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J. Biol. Chem., Vol. 277, Issue 37, 33736-33741, September 13, 2002
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From the
Received for publication, January 30, 2002, and in revised form, June 30, 2002
The extracellular calcium
(Ca2+o)-sensing receptor (CaR) can be
potentiated by allosteric activators including calcimimetics and
L-amino acids. In this study, we found 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. 10 mM
L-phenylalanine and 1 µM NPS R-467,
submaximal doses of the two agents, each elicited similar modulation of
R185Q. However, there are different relative potencies for these two
modulators with some receptors being more responsive to
L-phenylalanine and others being more responsive to NPS
R-467. The responsiveness of the CaR to Ca2+o
appears to be essential to observe the potentiating action of
L-phenylalanine but not 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. The two-site model is further
supported by the observation that these two classes of modulators exert
a synergistic effect on CaRs with inactivating mutations that are
responsive to both modulators.
The extracellular calcium (Ca2+o)-sensing
receptor (CaR)1 plays a key
role in mineral ion homeostasis by sensing small perturbations in the
level of Ca2+o and modulating the functions of the
parathyroid and the kidney so as to restore Ca2+o
to its normal level (1). The CaR has been identified as an important
therapeutic target for disorders of systemic Ca2+o
homeostasis such as primary and secondary hyperparathyroidism. Earlier
studies have documented that the CaR can be activated allosterically by
L-amino acids and the phenylalkylamine calcimimetics in the
presence of Ca2+o at millimolar levels (2). In
turn, these allosteric modulators stereoselectively enhance the
sensitivity of the CaR to its polycationic agonists such as
Ca2+o and spermine. It has been documented that
calcimimetics act through the transmembrane domain of the receptor
(3).
The CaR is a G protein-coupled receptor in the same subfamily C
as the metabotropic glutamate receptors (mGluRs) 1 through 8 (4-6).
Five of the residues in the glutamate binding site of mGluR1a in the
amino-terminal extracellular domain are conserved in the CaR. According
to Zhang et al. (9), mutations at these sites
significantly attenuate the responsiveness of the receptor to its
principal physiological agonist, Ca2+o, suggesting
possible homology between the glutamate binding site of the mGluRs and
the calcium binding site of the CaR. Ser-170 whose conserved residue,
Thr-188, in the mGluRs is involved in glutamate binding is also
important for the modulation of the CaR by L-phenylalanine.
Alanine substitution of Ser-170 along with substitutions of two other
serines at positions 169 and 171 was found to completely block the
response of the CaR to L-phenylalanine. Therefore,
L-phenylalanine appears to act through a site in the
extracellular domain of the CaR to enhance receptor function.
In the present studies, we examined the effects of a calcimimetic, NPS
R-467, on several mutant receptors and compared the results with the
effects of L-phenylalanine or both agents together on the
same CaRs to functionally demonstrate that these two types of
allosteric modulators act on the CaR through distinct sites.
Transient Expression of CaRs in Human Embryonic Kidney (HEK) 293 Cells--
The DNA for transfection was prepared using the plasmid
Midi Kit (Qiagen) (7). LipofectAMINE (Invitrogen) was employed as a DNA
carrier for transfection. The HEK293 cells used for transient transfection were provided by NPS Pharmaceuticals (Salt Lake City, UT)
and cultured in Dulbecco's modified Eagle's medium (Invitrogen) with
10% fetal bovine serum (Hyclone). The DNA-liposome complex was
prepared by mixing DNA and LipofectAMINE in Opti-MEM I reduced serum
medium (Invitrogen) and incubating the mixture at room temperature for
30 min. The DNA-LipofectAMINE mixture was then diluted with Opti-MEM I
reduced serum medium and added to 90% confluent HEK293 cells plated on
13.5 × 20.1-mm glass coverslips using 2.5 µg of DNA. After a
5-h incubation at 37 °C, equivalent amounts of Opti-MEM I reduced
serum medium with 20% fetal bovine serum were added to the medium
overlying the transfected cells, and the medium was replaced with fresh
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
at 24 h after transfection. The expressed CaR protein was assayed
48 h after the start of transfection. To co-express two receptors,
1.25 µg of each of the two cDNAs was mixed and used to transfect
HEK293 cells.
Measurement of Ca2+i by Fluorimetry in
Cell Populations--
Coverslips with HEK293 cells previously
transfected with the appropriate CaR cDNAs were loaded for 2 h
at room temperature with Fura-2/AM in 20 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 1.25 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 0.1% bovine serum
albumin, and 0.1% dextrose and then washed once with a bath solution
(20 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% dextrose, and 0.1% bovine
serum albumin) at 37 °C for 20 min. The coverslips were then placed diagonally in a thermostated quartz cuvette containing the bath solution using a modification of the technique employed previously in
this laboratory (7). The L-phenylalanine and/or NPS R-467 were added into the bath solution and preincubated for 50 s.
Concentrated Ca2+o at 100 mM or 1 M was added stepwise to an experimental solution containing
0.5 mM Ca2+ to achieve the following
concentrations: 1.5, 2.5, 3.5, 4.5, 5.5, 10, 15, 20, 30, 40, and 50 mM. Excitation monochrometers were centered at 340 and 380 nm, and emission light was collected at 510 ± 40 nm through a
wide band emission filter. The 340/380 excitation ratio of emitted
light was used to estimate Ca2+i as described
previously (7).
Statistics--
The activities of the wild-type and mutant CaRs
were determined in response to increasing concentrations of
Ca2+o in the presence or absence of modulators. The
mean responses at various concentrations of Ca2+o
were calculated from individual experiments and were expressed with the standard error of the mean (±S.E.) as the index of
dispersion. A comparison of responses of various receptors at 50 mM Ca2+o was performed using ANOVA or
Duncan's multiple comparison test (p We compared the modulatory effects of a phenylalkylamine
calcimimetic and an L-amino acid on the wild-type and
various mutant CaRs. Our initial studies examined the effects of 1 µM NPS R-467 and 10 mM phenylalanine on the
wild-type receptor. NPS R-467 decreased the EC50 value of
the wild-type CaR by 39%, whereas L-phenylalanine decreased it by 16% (Fig. 1A
and Table 1). However, NPS R-467 markedly decreased the maximal responses elicited by high
concentrations of Ca2+o (Table I).
Thus, the extent of receptor activation in the presence of 1 µM NPS R-467 or 10 mM
L-phenylalanine was similar at physiological concentrations
of Ca2+o. Unlike phenylalanine, NPS R-467 also
significantly reduced the Hill coefficient of the wild-type receptor
(Table I), suggesting that the requirement for
Ca2+o-binding is different for phenylalanine and
NPS R-467.
Similar to phenylalanine as demonstrated by Zhang et al.
(9), NPS R-467 significantly increased the
Ca2+o-elicited maximal responses of several mutant
receptors. For instance, NPS R-467 and L-phenylalanine each
increased the maximal response of R185Q to a similar extent ~1.4-fold
(Fig. 1B). NPS R-467 and L-phenylalanine also
substantially decreased the EC50 value of R185Q to a
greater extent than that seen for the wild-type CaR. There appears to
be a limit to the positive modulation of the wild-type CaR that can be
elicited by an addition of the calcimimetic or
L-phenylalanine. Mutant receptors with less sensitivity to
polycationic agonists and reduced maximal activation often showed
greater potentiation by the two modulators than the wild-type receptor.
The effects of NPS R-467 or L-phenylalanine were not always
identical when other mutant receptors were studied as described below.
Many mutant receptors with substitutions of conserved amino acid
residues within the putative amino acid binding site in the extracellular domain remained responsive to
L-phenylalanine. S170A and Y218S each responded to
L-phenylalanine with significant increases in the magnitude
of the response at all of the tested concentrations of
Ca2+o. With NPS R-467, all of the responses of the
two receptors were further increased to even greater levels (Fig.
2), and sensitivities of the respective
receptors to Ca2+o were also enhanced as indicated
by the significant declines in the EC50 values (Table I).
In contrast, S147A, D190A, and E297K showed greater increases in their
responses to L-phenylalanine than to NPS R-467 (Fig.
3). In the presence of either
L-phenylalanine or NPS R-467, the EC50 values
for these three mutant receptors were also significantly lowered (Table
I). Therefore, the responsiveness of the mutant receptors to
L-phenylalanine and NPS R-467 were not uniformly affected
by mutations, strongly suggesting that these two modulators act through
distinct binding sites.
L-Phenylalanine and NPS R-467 Synergistically
Potentiate the Function of the Extracellular Calcium-sensing Receptor
through Distinct Sites*
,,,¶
Department of Medicine,
Endocrine-Hypertension Division and Membrane Biology Program,
Brigham and Women's Hospital and Harvard Medical School, Boston,
Massachusetts 02115 and § NPS Pharmaceuticals, Inc.,
Salt Lake City, Utah 84108
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.05) (8).
Each of the experiments presented in the results was performed at least
three times. To calculate the EC50, the maximal response,
and the Hill coefficient, we used the MacFitCurve program and the Hill
equation F(X) = a·X
/(e + X) where "F" is a variant as a function
of X, "F(X)" is the response at a
given concentration with agonist (X), "a" is maximal
response, "e" is EC50, and "h" is Hill coefficient
to fit the experimental data.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 1.
Allosteric modulation of the wild-type
receptor and the mutant receptor R185Q. HEK293 cells were
transfected with the wild-type receptor (A) or R185Q
(B) and loaded with Fura-2. Changes in the emission ratio
(340/380 excitation) were measured to assess
Ca2+o-evoked Ca2+i responses.
Before the additions of Ca2+o, the cells were not
exposed (circle), exposed to 1 µM NPS
R-467 (square), or exposed to 10 mM Phe
(triangle). Responses are normalized to the maximal
cumulative Ca2+i responses of the wild-type
receptor in untreated cells. Each point is the mean value of the number
of measurements indicated in Table I. Standard errors of the mean
(±S.E.) are indicated with vertical bars through each
point. Some error bars are smaller than the symbol. The data
were fitted using MacCurveFit as described under "Experimental
Procedures."
EC50 values and Hill coefficients predicted using Hill equation
for the wild-type and mutant CaRs
View larger version (19K):
[in a new window]
Fig. 2.
Allosteric modulation of mutant receptors
S170A, Y218S, and S169A/S170A/S171A. HEK293 cells were transfected
with S170A (A) or Y218A (B).
Ca2+o-evoked Ca2+i responses
were measured as described in Fig. 1. Before the additions of
Ca2+o, the cells were not exposed
(circle), exposed to 1 µM NPS R-467
(square), or exposed to 10 mM Phe
(triangle). Responses are normalized to the maximal
cumulative Ca2+i responses of the wild-type
receptor in untreated cells. Each point is the mean value of the number
of measurements indicated in Table I. Standard errors of the mean
(±S.E.) are indicated with vertical bars through each
point. Some error bars are smaller than the symbol. The data
were fitted using MacCurveFit as described under "Experimental
Procedures."
View larger version (17K):
[in a new window]
Fig. 3.
Allosteric modulation of mutant receptors
S147A, D190A, and E297K. HEK293 cells were transfected with S147A
(A), D190A (B), or E297K (C).
Ca2+o-evoked Ca2+i responses
were measured as described in Fig. 1. Before the additions of
Ca2+o, the cells were not exposed
(circle), exposed to 1 µM NPS R-467
(square), or exposed to 10 mM Phe
(triangle). Responses are normalized to the maximal
cumulative Ca2+i responses of the wild-type
receptor in untreated cells. Each point is the mean value of the number
of measurements indicated in Table I. Standard errors of the mean
(±S.E.) are indicated with vertical bars through each
point. Some error bars are smaller than the symbol. The data
were fitted using MacCurveFit as described under "Experimental
Procedures."
The mutant receptors G143E, R795W, S169A/S170A/S171A showed weak
or no activation by the physiological agonist Ca2+o
and also showed no potentiation by L-phenylalanine. In
contrast, NPS R-467 enhanced the responses of each of these mutant
receptors to Ca2+o (Fig.
4). G143E is a special case where there
was no activation by Ca2+o unless NPS R-467 was
also present, indicating that the responsiveness of the CaR to agonist
stimulation is not essential for its modulation by NPS R-467 and that
these two allosteric activators act on the receptor through different
mechanisms.
|
Heterodimeric receptors were also affected differently by these two
types of modulators in some cases. For instance, heterodimers formed in
cells co-transfected with G143E and the inactive mutant CaR, A877Stop,
or in those cells co-transfected with S169A/S170A/S171A and A877Stop
that recovered 60% of their Ca2+i responses to
Ca2+o hardly responded to
L-phenylalanine (Fig. 5). In
contrast, NPS R-467 substantially reduced the EC50 values
of these heterodimers while eliciting modest increases in their maximal
responses (Table I).
|
To further demonstrate that L-phenylalanine and NPS R-467
act on the receptor through different sites, we examined whether these
two allosteric activators affected the function of the receptor synergistically when added together. The actions of both activators combined on the wild-type receptor resembled that of NPS R-467 alone
(Fig. 6A). In contrast, the
mutant receptor E297K, which has a very attenuated response in the
absence of these two modulators, became significantly more active when
these two modulators were combined than when they were applied
individually at their maximal effective doses (Fig. 6B). The
EC50 was reduced by nearly half (18-11 mM) in
the presence of either 30 µM NPS R-467 or 30 mM L-phenylalanine, whereas the presence of
both agents resulted in a further reduction in the EC50 to
nearly 25% of its original level (18-5 mM). As shown in
Fig. 6, 30 µM NPS R-467 and 30 mM L-phenylalanine were the maximal doses for these
modulators; thus, doubling the concentration of either modulator had no
further effect on receptor activity. Consistent with the lack of
L-phenylalanine modulation of the mutant CaRs, G143E and
S169A/S170A/S171A, synergistic effects were not observed with the
mutant receptors G143E (Fig. 6C) and S169A/S170A/S171A (data
not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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In this study, we compared the modulatory effects of NPS R-467 and L-phenylalanine, two allosteric activators of the CaR. Both compounds were found to potentiate the activation of the wild-type receptor as well as many mutant receptors with inactivating mutations. With the wild-type receptor, the primary effect observed with these modulators is a decrease in the EC50 for activation of the CaR by Ca2+o. Most mutant receptors also showed a decrease in EC50. In addition, many of these receptors demonstrated increases in their maximal responses to agonist activation, which was not found in the wild-type CaR. Interestingly, NPS R-467 consistently decreased the EC50 for the mutant receptors, whereas L-phenylalanine often showed little or no effect on EC50 despite producing significant increases in the maximal responses of many mutant receptors. Another difference between NPS R-467 and L-phenylalanine is that NPS R-467 significantly decreased the maximal response of the wild-type receptor, whereas L-phenylalanine had no effect on this parameter in the wild-type receptor. The presence of the physiological polycationic CaR agonist Ca2+o is essential for the actions of both NPS R-467 and L-phenylalanine on the wild-type and mutant receptors.
We chose 1 µM NPS R-467 and 10 mM L-phenylalanine as our experimental doses for the two modulators because these concentrations gave submaximal but substantial effects on the wild-type CaR. Using these two doses, we found that the efficacies of the two modulators were quite different depending on the mutant CaR that was tested. In most cases, NPS R-467 gave more pronounced changes in maximal response and EC50 than did L-phenylalanine. In fact, there were several mutant CaRs that were responsive to NPS R-467 but not to L-phenylalanine. The most obvious exception to this observation is the wild-type CaR, which shows an unexplained decline in its maximal activity in the presence of NPS R-467. In addition, there were also several mutants that appeared to be more responsive to L-phenylalanine such as S147A and E297K. No mutant receptors were found to be responsive to L-phenylalanine but were found to be insensitive to NPS R-467. This difference in the efficacies of NPS R-467 and L-phenylalanine with mutant receptors suggests separate sites of action for these two allosteric modulators. Corroborating this point was the synergistic behavior of NPS R-467 and L-phenylalanine. Each modulator had additional potentiating effects on some mutant receptors in the presence of a maximal concentration of the other modulator.
NPS R-467 can be effective with mutant CaRs that are not normally responsive to Ca2+o. For example, the responsiveness of G143E is restored to 26% of that of the wild-type receptor in the presence of 1 µM NPS R-467, whereas the mutant receptor is completely inactive in the absence of NPS R-467 (see Fig. 4). In contrast, L-phenylalanine appears to need some degree of Ca2+o responsiveness to positively modulate the activity of CaR as suggested by the lack of positive modulation of G143E and G549R by L-phenylalanine. The difference may be attributed to the mechanism of action of NPS R-467, which may allow it to magnify subthreshold activation of the CaR.
L-Phenylalanine had little effect on Hill coefficients of the wild-type and mutant CaRs, whereas NPS R-467 often had a negative impact on the Hill coefficients of the wild-type and some mutant receptors (Table I). These findings suggest that the action of NPS R-467 appears to be less dependent on the Ca2+o binding sites. It is probable that the interaction of the CaR with NPS R-467 increases the affinity of the receptor for its cognate G proteins or enhances signal transduction from the head of the CaR to its intracellular domains. When taken together with previous studies on the probable binding sites of NPS R-467 and L-phenylalanine, our data are consistent with the findings that NPS R-467 acts at the transmembrane region of the CaR (3).
In conclusion, these two types of allosteric modulators act
on the receptor not only through distinct sites but also through distinct mechanisms. Our findings provide a solid base for developing novel therapeutic agents that interact with the CaR with high affinities through the amino acid binding site. In addition, the synergistic effect of these two types of allosteric modulators on the
CaR potentially provides a new therapeutic approach to the management
of severe hypercalcemia associated with various types of hyperparathyroidism.
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ACKNOWLEDGEMENT |
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We thank Dr. Edward M. Brown for critical review of the paper.
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FOOTNOTES |
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* This work was supported National Institutes of Health Grant DK54934 (to M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Endocrine-Hypertension Division, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-732-4093; Fax: 617-732-5764.
Published, JBC Papers in Press, July 11, 2002, DOI 10.1074/jbc.M200978200
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ABBREVIATIONS |
|---|
The abbreviations used are: CaR, Ca2+o-sensing receptor; mGluRs, metabotropic glutamate receptors; HEK, human embryonic kidney; ANOVA, analysis of variance.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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