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Metabolic Diseases Branch (O.M.H., J.H., K.R., A.M.S.), Laboratory of Bioorganic Chemistry (R.X., K.A.J.), NIDDK, NIH, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Allen M. Spiegel, NIH, Building 31, Room 9A-52, Bethesda, Maryland 20892. E-mail: spiegela{at}extra.niddk.nih.gov
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Between VFT domain and 7
transmembrane (TM) domain, a cysteine-rich region with nine highly
conserved cysteines in a closely spaced (about 60 amino acids long)
sequence (Fig. 1) is present in all the members of family 3 except
GABABR1.
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While both gain of function mutations identified in ADH and R-568 have been shown to increase CaR sensitivity to Ca2+, their mechanism of action is unclear. As an initial step in understanding how such mutations enhance receptor sensitivity, we analyzed the effects of various gain of function mutations situated in the ECD on CaR response to Ca2+. We then studied the effects of R-568 on these activating mutants to see if the drug could further enhance sensitivity to Ca2+ of the mutant receptors. Further, by using truncation and deletion mutants of the CaR and different CaR/mGluR1 chimeras, we explored the site of action of R-568 and the mechanism of action of a potent left-shifting mutation (L125P). These studies reveal important differences between the activation mechanisms of R-568 and ADH mutations such as L125P, define the CaR 7TM as the specific site of action of R-568, and suggest that the 7TM domain of the closely related mGluR1 is more permissive for activation than that of the CaR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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strain). The mutations were confirmed by automated
DNA sequencing using a Taq DyeDeoxy Terminator Cycle Sequencing kit and
ABI prizm-377 DNA sequencer (PE Applied Biosystems, Foster
City, CA). Double mutants were created by using a single mutant DNA as
a template in the next round of mutagenesis.
Construction of truncated and deletion mutants and chimeras between
CaR and mGluR1
A detailed description of the construction of the deletion CaR
mutant and of chimeras between CaR and mGluR1 has been published
(16). The VFT, cysteine-rich domain and 7TM domain of the
CaR are indicated schematically in Fig. 1
. The CaR constructs included
in this study are designated as follows: Ca-//-Ca, which lacked the
cysteine-rich region of the CaR ECD, Ca-Glu-Ca, which involved
replacing the cysteine-rich region of the CaR with that of the mGluR1,
Ca-Ca-Glu, which involved replacing the 7TM and C-tail domains of the
CaR with that of mGluR1, and Ca-Glu-Glu, which involved replacing both
the cysteine-rich domain and 7TM and C-tail domains of the CaR with
that of mGluR1. The N-terminal-truncated version of the human CaR
(Rho-C-hCaR) lacked the majority of the ECD and included a rhodopsin
epitope tag (corresponding to 20 amino acids of the N terminus of
rhodopsin; see Fig. 1) and the amino acids residues 599–903 of the CaR
(7).
Transient transfection of wild-type and mutant receptor cDNAs in
HEK-293 cells
Receptor cDNAs in pCR3.1 were prepared with a
QIAGEN maxi Plasmid Maxi DNA preparation kit
(QIAGEN Inc., Chatsworth, CA) and were introduced into
HEK-293 cells by the Lipofectamine transfection method (Life Technologies, Inc., Gaithersburg, MD). For transfection, a given
amount of the plasmid DNA was diluted in DMEM (Biofluids
Inc., Rockville, MD) and mixed with diluted Lipofectamine, and the
mixture was incubated at room temperature for 30 min. The
DNA-Lipofectamine complex was further diluted in serum-free DMEM and 12
µg of DNA was added to 80–90% confluent HEK-293 cells plated in 75
cm2 flasks. After 5 h of incubation, equal
volume of DMEM containing 10% FBS (Biofluids Inc.,
Rockville, MD) was added, and the media were replaced 24 h after
transfection with complete DMEM containing 10% FBS. Membrane protein
extraction for immunoblotting and phosphoinositide hydrolysis assay
were performed 48 h after transfection.
Immunoblotting analysis with detergent-solubilized whole cell
extracts
Confluent cells in 75 cm2 flasks or 6-well
plates were rinsed with ice-cold PBS and scraped on ice in Buffer-B
containing 20 mM Tris-HCl (pH 6.8), 150 mM
NaCl, 10 mM EDTA, 1 mM EGTA, 1% Triton X-100
with freshly added protease inhibitors cocktail (Complete, Roche Molecular Biochemicals, Indianapolis, IN). To prevent
nonspecific disulfide bond formation during protein extraction, the
intact cells were incubated and washed in PBS containing 50
mM iodoacetamide and 10 mM iodoacetamide was
included in the lysis buffer. Proteins were eluted with gel loading
sample buffer containing ß-mercaptoethanol as reducing agent and run
on SDS-PAGE and then analyzed on immunoblots stained with anti-hCaR
monoclonal antibody ADD to detect total hCaR immunoreactive species.
The protein content of each sample was determined by the modified
Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA)
and 40–60 µg of protein per lane was separated on 6% SDS-PAGE. The
proteins on the gel were electrotransferred to nitrocellulose membrane
and incubated for 90 min with protein A-purified mouse monoclonal
anti-hCaR antibody ADD (raised against a synthetic peptide
corresponding to residues 214–235 of hCaR protein at a dilution of
1:10000). After washing 3 times for 15 min with Tris-buffered saline
with Tween (TBST, containing 0.05 M Tris, pH 8.0, 0.05
M NaCl with 0.1% Tween 20), the membrane was incubated for
90 min with a secondary goat antimouse antibody conjugated to
horseradish peroxidase (Kirkegaard & Perry Laboratories,
Gaithersburg, MD) at a dilution of 1:5000. After washing 3 times for 15
min with TBST and once for 15 min with Tris-buffered saline (TBS), the
hCaR protein was detected with an enhanced chemiluminescence system
(ECL) (Amersham Pharmacia Biotech, Arlington Heights,
IL).
Phosphoinositide (PI) hydrolysis assay
PI hydrolysis assay has been described (15, 17).
Briefly, 24 h after transfection, transfected cells from a
confluent 75 cm2 flask were replated in a 12-well
plate in medium containing 3.0 µCi/ml of
3H-myoinositol (NEN Life Science Products, Beverly, MA) in complete DMEM for another 24 h,
followed by 1 h incubation with PI buffer (120 mM
NaCl, 5 mM KCl, 5.6 mM glucose, 0.4
mM MgCl2, 20 mM LiCl in
25 mM PIPES buffer, pH 7.2). After removal of PI buffer,
cells were incubated for an additional 1 h with different
concentrations of [Ca2+]o
in PI buffer, with or without 1 µM of R-568. The
reactions were terminated by addition of 1 ml of acid-methanol (167
µl of HCl in 120 ml of methanol). Total inositol-phosphates were
purified by chromatography on Dowex 1-x8 columns.
Synthesis of calcimimetic agent, R-568
The calcimimetic agent,
(R)-N-(3-methoxy--phenylethyl)-3-(2'-chlorophenyl)-1-propylamine
hydrochloride) or R-568 was synthesized by a novel route. Racemic
1-(3-methoxyphenyl)ethylamine was prepared by reductive amination of
3-methoxyacetophenone with ammonium acetate (18), and the
enantiomerically pure R-isomer isolated through crystallization with
R-mandelic acid (19). The final products were then
obtained by reductive amination of 3-(2-chlorophenyl)propionaldehyde
with the free base of R-1-(3-methoxyphenyl)ethylamine using sodium
cyanoborohydride in tetrahydrofuran containing a trace of acetic acid,
to provide R-568. The amine was isolated as the hydrochloride salt
following treatment of the free base with anhydrous hydrochloric acid
gas in dry ether. The specific rotations obtained were shown to be
equivalent to the reported values (12). Proton NMR
spectra, high resolution mass spectra, and CHN analyses of the final
product were consistent with the assigned structure.
Statistical analysis
Data were calculated as mean ± SE. For
comparisons, paired t test was used and P <
0.05 was considered significant. The statistical program used was
SigmaStat 1.0 (Jandel Corp., San Rafael, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Of these, A116T (20), and L125P
(21), had not been previously functionally characterized,
whereas F128L (8) and F612S (22) had been
shown to cause increased Ca2+ sensitivity
("left shift"). First, we characterized the pattern of activation
of these mutants by comparing them to the WT hCaR’s entire dose
response range to calcium (Fig. 2, insets). To focus on the most relevant concentration range,
we performed all subsequent PI hy-drolysis assays at concentrations
of Ca2+ varying from 0 mM
to 4.0 mM (Fig. 2). This concentration range was
most informative for comparisons of WT hCaR and various left-shifted
mutant receptors. We first confirmed that F128L and F612S mutant
receptors show a significant left-shifted response to
Ca2+ and found that to be true for the A116T and
L125P mutants as well (Fig. 2, A–D). The most striking increase in
Ca2+ sensitivity was seen with the L125P mutant,
especially at low doses of calcium (Fig. 2B); the three other mutants
each showed a similar increase in sensitivity compared with WT, but
this was not as marked as seen with L125P. Even the basal levels for
L125P were significantly higher than the wild-type CaR’s basal levels.
In distinction to Ca2+ sensitivity, which was
highest for the L125P mutant, maximum response for both the L125P and
F128L mutants were lower than that of WT, whereas maximum response for
the A116T and F612S mutants were equivalent to WT (Fig. 2).
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, under reducing
conditions, ADD antibody detected two monomeric forms for the wild-type
hCaR and for the mutants studied. Previous studies demonstrated that
the monomeric 150-kDa band represents hCaR forms expressed at the cell
surface and modified with N-linked, complex carbohydrates; the 130-kDa
band represents high-mannose-modified forms, trapped intracellularly
and sensitive to Endo-H digestion. Therefore, we can see that all
single activating mutants herein studied are well expressed on the cell
surface. Differences observed regarding levels of expression among the
mutants studied did not correlate with the degree of left-shift
observed, but the relatively lower expression for the L125P and F128L
mutants compared with WT may relate to the lower maximum response to
Ca2+ observed for these mutants (Fig. 2).
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). To test if the response to
[Ca2+]o of mutant CaRs
that are already left-shifted could be further enhanced by R-568, we
incubated cells transfected with mutant CaR cDNAs with various
concentrations of [Ca2+]o
with and without simultaneous incubation with a near maximal
concentration (12) of R-568. A dose curve ranging from 0
to 10 µM of R-568 was performed (data not shown) and
based on that, 1 µM of R-568 was used in our experiments.
As shown in Fig. 4, B–E, the calcimimetic agent was capable of
significantly further enhancing the response to
[Ca2+]o at concentrations
up to 2.0 mM for the A116T, F128L, F612S mutants; for the
L125P mutant, R-568 enhanced response significantly only at 0
mM of
[Ca2+]o. We also created
two double activating mutants including the mutation L125P (A116T +
L125P and L125P + F128L). Both double-mutants that included L125P did
not show a significant increase in sensitivity over L125P alone (data
not shown).
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. The
responses of these receptor constructs to calcium with and without
R-568 is shown at Fig. 6. R-568
significantly increased the sensitivity to
[Ca2+]o of wild-type CaR
at calcium concentrations of 2.0 and 4.0 mM. The chimera
that contains the mGluR cysteine rich region (Ca-Glu-Ca) is less
expressed at the cell surface and its mobility is altered on a SDS-PAGE
gel when compared with the WT hCaR. However, it still responds to
calcium, this response being 
25% of the WT hCaR’s response
(16). Addition of R-568 results in a dramatic and
significant increase of both the sensitivity of this chimera to
[Ca2+]o and the maximum
response achieved (Fig. 6B). Both chimeras that contain the mGluR
transmembrane domain (Ca-Ca-Glu and Ca-Glu-Glu) were well expressed at
the cell surface with slower mobility on SDS-PAGE when compared with
the wild-type CaR (16). This altered mobility may be
explained by the 141 amino acid longer 7 TM plus C-tail of mGluR1. Both
Ca-Ca-Glu and Ca-Glu-Glu were almost comparable to the WT hCaR in
Ca2+ sensitivity, but maximal responses were
approximately 80% and 60%, respectively, of the WT hCaR’s maximal
response. The calcimimetic compound did not change the
Ca2+ sensitivity or maximal response for either
of these chimeras (Figs. 6, C and D). Ca-//-Ca has a faster mobility
on SDS-PAGE due to the lack of the cysteine-rich domain. This deletion
mutant is less well expressed but a significant proportion of the
mutant receptor does reach the cell surface as reflected in the upper,
fully processed band on immunoblot (Fig. 5) (16). The
Rho-C-CaR truncation mutant is well expressed at the cell surface as
demonstrated previously using a rhodopsin N-terminal epitope monoclonal
antibody (7). Both these constructs, Ca-//-Ca and
Rho-C-CaR, which by themselves are unable to respond to
[Ca2+]o but still have
their CaR 7TM domains intact, became sensitive to
[Ca2+]o in the presence
of R-568 and show a maximal response approximately 30% that of WT
(Fig. 6, E and F).
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). This mutation is located in the ECD in a region
where other four neighboring activating mutations are present. When the
L125P mutation was placed into the context of the Ca-Glu-Ca chimera,
this resulted in a significant left-shift for calcium concentrations
ranging from 0 to 8.0 mM, but the maximal response was not
increased (Fig. 7B). The deletion mutant,
which does not have the CaR cysteine-rich domain (Ca-//-Ca) is not
activated by this mutation (Fig. 7C). On the other hand, both chimeras
that share mGluR1 7TM domains had their response to calcium
significantly potentiated by the L125P mutation (for the Ca-Ca-Glu
chimera, activation of the L125P mutant was significantly higher from 0
to 8.0 mM calcium, whereas for the Ca-Glu-Glu chimera the
L125P mutant was significantly more activated at all calcium
concentrations) (Fig. 7, D and E). Strikingly, for both of these
chimeras adding the L125P mutation resulted in extremely high basal
levels (60% of the WT maximal response).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we tested the effect of various ADH mutations, alone and in combination, as well as with the drug, R-568, to see if these combinations would show additional increases in Ca2+ sensitivity. If R-568 and certain mutations enhance sensitivity by the same mechanism, combining these might not lead to further increases in Ca2+ sensitivity. In contrast, combinations of mutations or drug plus mutation that involved different sites and mechanisms of action might lead to further increases in Ca2+ sensitivity.
We studied three neighboring activating mutations A116T (20), L125P (21), and F128L (9) from a portion of the ECD that is a "hot spot" for ADH mutations, harboring five (together with N118K and E127A) of the nine reported ADH mutations located in the ECD; and a mutation, F612S (22), at the junction of the ECD and first transmembrane domain. Each mutation enhanced CaR sensitivity to Ca2+ with L125P being the most effective, but L125P and F128L mutants showed a lower maximal response than WT CaR, possibly related to the lower levels of cell surface expression observed for these mutants on immunoblot. R-568 further enhanced the Ca2+ sensitivity of ECD mutant receptors such as A116T, F128L, and F612S but had minimal additional effect on L125P. With the double mutants, we also noted that combining other mutations with L125P led to minimal increases in Ca2+ sensitivity.
Thus, the principal determinant of further increases in Ca2+ sensitivity was the degree of left-shift caused by any individual mutation. It appears that certain modifications of the CaR, such as the L125P mutation alone lead to a maximal state of Ca2+ sensitivity that cannot be further left-shifted. Whether such mutant receptors could be right-shifted by compounds acting in the opposite way to R-568 is not only of theoretical interest but could have practical applications in treatment of subjects with ADH and in other states in which increases in PTH secretion are desirable. It is also interesting that the L125P mutation was shown to be a de novo mutation not present in either parent of the severely affected neonate in whom the mutation was identified (21). The severity of the clinical manifestations are compatible with our in vitro results showing that the L125P appears to cause a maximal left-shift of the CaR.
The site of action of R-568 appears to be within and specific for the 7TM domain of the CaR because R-568 had no effect on chimeras such as Ca-Ca-Glu and Ca-Glu-Glu lacking the CaR 7TM domain. Similar results using closely related receptors such as mGluRs and using different chimeras tested in microinjected Xenopus oocytes have previously been reported (5, 12). The effects of R-568 on the Ca-Glu-Ca chimera and on the Ca-//-Ca deletion and Rho-C-CaR truncation mutants were of particular interest. The Ca-Glu-Ca chimera was previously shown to be significantly impaired in Ca2+ response both in terms of EC50 and maximal activation (16). R-568 dramatically enhanced both parameters. These data are compatible with the idea that binding of R-568 to the CaR 7TM amplifies signals coming from the ECD to the 7TM domain. The Ca-//-Ca and Rho-C-CaR constructs fail to respond to Ca2+ despite a significant degree of cell surface expression. R-568 revealed a Ca2+ response of both of these constructs, albeit one that is much lower than that of wild-type CaR. This suggests that the 7TM domain may contain a very low affinity Ca2+ binding site that by itself, e.g. in constructs such as Rho-C-CaR lacking the ECD or Ca-//-Ca lacking the cysteine-rich region to transmit signal from the VFT to 7TM domain, is incapable of activating the CaR. R-568 action on the 7TM to amplify its response unmasks the Ca2+ responsiveness of constructs such as Ca-//-Ca and Rho-C-CaR.
The effects of R-568 contrast with those of the L125P activating mutation. The latter’s effect on the Ca/Glu/Ca chimera are modest by comparison with R-568, presumably because L125P acts proximal to the impaired cysteine-rich region of the chimera, whereas R-568 acts distally. For the same reason, the L125P mutant fails to activate the Ca-//-Ca deletion mutant at all (16). An unexpected effect of the L125P mutation, namely strikingly elevated basal activity (with basal defined as no added calcium in the PI buffer), was evident in the Ca-Ca-Glu and Ca-Glu-Glu chimeras. The same chimeras without the L125P mutation showed only slight (<20% of WT maximum response) increased basal activity. We interpret these results to suggest that the 7TM domain of the mGluR1 in the context of a chimeric receptor with the CaR VFT part of the ECD is more permissive for activation than that of the WT CaR. Interestingly, addition of R-568 to the CaR with an L125P mutation, while not causing a further left-shift in Ca2+ response, increases basal activity. This further supports our conclusions regarding the distinct sites and mechanisms of activation of the L125P mutant and the calcimimetic compound.
Our results lead us to suggest a tentative model for CaR activation. Ca2+ binding to site(s) on the ECD VFT domain causes a conformational change that is transmitted through the ECD cysteine-rich region to the 7TM domain and leads to receptor activation. The precise nature of the signal from ECD to 7TM domain, e.g. a direct, protein-protein interaction between ECD and 7TM domain, is presently unknown. ECD mutations such as occur in ADH facilitate the conformational change of the VFT domain resulting in transmission of a more powerful signal to the 7TM domain for any given concentration of Ca2+. This is particularly likely to be true for mutations such as L125P that occur in an ADH mutation "hot-spot" in the ECD, the loop 2 dimerization interface in a model depicted in Ref. 5 . R-568 in contrast binds to the 7TM and facilitates its activation by any signal transmitted from the ECD, as well as by high concentrations of Ca2+ binding directly to the 7TM domain. Further studies of CaR structure and function are needed to define the Ca2+ binding site(s) and to elucidate the precise mechanism of activation. Studies comparing the structure/conformation of a mutant CaR such as L125P which shows dramatically increased Ca2+ sensitivity, with that of the WT CaR could provide a useful approach.
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Acknowledgments |
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Footnotes |
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Received April 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 R. Vargas-Poussou, C. Huang, P. Hulin, P. Houillier, X. Jeunemaitre, M. Paillard, G. Planelles, M. Dechaux, R. T. Miller, and C. Antignac Functional Characterization of a Calcium-Sensing Receptor Mutation in Severe Autosomal Dominant Hypocalcemia with a Bartter-Like Syndrome J. Am. Soc. Nephrol., September 1, 2002; 13(9): 2259 - 2266. [Abstract] [Full Text] [PDF]
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K. Ray and J. Northup Evidence for Distinct Cation and Calcimimetic Compound (NPS 568) Recognition Domains in the Transmembrane Regions of the Human Ca2+ Receptor J. Biol. Chem., May 17, 2002; 277(21): 18908 - 18913. [Abstract] [Full Text] [PDF]
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L. Gama, S. G. Wilt, and G. E. Breitwieser Heterodimerization of Calcium Sensing Receptors with Metabotropic Glutamate Receptors in Neurons J. Biol. Chem., October 12, 2001; 276(42): 39053 - 39059. [Abstract] [Full Text] [PDF]
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