyr or His for Phe-706 (but not substitution of Ala, Leu, Val, Glu, or Trp) partially restored the
ability of high Ca2+
o to activate PLC. Among 8 residues examined in the i3 of 13 residues, Leu-797 and Phe-801 were
shown to be most critical to the activation of PLC, while
Glu-803 was shown to be essential for efficient cell-surface
expression of the CaR. However, it is not clear whether the
residues critical to PLC activation are also important for receptor coupling to other signaling pathways, such as inhibition of AC. Sequence alignment of the CaR and mGluRs
M. Bai / Cell Calcium 35 (2004) 197–207
suggests that Phe and Glu at positions 706 and 707 in the i2
of the CaR could be important for coupling of the CaR to Gi .
8. The intact C-tail of the CaR is important for normal
function of the receptor
Like many other members of the family C GPCRs, the
CaR possesses a large C-tail that contains 216 amino acid
residues, beginning with the lysine at amino acid position
863 [3]. Even though the overall amino acid sequence of the
CaR is extraordinarily conserved among human, bovine, rat,
rabbit, mouse, and chicken, the amino acid residues in the
signal peptide and those in the C-tail beyond position 946
are quite diverse. Therefore, amino acid residues between
positions 863 and 946 in the C-tail are likely to be important
for the normal function of the receptor. Indeed, truncations
at various positions within this region have been reported
to cause either familial hypocalciuric hypercalcemia (FHH)
or autosomal dominant hypocalcemia (ADH) due to loss or
gain of function of the receptor [48,60].
It has been reported that mutant CaRs truncated at positions 863, 865, 874, or 877 showed no response to Ca2+
o ,
despite exhibiting near-normal or higher-than-normal expression levels [32,48,61]. In contrast, the truncation at
position 888 inactivates the PLC pathway, leading to intracellular store release but not CaR-mediated Ca2+ influx
[62]. On the contrary, the CaRs with a stop codon at position 892 or with an in-frame deletion of amino acid residues
894–1075, respectively, are significantly more active than
the wild-type receptor in eliciting the release of calcium
from intracellular stores [32,60]; both receptors have increased levels of cell-surface expression when compared
with the wild-type CaR. The in-frame deletion is associated
with ADH [60]. Thus, this experiment in nature provides
strong additional support for the hypothesis that there are
structural elements within regions of the C-tail of the receptor distal to residue 891 that reduce its cell-surface
expression and biological activity in some manner.
It has been suggested that the CaR C-tail not only is
important for the characteristic positive cooperativity of
the receptor but also influences the rate at which the CaR
desensitizes after repeated exposures to Ca2+
o [63]. However, in this study, the authors showed that the receptor
with a truncation at position 868 is active but less sensitive
to Ca2+
o , contradicting the findings by two other groups
that truncations at 863, 865, 874, and 877 cause complete
inactivation of the receptor [32,48,61].
9. The CaR can be negatively regulated by PKC
phosphorylation
It has been shown that high Ca2+
o -evoked suppression
of PTH secretion and the concurrent increases in IPs and
Ca2+
in parathyroid cells can be regulated negatively by
i
203
activation of PKC [64–72]. It has been suggested that such
negative regulation is involved in the reduced responsiveness
of adenomatous or hyperplastic parathyroid glands to Ca2+
o ,
owing to an increase in membrane-associated PKC [73,74],
although there is also reduced expression of the CaR in these
pathologic glands [75,76]. Likewise, PKC may contribute to
age-related changes in the regulation of PTH secretion by
Ca2+
o in rats [77].
Similar effects of PKC on CaR-mediated increases in
Ca2+
i have been demonstrated in CaR-transfected HEK293
cells [78]. Moreover, we demonstrated that several PKC activators exert inhibitory effects on CaR-mediated increases
2+ from intracellular stores but
in Ca2+
i due to release of Ca
do not exert these effects on the increases resulting from
Ca2+ influx. Consistent with the mediation of this effect
by activation of PKC, the inhibitory effect of PKC activators on Ca2+ release can be blocked effectively by a PKC
inhibitor. Moreover, inhibition of PKC converted oscillatory Ca2+
i responses to transient, non-oscillatory responses,
but without decreasing the overall response to increase in
Ca2+
o [79]. In another study, inhibition of PKC resulted in
a five-fold enhancement of IP3 signaling [80]. Even though
blocking protein kinase A (PKA) alone had almost no effect
on CaR-mediated IP3 signaling, the presence of a PKA inhibitor significantly potentiates the effect of a PKC inhibitor
on IP3 signaling.
Of five putative PKC phosphorylation sites in the intracellular domains of the CaR, one each within i2 and i3 and
three within the C-tail, Thr-888 in the C-tail is the major
PKC site mediating the inhibitory effect of PKC activators on
Ca2+ mobilization [78]. The effect of PKC activation could
be maximally blocked by mutating three PKC sites—T888,
S895, and S915—or all five PKC sites. The residual negative effect (30%) of PMA that remains after alteration of all
of the PKC sites of the CaR suggests that PKC can phosphorylate other sites on the CaR and/or regulate other components within this pathway (e.g. G proteins and/or PLC␤s). In
vitro phosphorylation showed that Thr-888 could be phos2+
phorylated readily by PKC and Ca2+
oso -stimulated Ca i
cillations produced by the CaR require negative feedback by
PKC phosphorylation of Thr-888 [79].
Our recent studies have shown that the truncation at 888
mimics the effects of PKC activators on CaR-mediated IP
and Ca2+
responses [62]. This finding suggests that PKC
i
phosphorylation of the CaR at 888 prevents G protein subtypes from interacting with the region of the receptor critical for releasing Ca2+ stores that is missing in the truncated
receptor T888Stop.
10. Identification and characterization of naturally
occurring CaR mutations provides insights into the
structure–function relationships of the receptor
The identification of inactivating and activating mutations in the CaR has given us unequivocal evidence for the
204
M. Bai / Cell Calcium 35 (2004) 197–207
physiological relevance and structural information on the
function of the receptor. So far, over 100 naturally occurring mutations that have a functional impact on the CaR
have been identified [46,81–95]. More than one half of
them are located in the ECD; the rest are located through
the regions of the TMD (except TM 1) and C-tail. Missense
mutations (substitution of a different amino acid residue for
the one normally coded for) account for most of the naturally occurring mutations. Several other types of mutations
with functional consequences have also been identified in
the CaR. These include nonsense mutations (e.g. point mutations changing an amino acid residue to a stop codon),
thereby producing a truncated protein, and frameshift mutations (loss or gain of one or more nucleotides) with a resultant change in the downstream coding sequence, usually
producing a premature stop codon.
We and others have characterized a number of mutant
CaRs bearing inactivating or activating mutations in transiently transfected HEK293 cells [30,46,48,87,91,94–96].
Consistently, inactivating mutations are associated with FHH
and occasionally with NSHPT and activating mutations are
associated with ADH. These studies also revealed several
mechanisms through which CaR mutations alter the function
of the receptor and support findings derived from studies directed at understanding the structure–function relationship
of the CaR, as exemplified below.
Consistent with the notion that the ECD of the receptor
is the site for Ca2+
o -binding, most naturally occurring mutations in the ECD affect only the apparent affinity of the receptor for Ca2+
o . It is possible that these mutations alter the
amino acid residues in direct contact with Ca2+
o or the mi-binding
sites.
We also
croenvironment of one or more Ca2+
o
observed that some mutations in the ECD could cause more
gross alteration of the receptor structure, reflected by substantial decreases in receptor expression on the cell surface.
Characterization of the mutant receptor that has a
single-base deletion and transversion in codon 747 revealed
that some TMs may be important for the dimerization of
the receptor. The resultant truncated protein, which lacks
the last four TMs, does not form the high molecular species
corresponding to oligomeric CaRs [96] that can be detected
in cells transfected with the wild-type and tail-truncated
mutant receptors under the same experimental conditions.
This result suggests that some dimerization motifs might
be present in the missing portion of the receptor, perhaps
one in TM5, which has a consensus sequence important for
dimerization of the ␤-adrenergic receptor [35].
It has previously been found that inactivating mutations
in heterozygous states cause non-uniform elevations in the
serum calcium concentration ranging from 0.3 to 0.76 mM
higher than the normal level of 2.40 ± 0.02 mM (n = 43)
[30], despite the presence in all heterozygotes of one normal
allele of the CaR. Consistently, cotransfected mutant receptors have different negative effects on the wild-type receptor.
For instance, the elevation in serum calcium concentration
in vivo is less (≤0.42 mM) for mutant receptors, such as
R66C, G143E and E297K, with relatively low levels of the
cell-surface form of the CaR protein in vitro [30,32]. Among
them, R66C, which shows the least surface expression, has
little, if any, effect on the wild-type receptor [30]. In this
case, the mild elevation of serum calcium may result simply
from the decrease in the dose of the normal CaR gene. Conversely, R185Q and R795W, both of which show normal
receptor expression, result in the highest elevated serum
calcium concentrations in affected members of families harboring these mutations [46]. These studies suggest that the
presence of a significant amount of relatively inactive mutant receptors on the cell surface may reduce significantly
the number of G proteins available for the wild-type receptor
prior to activation. Alternatively, these mutant receptors may
significantly affect the function of the coexpressed wildtype receptor through direct interaction in a heterodimeric
complex.
11. Conclusion
Biochemical and functional characterization of the cloned
CaR and various mutant CaRs, including those carrying
naturally occurring mutations, provides insights into the
structure–function relationship of this receptor. These studies also delineate how this subfamily of GPCRs might
function, including the domains involved in agonist binding
and consequences of intermolecular interactions within a
dimeric GPCR. However, much remains to be learned about
the structural elements directly involved in agonist binding,
G protein-coupling, and dimerization.
For instance, the residues involved in Ca2+
o binding remains unclear, although they could be noncharged (i.e. serine and threonine) and negatively charged (i.e. glutamate and
aspartate) amino acid residues within the ECD. The acidic
regions have been found in other low-affinity Ca2+ -binding
proteins, including calreticulin and calsequestrin [1]. There
is also a short, highly acidic region in the second extracellular loop of the CaR (four of five consecutive residues
are either glutamate or aspartate) that might be involved in
mediating interactions of the agonist-bound ECD with the
transmembrane segments of the receptor that are presumably involved in receptor-effector coupling. The ECD of the
receptor does not possess high-affinity Ca2+
o -binding motifs, such as EF hands, which would not function properly
in Ca2+
o -sensing in any event because they would be persistently occupied at ambient, millimolar levels of Ca2+
o .
Further work is needed to understand how the CaR activates its respective G proteins. It has been shown that the
phenylalanine at residue 706 within the i2 appears critical
for activation of PLC, presumably via Gq/11 , as do several
residues within the i3 [59]. Additional studies are needed to
identify the residues within i2 as well as those in the C-tail
that are important for differential activation of various signaling pathways, Gq/11 and Gi , and to understand how the
dimeric receptor activates G proteins.
M. Bai / Cell Calcium 35 (2004) 197–207
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