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From the
Parathyroid cells express a cell surface receptor, coupled to
the mobilization of intracellular Ca
The secretion of parathyroid hormone (PTH)
Alterations in parathyroid cell
CaR function have been implicated in at least two different human
disease states. In primary hyperparathyroidism (1
A bovine parathyroid
cell CaR cDNA was recently isolated by expression cloning in
Xenopus oocytes
(18) . This clone encoded a novel
receptor protein with limited sequence homology to the family of G
protein-coupled receptors (GPRs). We have used this bovine clone to
demonstrate the presence of CaR-related transcripts in a human
adenomatous parathyroid gland removed from an individual diagnosed with
primary hyperparathyroidism. Full-length CaR cDNA clones were isolated
from a cDNA library prepared from this adenoma, the sequence of the CaR
obtained from the adenomatous gland was compared to the sequence of the
normal human CaR gene, and the function of the encoded CaR has been
examined by expression in Xenopus oocytes. The human
parathyroid adenoma CaR cDNA clones encode a CaR, coupled to the
mobilization of intracellular Ca
DNA sequencing was performed on double-stranded plasmid
DNA using the chain termination method
(24) with Sequenase
enzyme (United States Biochemical Corp., Cleveland, OH). Much of the
sequencing was done using ordered deletions across the cDNA
inserts
(25) , with remaining regions sequenced using specific
oligonucleotide primers. All regions were sequenced on both strands.
Nucleotide sequences were analyzed with the MacVector (IBI-Kodak, Inc.)
package of software, including the most recent release (June, 1994) of
the NCBI sequence data bases.
A second
cDNA clone with an insert of
The 5.2 and 4.0 clones
also differ at the 3` ends of the cDNA inserts, again in non-coding
sequence. The 4.0 clone terminates in a run of A residues, but this is
not preceded by a typical poly(A) addition sequence. The 5.2 clone is
identical in sequence with the 4.0 clone at its 3` end, but extends
beyond the 4.0 clone 3` terminus another 1138 bp. This extension
corresponds to the long 3`-UTR on the bovine CaR cDNA clone. The 5.2
cDNA terminates in a poly(A) tail at its 3` end, and this is preceded
by a polyadenylation signal. The 5.2 sequence shows a cluster of A
residues at the point where the 4.0 clone terminates. The cDNA
synthesis was primed with oligo(dT), and it is possible that the 4.0
poly(A) tail represents priming at this internal cluster of A residues
rather than at a true poly(A) tail on a mRNA; the absence of a
polyadenylation signal preceding the end of the 4.0 clone would support
this conclusion. Of 16 clones analyzed, five had 3` ends corresponding
to the 5.2 clone, 10 had 3` ends corresponding to the 4.0 clone, and a
single clone had a poly(A) tail beginning at 4001 bp on the 5.2
sequence (Fig. 2). There is a close approximation of the
consensus polyadenylation sequence preceding the poly(A) tail on this
latter clone, and it would appear that it is a polyadenylation site
that is infrequently utilized.
Within the coding sequence the two
human clones show three differences. Between amino acids 536 and 537 on
the 4.0 sequence, the 5.2 sequence has an insertion of 10 amino acids
(30 bp) (Fig. 1). In comparing the human to the bovine CaR in
this region, the bovine CaR protein is an exact match to the 4.0
sequence. None of the other cDNA clones analyzed have this 30 bp
insertion, and the significance of this cDNA variant remains unknown.
Comparison of the 4.0 and 5.2 sequences show two single base pair
differences, with each resulting in an amino acid difference. At amino
acid 926, the 4.0 cDNA encodes Gln and the 5.2 cDNA encodes Arg; the
bovine cDNA encodes Gln at this position. At amino acid 990, the 4.0
cDNA encodes Gly and the 5.2 cDNA encodes Arg; the bovine cDNA encodes
Arg at this position. This latter variant has been shown to be a benign
polymorphism present in the human population.
We have cloned human parathyroid CaR cDNAs from an
adenomatous gland. The encoded CaR protein displays several of the
features predicted by functional studies of this novel extracellular
cation receptor. When expressed in oocytes, the CaR is activated by
known CaR agonists with the same rank order of potency seen in
parathyroid cells and is coupled to the mobilization of intracellular
Ca
The CaR
sequence predicts a cluster of seven hydrophobic segments, a feature
common to all known GPRs. Based on this feature, the structure of the
CaR is proposed to consist of an unusually large
NH
NH
Activation of protein kinase C
decreases the sensitivity of parathyroid cells to regulation by
extracellular Ca
The
human CaR cDNA clones were isolated from an adenomatous parathyroid
gland removed from a patient with 1° HPT. In 1° HPT,
parathyroid cells show an elevation in the Ca
One of the differences between the two cDNA clones
sequenced, a 10 amino acid insertion in the NH
It is possible that the set-point increase characteristic of
1° HPT is not due to an alteration of the primary sequence of the
receptor, but reflects a change in the amount of receptor protein
relative to the increased mass of the hypercellular adenoma. The severe
impairment of parathyroid function in neonatal severe
hyperparathyroidism, the homozygous form of FHH, suggests that the
levels of functional CaR protein are an important determinant of proper
extracellular Ca
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank
Volume 270,
Number 21,
Issue of May 26, pp. 12919-12925, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
, that is
activated by increases in the concentration of extracellular
Ca
and by a variety of other cations. This
``Ca
receptor'' (CaR) serves as the primary
physiological regulator of parathyroid hormone secretion. Alterations
in the CaR have been proposed to underlie the increases in
Ca
set-point seen in primary hyperparathyroidism due
to parathyroid adenoma. We have isolated human CaR cDNAs from an
adenomatous parathyroid gland. The cloned receptor, expressed in
Xenopus oocytes, responds to extracellular application of
physiologically relevant concentrations of Ca
and
other CaR agonists. The rank order of potency of CaR agonists displayed
by the native receptor (Gd
> neomycin B >
Ca
> Mg
) is maintained by the
expressed receptor. The nucleotide sequence of the human CaR cDNA
predicts a protein of 1078 amino acids with high sequence similarity to
a bovine CaR, and displays seven putative membrane-spanning regions
common to G protein-coupled receptors. The deduced protein sequence
shows potential sites for N-linked glycosylation and
phosphorylation by protein kinase C and has a low level of sequence
similarity to the metabotropic glutamate receptors. Comparison of the
cDNA sequence to that of the normal human CaR gene showed no alteration
in the coding region sequence of the CaR in this particular instance of
parathyroid adenoma. Human cDNA clones with differing 5`-untranslated
regions were isolated, suggesting alternative splicing of the
parathyroid CaR mRNA. A rare variant cDNA clone representing a 10 amino
acid insertion into the extracellular domain was also isolated.
Northern blot analysis of normal and adenomatous parathyroid gland mRNA
identified a predominant transcript of
5.4 kilobases, and less
abundant transcripts of
10, 4.8 and 4.2 kilobases in RNA from the
adenoma. While there is no evidence for alteration of the primary amino
acid sequence of the CaR in this adenoma, modulation of CaR
biosynthesis through alternative RNA processing may play a role in
set-point alterations.
()
by the parathyroid gland is regulated by the circulating
level of Ca, with elevation of the serum
Ca
concentration leading to an inhibition of PTH
secretion. Functional studies of parathyroid cells have suggested the
presence of a cell surface, G protein-coupled ``Ca
receptor'' (CaR) capable of detecting physiological changes
in the concentration of extracellular Ca
and
modulating PTH secretion
(1, 2) . Increases in the
concentration of extracellular Ca
lead to increased
formation of inositol 1,4,5-trisphosphate
(3) and diacylglycerol
(4) and a rapid elevation in levels of cytosolic Ca
which results from both the mobilization of intracellular
Ca
and influx of extracellular
Ca
(1, 2, 5) . In addition,
parathyroid cells exposed to high extracellular Ca
show an inhibition of adenylate cyclase activity, and this
inhibition can be blocked by pretreatment with pertussis
toxin
(6) . These observations suggest that either a single CaR
is capable of effecting both mobilization of intracellular
Ca
and inhibition of adenylate cyclase, or that
isoforms of the CaR, coupled to differing G protein-mediated signaling
pathways, exist. Although Ca
is the endogenous ligand
for the CaR, in vitro a variety of di-, tri-, and polyvalent
cations (neomycin, spermine, protamine, La
,
Gd
, Ba
, Sr
,
Mg
) have been shown to activate this
receptor
(7, 8) . Studies on cultured bovine parathyroid
cells suggest that the CaR is a cell surface glycoprotein
(9) and that protein kinase C activation inhibits CaR
signaling
(10, 11) .
HPT),
parathyroid cells show an elevated Ca ``set-point'' for PTH suppression
(12) , possibly
reflecting an alteration in CaR function. Recently, mutations in the
CaR gene were identified in kindreds with familial hypocalciuric
hypercalcemia (FHH)
(13) , an inherited syndrome in which
affected individuals display an elevated Ca
set-point
(14) . A CaR-specific agonist, NPS R-568, has
been shown to reduce serum PTH levels in vivo(15) ,
further demonstrating the role of the CaR in modulating PTH secretion.
Altogether, there is compelling evidence that the parathyroid CaR
serves a central role in maintaining bodily Ca
homeostasis. Cell types in addition to the parathyroid cell are
responsive to changes in extracellular Ca
, suggesting
that the same or a related CaR may modulate the function of a variety
of cells. Calcitonin secretion by thyroid C cells
(16) and renin
secretion by juxtaglomerular cells
(17) are well documented
examples in which hormone secretion is modulated by extracellular
Ca
. To facilitate the study of human CaR function in
normal and disease states and aid in the identification of novel cell
types expressing the CaR, we undertook the isolation of human CaR cDNA
clones from an adenomatous parathyroid gland.
, with a
pharmacological profile similar to that seen with normal parathyroid
cells.
RNA Preparation and Northern Blot Analysis
Human
tissue samples were frozen on dry ice and stored at -90° C.
Total RNA was prepared by the guanidinium thiocyanate/acid phenol
extraction procedure
(19) . Poly(A) RNA was
obtained by oligo-(dT) cellulose affinity chromatography. RNA was size
fractionated on 1.2% agarose/formaldehyde gels and transferred to nylon
membranes. The 5.3-kbp cDNA insert of a bovine CaR cDNA clone
(pBoPCaR1) was excised from the pSport vector by SalI +
NotI digestion, gel-purified, and labeled by random-primed
synthesis with [
P]dCTP
(20) to a specific
activity of >10
counts/min/µg. Nylon membranes were
hybridized in a solution consisting of 5 SSC (1
SSC
= 0.15 M NaCl, 0.015 M sodium citrate, pH
7.2), 400 mM NaPO
, 5% SDS, 1 mg/ml bovine serum
albumin, 100 µg/ml sonicated salmon testis DNA, and 35% formamide
at 42 °C, with BoPCaR1 insert probe at a concentration of 2
10
counts/min/ml. Following hybridization, blots were
washed in 2 SSC, 0.5% SDS at 50° (moderate stringency) or
0.1
SSC, 65 °C (high stringency).
cDNA Cloning and DNA Sequence Analysis
Human
adenomatous parathyroid mRNA was used in cDNA synthesis
(21) using Superscript (Life Technologies, Inc.) reverse
transcriptase. cDNA was synthesized with a NotI/dT primer at
the 3` end and a EcoRI adaptor at the 5` end. cDNA in the
3-6 kbp size range was gel-isolated and ligated into
EcoRI/NotI digested Zap II cloning vector.
In vitro packaged phage were plated (440,000 primary
recombinants) and screened by standard plaque hybridization
techniques
(22) , using full-length BoPCaR1 insert probe under
the hybridization and moderate stringency wash conditions listed for
Northern blot analysis. The same filters were stripped and hybridized
with a 780-bp EcoRI fragment probe from the 5` end of the
bovine cDNA, to identify human clones with long inserts. Selected
hybridizing plaques were taken through secondary and tertiary rounds of
purification, and isolated phage clones were used for in vivo rescue of cDNA inserts in the pBluescript SK
plasmid vector
(23) . Preliminary restriction mapping of
cDNA inserts indicated that two classes of inserts were represented,
sharing a common central region and differing at the ends. Two cDNA
clones, with inserts of
4 and
5 kbp, were selected for
sequencing.
RNA Transcription and Oocyte Expression
The
pBluescript vector bearing the CaR insert was linearized by
NotI digestion, and capped, sense-strand cRNA was synthesized
by T7 RNA polymerase transcription
(26) . In vitro transcribed RNA was concentrated by ethanol precipitation, and the
size and integrity of the RNA were assessed on denaturing agarose gels.
CaR cRNA was injected into Xenopus oocytes (typically 12
ng/oocyte), and oocytes were maintained for 3-5 days prior to
assay of CaR function. Two-electrode voltage clamp recording was used
to monitor the endogenous Ca-dependent Cl
current in response to extracellular application of CaR agonists.
Details of oocyte manipulations and electrophysiological procedures
were as described previously
(27) .
Northern Blot Analysis of Human Parathyroid
RNA
An adenomatous parathyroid gland was removed from a
39-year-old male diagnosed with primary hyperparathyroidism. Northern
blot analysis of poly(A) RNA prepared from the
adenoma, utilizing the bovine CaR cDNA as hybridization probe,
identified a prominent transcript of
5.4 kb and more faintly
hybridizing bands at
10, 4.8, and 4.2 kb (Fig. 1). All of
these hybridizing bands persisted after high stringency washes. Normal
parathyroid gland mRNA was prepared from tissue obtained at autopsy
from a 65-year-old female. Although the RNA prepared from this
post-mortem tissue was somewhat degraded, Northern blot analysis
identified a faint CaR transcript at 5.4 kb (Fig. 1), suggesting
that this is the primary size of the human CaR transcript in both
normal and adenomatous parathyroid glands. Xenopus oocytes
expressing the human adenoma mRNA responded to extracellular
application of CaR agonists with oscillatory inward Cl
currents, indicating that the adenoma RNA encoded a functional
CaR (data not shown).
Figure 1:
Northern blot analysis of human CaR
mRNA in the parathyroid gland. Poly(A) RNA from an
adenomatous gland (2 µg, lane A) or a normal gland (3
µg, lane B), hybridized with the bovine CaR cDNA. The blot
was hybridized and washed under moderate stringency conditions. The
sizes of the hybridizing transcripts are
indicated.
Cloning and Sequence of the Human CaR
The human
adenoma mRNA was used to construct a cDNA library enriched for inserts
in the 3-6 kbp size range. This library (440,000 clones) was
screened at moderate stringency using the full-length bovine CaR cDNA
as hybridization probe. Approximately 600 plaques (0.14%) in this
size-selected library were hybridization-positive. Eighteen clones that
hybridized with a probe derived from the 5` end of the bovine CaR clone
were plaque-purified and characterized by restriction mapping. Clones
could be separated into two classes, with inserts in one class of
4 kbp and in the other
5 kbp. Restriction mapping showed both
classes to share a similar central region, with differences at the 5`
and 3` ends of the inserts. Clones representing the
4 kbp insert
size (phPCaR-4.0) and the
5 kbp insert size (phPCaR-5.2) were
completely sequenced. The sequence of the phPCaR-4.0 clone has a large
open reading frame encoding a protein of 1078 amino acids
(Fig. 2), with 93% identity to the CaR protein predicted by the
bovine CaR cDNA. The sequence surrounding the initiation codon of this
open reading frame is a good match to the Kozak consensus sequence for
translational initiation
(28) , and the 5` sequence preceding
this start codon contains stop codons in all frames. Hydrophobicity
analysis of the predicted human CaR protein shows a cluster of seven
hydrophobic regions 20-25 amino acids in length, reminiscent of
the seven membrane-spanning regions displayed by all known GPRs. By
analogy with the general structure of the GPR superfamily, the human
CaR can be proposed to consist of a very large amino-terminal
extracellular domain (amino acids 1-612), a central membrane
spanning region (amino acids 613-862), and a relatively long
carboxyl-terminal intracellular domain (amino acids 863-1078). The
extracellular domain contains 11 potential sites of N-linked
glycosylation, 10 of which are conserved in the bovine CaR. The bovine
CaR contains highly acidic regions in the NH
-terminal
extracellular domain (amino acids 216-251 and 557-611) and
in the second extracellular loop. These clusters of acidic residues
have been proposed to serve as sites of low affinity interaction with
cationic ligands
(18) . All of these acidic amino acids are
conserved between the human and bovine CaR proteins. There are five
potential sites for protein kinase C phosphorylation located in the
intracellular loop regions and the COOH-terminal intracellular domain
of the human CaR. Four of these sites are present in the bovine CaR.
The human CaR has two potential sites for cAMP-dependent
phosphorylation (Ser-899 and Ser-900). Neither of these protein kinase
A phosphorylation sites is present in the bovine CaR.
Figure 2:
Nucleotide and deduced amino acid sequence
of the human parathyroid CaR cDNAs. The sequence of the phPCaR-4.0
clone is presented, along with the sequence of the alternative 5` end
and the additional 3` end sequence of the phPCaR-5.2 clone. The
putative membrane spanning regions (TM-1 to TM-7)
are underlined. The coding region differences in the
phPCaR-5.2 clone (10 amino acid insertion at amino acid 536, single
codon changes at amino acids 929 and 990) are shown. Polyadenylation
signals (AATAAA) are indicated and sites of polyadenylation marked
(). Potential sites of N-linked glycosylation (
),
protein kinase C phosphorylation (), and protein kinase A
phosphorylation (
) are indicated, as are the acidic amino
acids (∪) and the cysteine residues that are conserved with the
mGluRs (C).
In general,
there is a high level of sequence conservation between the bovine and
human CaR proteins (Fig. 3). The only region displaying extensive
sequence divergence is the COOH-terminal end of the intracellular
domain. Of the 74 amino acid differences found between the two species,
43 are located in the region from amino acids 920 to 1078. The few
other differences are distributed throughout the remainder of the
protein, and many are conservative amino acid substitutions.
Figure 3:
Optimized amino acid sequence alignment of
human and bovine CaRs with rat mGluR-1. The seven putative
transmembrane regions are underlined. Residues showing
identity in the three proteins are enclosed in
boxes.
The CaR
shows limited, but significant homology to the metabotropic glutamate
receptors (mGluRs). An alignment of human and bovine parathyroid CaRs
to the rat mGluR1 protein is presented (Fig. 3). The proposed
structure of the CaR, with large extracellular and intracellular
domains and relatively short intracellular loops, is very similar to
that proposed for the mGluRs, although overall the human CaR shows only
18-24% identity to the various rat mGluR proteins. Similarity
between the CaR and mGluR sequences is maintained throughout the
NH-terminal extracellular and membrane-spanning domains,
with little conservation seen in the COOH-terminal intracellular
domain. A hydrophobic region in the extracellular domain (amino acids
137-160), as well as the first and third intracellular loops,
show particularly strong conservation (46, 50, and 75% identities,
respectively) between the human CaR and rat mGluR1. Within the
extracellular and membrane-spanning domains, the relative positions of
20 cysteine residues are maintained between the mGluRs and both the
human and bovine CaRs (Fig. 2, and 3), suggesting conservation of
structural conformation. Searches of a protein data base identified a
low level of sequence similarity between amino acids 150-430 of
the CaR and amino acids 110-370 of mouse and rat
N-methyl-D-aspartate receptor subunits (data not
shown). Similarity between the corresponding region of rat mGluR1 and
the 
-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate
receptor subunits has been noted previously
(29) .
5 kbp (phPCaR-5.2) was sequenced and
shown to have several differences from the phPCaR-4.0 clone. In the
5`-untranslated region (UTR), both clones show identical sequence from
the start codon to -242 bp, at which point the sequences completely
diverge (Fig. 1). The 4.0 sequence extends 132 bp 5` of this
point, while the 5.2 sequence extends another 198 bp. In comparing
these two human 5`-UTRs to the 5`-UTR of the bovine CaR cDNA, the human
and bovine clones show related sequence from the initiation codon to
the point at -242 bp after which the bovine clone shows no
relationship to either of the two human cDNA sequences. It appears that
alternative 5`-UTR exons have been spliced onto a common coding region
in the human CaR message. Of 16 full-length cDNA clones analyzed, nine
corresponded to the 4.0 clone 5` end and seven to the 5.2 clone 5` end.
None of these additional clones had 5` sequence extending significantly
further than that presented in Fig. 1.
()
The former single base pair variant is most likely an error
introduced during cDNA synthesis or cloning procedures. The two cDNAs
both give rise to functionally similar proteins following expression in
oocytes (see following data), and it appears that these amino acid
variations in the phPCaR-5.2 clone have no discernible functional
consequence.
Expression of the Human CaR
The functional
properties of exogenously expressed Ca mobilizing
receptors in Xenopus oocytes can be monitored through the
activity of an endogenous Ca
-dependent Cl
current. Oocytes injected with in vitro transcribed
human CaR cRNA respond to extracellular application of Ca
(3-10 mM) with large inward Cl
currents (Fig. 4A), while water-injected control
oocytes show no such response. Although in vitro transcribed
RNA from both clones phPCaR-4.0 and 5.2 gave a CaR response following
2-4 days incubation in the oocyte, both human clones routinely
gave smaller responses than an equivalent amount of CaR cRNA
transcribed from the bovine clone. Deleting the 5`-UTR up to a point 26
bp 5` of the initiation codon gave much stronger (5-10-fold
larger Cl
current amplitudes) functional responses
for the human CaR cRNA (data not shown). Human CaR clones phPCaR-4.0 or
5.2 expressed in oocytes respond to extracellular Ca
with half-maximal effective concentrations (EC
) of
7.05 ± 0.19 and 7.51 ± 0.12 mM, respectively
(Fig. 4B), somewhat elevated from the Ca
set point measured in vitro on human parathyroid cells.
The bovine CaR, expressed in oocytes under identical conditions, also
had a somewhat elevated EC
for Ca
of
4.5 mM. The EC
values of the CaR agonists
Gd
, neomycin, and Mg
determined for
the human CaR expressed in oocytes (
50 µM,
100
µM, and
12 mM, respectively) are similar to
those seen with bovine parathyroid cells
(7, 8) . All of
these CaR agonists elicit a response in the absence of extracellular
Ca
, indicating that the activation of the
Ca
-dependent Cl
current is due to
release of intracellular Ca
stores and not influx of
extracellular Ca
.
Figure 4:
Expression of human CaR cRNA in
Xenopus oocytes. A, Inward Cl currents induced by extracellular application of
Ca
, recorded in a oocyte injected 3 days prior with
10 ng of phPCaR-4.0 cRNA. The time span between application of 3.00 and
5.62 mM Ca
was 5 min. B,
concentration-response analysis of extracellular Ca
activation of phPCaR 4.0 (
) and 5.2 (
) clones
expressed in oocytes; n = 4 for each data
point.
. The recent findings that mutations in the CaR
gene result in FHH and neonatal severe hyperparathyroidism
(13) ,
and that pharmacological activation of this receptor elicits
hypocalcemia
(15) , provides further evidence that this cloned
CaR protein plays a central role in modulating parathyroid gland
function and bodily Ca
homeostasis.
-terminal extracellular domain, a membrane-spanning
domain with seven transmembrane regions, and a relatively large
COOH-terminal intracellular domain. Searches of protein and nucleic
acid sequence data bases show the CaR to share sequence similarity with
the rat mGluRs, but no other known GPRs. Together, the CaR and mGluRs
define a novel subfamily within the large group of GPRs. The overall
domain structure of the CaR and mGluRs, with large
NH-terminal extracellular regions and membrane-spanning
domains with relatively short extracellular and intracellular loops, is
nearly identical (the proteins can be aligned with very few gaps
introduced). Although the sequence similarity between the CaR and
mGluRs is low (18-24% identity overall), it extends throughout
the extracellular and membrane-spanning domains, with localized regions
of identity that are quite high. The putative extracellular domains of
both human and bovine CaR contain nine conserved sites for
N-linked glycosylation, supporting the extracellular
assignment of this region and consistent with previous findings
suggesting that the CaR is a glycoprotein
(9) . Both the human
and bovine CaR proteins contain 20 cysteine residues, located in the
NH-terminal extracellular domain and extracellular loops,
whose positions are conserved between the CaRs and mGluRs. This
observation, coupled with the extensive sequence homology throughout
the extracellular domains, suggests that the CaR and mGluRs may share a
common structural conformation in this region.
-terminal extracellular domain of mGluR1 has been
shown to function in ligand selectivity
(30) . The extracellular
domains of the mGluRs and ionotropic GluRs (iGluRs) share low level
sequence homology with each other, and with bacterial periplasmic amino
acid-binding proteins (PBPs). The conformation of the ligand-binding
sites of mGluRs and iGluRs have been modeled on the known structures of
the homologous bacterial PBPs, with the suggestion that the sequence
conservation reflects a relationship to a common ancestral amino
acid-binding structure
(31, 32) . In addition to the
homology to mGluRs, the extracellular domain of the CaR protein shows a
low level of sequence similarity to an extracellular domain of the
N-methyl-D-aspartate receptor (an iGluR). This
sequence conservation suggests that the CaR extracellular domain may
serve as a ligand interaction site, with structural homology to the
ligand-binding domains of the glutamate receptors and PBPs. The ligand
for the CaR is not an amino acid, but Ca, and
glutamate receptor agonists do not activate the CaR when expressed in
oocytes
(18) . The CaR functions in ambient Ca
concentrations in the low millimolar range, and consistent with
this the CaR protein does not contain any of the consensus sequences
proposed for high affinity Ca
binding
(33) .
Low affinity Ca
binding has been associated with
stretches of acidic residues
(34) , and the CaR does contain
acidic regions in the NH
-terminal extracellular domain
(amino acids 215-251 and 556-610) and second extracellular
loop (ELEDE). These acidic regions are conserved between the human and
bovine CaRs, but not in the mGluRs. The CaR extracellular region may
represent a ligand-binding domain with a conformation structurally
homologous to that of the PBPs/GluRs, but modified for low affinity
interaction with cationic compounds.
, and it has been suggested that this
effect may result from direct phosphorylation of the
CaR
(10, 11) . The bovine and human CaRs show potential
sites for protein kinase C phosphorylation in the first and third
intracellular loops and in the intracellular C-terminal domain, thus
providing sites on the CaR consistent with this view. The COOH-terminal
intracellular domain is the only region displaying extensive sequence
divergence between the human and bovine CaRs, and the human protein has
one additional protein kinase C site in this region, as well as
potential sites for protein kinase A phosphorylation. The intracellular
regions of GPRs are known to function in modulating receptor
interactions with cellular signal transduction pathways, and variations
in this region raise the possibility of species differences in the
coupling of CaR activation to intracellular signaling pathways.
concentration at which PTH secretion is half-maximally suppressed
(the Ca
set-point), and alterations in the CaR have
been proposed to underlie this set-point increase
(12) , just as
a mutation in the CaR has been shown to account for the decreased
sensitivity to extracellular Ca
seen in one instance
of FHH
(13) . Somatic mutation leading to alteration of the CaR
protein sequence expressed in the adenomatous gland is a possibility,
but it is unlikely to be the case in the particular tumor under study.
The phPCaR-4.0 and 5.2 clones do have three sequence differences in the
coding region, yet extracellular Ca
is equipotent in
activating either receptor. The EC
for Ca
activation of both CaR clones expressed in the oocyte is elevated
relative to that seen in parathyroid cells, but that is likely a
function of expression in the Xenopus system. Elevated
EC
values for Ca
activation of the CaR
were seen in oocytes expressing either bovine parathyroid gland mRNA
(27) or the pBoPCaR1 cRNA, suggesting that the apparent
elevation of the CaR set-point is inherent in this exogenous expression
system.
-terminal
extracellular domain of the phPCaR-5.2 clone, arises from alternative
splice site selection, and is a rare occurrence in parathyroid
mRNA.
()
The phPCaR-4.0 coding region sequence is
an exact match to the normal human CaR gene, except for the change at
amino acid 990, and this change is a polymorphism present in the human
population and is not associated with parathyroid disease. The sequence difference at amino acid 926 in the phPCaR-5.2 clone
is likely due to a cloning artifact, and this clone does give rise to a
functional receptor. Thus, two independent CaR cDNA clones from the
adenomatous gland do not appear to bear coding region mutations that
could account for a set-point increase. A survey of CaR sequences from
a collection of adenomatous parathyroid glands will be required to
rigorously exclude the occurrence of CaR mutations leading to 1°
HPT.
sensing
(13, 30) . A
decrease in CaR levels in parathyroid adenomas could account for the
observed set-point increases. The parathyroid adenoma examined in this
study shows multiple CaR transcripts by Northern blot analysis, and
cDNA clones reveal alternative RNA processing of 5`- and 3`-UTRs. The
use of alternative promoters, or alternative processing to RNA species
that may differ in their stability or the efficiency with which they
are translated, are all events which could alter the levels of
functional CaR protein expressed in the disease tissue. A third
possibility is that CaR levels remain normal in a parathyroid adenoma,
but secondary modifications of the receptor protein, or an alteration
in the signaling pathways downstream of the receptor, results in the
observed set-point increase. For example, increased activity of protein
kinase C could result in a decreased sensitivity of the CaR to
extracellular Ca
. The isolation of these human CaR
cDNA clones will facilitate studies aimed at addressing these questions
of the involvement of the CaR in the etiology of 1° HPT.
/EMBL Data Bank with accession number(s)
U20759 and U20760.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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L. Gama and G. E. Breitwieser A Carboxyl-terminal Domain Controls the Cooperativity for Extracellular Ca2+ Activation of the Human Calcium Sensing Receptor. A STUDY WITH RECEPTOR-GREEN FLUORESCENT PROTEIN FUSIONS J. Biol. Chem., November 6, 1998; 273(45): 29712 - 29718. [Abstract] [Full Text] [PDF]
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 Y. Cao, B. C. Oh, and L. Stryer Cloning and localization of two multigene receptor families in goldfish olfactory epithelium PNAS, September 29, 1998; 95(20): 11987 - 11992. [Abstract] [Full Text] [PDF]
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Y. Oda, C.-L. Tu, S. Pillai, and D. D. Bikle The Calcium Sensing Receptor and Its Alternatively Spliced Form in Keratinocyte Differentiation J. Biol. Chem., September 4, 1998; 273(36): 23344 - 23352. [Abstract] [Full Text] [PDF]
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M. Bai, S. Trivedi, C. R. Lane, Y. Yang, S. J. Quinn, and E. M. Brown Protein Kinase C Phosphorylation of Threonine at Position 888 in Ca2+o-Sensing Receptor (CaR) Inhibits Coupling to Ca2+ Store Release J. Biol. Chem., August 14, 1998; 273(33): 21267 - 21275. [Abstract] [Full Text] [PDF]
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T. Yamaguchi, N. Chattopadhyay, O. Kifor, and E. M. Brown Extracellular Calcium (Ca2+o)-Sensing Receptor in a Murine Bone Marrow-Derived Stromal Cell Line (ST2): Potential Mediator of the Actions of Ca2+o on the Function of ST2 Cells Endocrinology, August 1, 1998; 139(8): 3561 - 3568. [Abstract] [Full Text] [PDF]
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T. Watanabe, M. Bai, C. R. Lane, S. Matsumoto, K. Minamitani, M. Minagawa, H. Niimi, E. M. Brown, and T. Yasuda Familial Hypoparathyroidism: Identification of a Novel Gain of Function Mutation in Transmembrane Domain 5 of the Calcium-Sensing Receptor J. Clin. Endocrinol. Metab., July 1, 1998; 83(7): 2497 - 2502. [Abstract] [Full Text]
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D. T. Ward, E. M. Brown, and H. W. Harris Disulfide Bonds in the Extracellular Calcium-Polyvalent Cation-sensing Receptor Correlate with Dimer Formation and Its Response to Divalent Cations in Vitro J. Biol. Chem., June 5, 1998; 273(23): 14476 - 14483. [Abstract] [Full Text] [PDF]
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L. G. Hammerland, J. E. Garrett, B. C. P. Hung, C. Levinthal, and E. F. Nemeth Allosteric Activation of the Ca2+ Receptor Expressed in Xenopus laevis Oocytes by NPS 467 or NPS 568 Mol. Pharmacol., June 1, 1998; 53(6): 1083 - 1088. [Abstract] [Full Text]
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 M. M. Mupanomunda, Y. Wang, and R. D. Bukoski Effect of chronic sensory denervation on Ca2+-induced relaxation of isolated mesenteric resistance arteries Am J Physiol Heart Circ Physiol, May 1, 1998; 274(5): H1655 - H1661. [Abstract] [Full Text] [PDF]
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E. F. Nemeth, M. E. Steffey, L. G. Hammerland, B. C. P. Hung, B. C. Van Wagenen, E. G. DelMar, and M. F. Balandrin Calcimimetics with potent and selective activity on the parathyroid calcium receptor PNAS, March 31, 1998; 95(7): 4040 - 4045. [Abstract] [Full Text] [PDF]
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 Y. Kubo, T. Miyashita, and Y. Murata Structural Basis for a Ca2+-Sensing Function of the Metabotropic Glutamate Receptors Science, March 13, 1998; 279(5357): 1722 - 1725. [Abstract] [Full Text]
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I. Cheng, M. E. Klingensmith, N. Chattopadhyay, O. Kifor, R. R. Butters, D. I. Soybel, and E. M. Brown Identification and Localization of the Extracellular Calcium-Sensing Receptor in Human Breast J. Clin. Endocrinol. Metab., February 1, 1998; 83(2): 703 - 707. [Abstract] [Full Text]
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S. E. McNeil, S. A. Hobson, V. Nipper, and K. D. Rodland Functional Calcium-sensing Receptors in Rat Fibroblasts Are Required for Activation of SRC Kinase and Mitogen-activated Protein Kinase in Response to Extracellular Calcium J. Biol. Chem., January 9, 1998; 273(2): 1114 - 1120. [Abstract] [Full Text] [PDF]
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N. Chattopadhyay, I. Cheng, K. Rogers, D. Riccardi, A. Hall, R. Diaz, S. C. Hebert, D. I. Soybel, and E. M. Brown Identification and localization of extracellular Ca2+-sensing receptor in rat intestine Am J Physiol Gastrointest Liver Physiol, January 1, 1998; 274(1): G122 - G130. [Abstract] [Full Text] [PDF]
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K. Ray, G.-F. Fan, P. K. Goldsmith, and A. M. Spiegel The Carboxyl Terminus of the Human Calcium Receptor. REQUIREMENTS FOR CELL-SURFACE EXPRESSION AND SIGNAL TRANSDUCTION J. Biol. Chem., December 12, 1997; 272(50): 31355 - 31361. [Abstract] [Full Text] [PDF]
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 R. D. Bukoski, K. Bian, Y. Wang, and M. Mupanomunda Perivascular Sensory Nerve Ca2+ Receptor and Ca2+-Induced Relaxation of Isolated Arteries Hypertension, December 1, 1997; 30(6): 1431 - 1439. [Abstract] [Full Text]
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F. Farnebo, U. Enberg, L. Grimelius, M. Backdahl, M. Schalling, C. Larsson, and L.-O. Farnebo Tumor-Specific Decreased Expression of Calcium Sensing Receptor Messenger Ribonucleic Acid in Sporadic Primary Hyperparathyroidism J. Clin. Endocrinol. Metab., October 1, 1997; 82(10): 3481 - 3486. [Abstract] [Full Text] [PDF]
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L. Gama, L. M. Baxendale-Cox, and G. E. Breitwieser Ca2+-sensing receptors in intestinal epithelium Am J Physiol Cell Physiol, October 1, 1997; 273(4): C1168 - C1175. [Abstract] [Full Text] [PDF]
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S. J. Quinn, C.-P. Ye, R. Diaz, O. Kifor, M. Bai, P. Vassilev, and E. Brown The Ca2+-sensing receptor: a target for polyamines Am J Physiol Cell Physiol, October 1, 1997; 273(4): C1315 - C1323. [Abstract] [Full Text] [PDF]
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M. Mailland, R. Waelchli, M. Ruat, H. G. W. M. Boddeke, and K. Seuwen Stimulation of Cell Proliferation by Calcium and a Calcimimetic Compound Endocrinology, September 1, 1997; 138(9): 3601 - 3605. [Abstract] [Full Text] [PDF]
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F. De Luca, K. Ray, E. E. Mancilla, G.-F. Fan, K. K. Winer, P. Gore, A. M. Spiegel, and J. Baron Sporadic Hypoparathyroidism Caused by de Novo Gain- of-Function Mutations of the Ca2+-Sensing Receptor J. Clin. Endocrinol. Metab., August 1, 1997; 82(8): 2710 - 2715. [Abstract] [Full Text] [PDF]
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M. Kobayashi, H. Tanaka, K. Tsuzuki, M. Tsuyuki, H. Igaki, Y. Ichinose, K. Aya, N. Nishioka, and Y. Seino Two Novel Missense Mutations in Calcium-Sensing Receptor Gene Associated with Neonatal Severe Hyperparathyroidism J. Clin. Endocrinol. Metab., August 1, 1997; 82(8): 2716 - 2719. [Abstract] [Full Text] [PDF]
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G. Fan, P. K. Goldsmith, R. Collins, C. K. Dunn, K. J. Krapcho, K. V. Rogers, and A. M. Spiegel N-Linked Glycosylation of the Human Ca2+ Receptor Is Essential for Its Expression at the Cell Surface Endocrinology, May 1, 1997; 138(5): 1916 - 1922. [Abstract] [Full Text] [PDF]
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 S. H.S. Pearce, C. Williamson, O. Kifor, M. Bai, M. G. Coulthard, M. Davies, N. Lewis-Barned, D. McCredie, H. Powell, P. Kendall-Taylor, et al. A Familial Syndrome of Hypocalcemia with Hypercalciuria Due to Mutations in the Calcium-Sensing Receptor N. Engl. J. Med., October 10, 1996; 335(15): 1115 - 1122. [Abstract] [Full Text] [PDF]
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M. Bai, S. Quinn, S. Trivedi, O. Kifor, S. H.S. Pearce, M. R. Pollak, K. Krapcho, S. C. Hebert, and E. M. Brown Expression and Characterization of Inactivating and Activating Mutations in the Human Ca2+o-sensing Receptor J. Biol. Chem., August 9, 1996; 271(32): 19537 - 19545. [Abstract] [Full Text] [PDF]
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J. Hu, G. Reyes-Cruz, P. K. Goldsmith, and A. M. Spiegel The Venus's-flytrap and Cysteine-rich Domains of the Human Ca2+ Receptor Are Not Linked by Disulfide Bonds J. Biol. Chem., March 2, 2001; 276(10): 6901 - 6904. [Abstract] [Full Text] [PDF]
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J. Hu, O. Hauache, and A. M. Spiegel Human Ca2+ Receptor Cysteine-rich Domain. ANALYSIS OF FUNCTION OF MUTANT AND CHIMERIC RECEPTORS J. Biol. Chem., May 19, 2000; 275(21): 16382 - 16389. [Abstract] [Full Text] [PDF]
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N. Solban, H.-P. Jia, S. Richard, S. Tremblay, A. M. Devlin, J. Peng, F. Gossard, D.-F. Guo, G. Morel, P. Hamet, et al. HCaRG, a Novel Calcium-regulated Gene Coding for a Nuclear Protein, Is Potentially Involved in the Regulation of Cell Proliferation J. Biol. Chem., October 6, 2000; 275(41): 32234 - 32243. [Abstract] [Full Text] [PDF]
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Z. Huang, S.-L. Cheng, and E. Slatopolsky Sustained Activation of the Extracellular Signal-regulated Kinase Pathway Is Required for Extracellular Calcium Stimulation of Human Osteoblast Proliferation J. Biol. Chem., June 8, 2001; 276(24): 21351 - 21358. [Abstract] [Full Text] [PDF]
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G. Hjalm, R. J. MacLeod, O. Kifor, N. Chattopadhyay, and E. M. Brown Filamin-A Binds to the Carboxyl-terminal Tail of the Calcium-sensing Receptor, an Interaction That Participates in CaR-mediated Activation of Mitogen-activated Protein Kinase J. Biol. Chem., September 7, 2001; 276(37): 34880 - 34887. [Abstract] [Full Text] [PDF]
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C.-L. Tu, W. Chang, and D. D. Bikle The Extracellular Calcium-sensing Receptor Is Required for Calcium-induced Differentiation in Human Keratinocytes J. Biol. Chem., October 26, 2001; 276(44): 41079 - 41085. [Abstract] [Full Text] [PDF]
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