Preparation of Calex 231 - Journal of Biological Chemistry · E837A7.39, caused a loss of Calex 231’s ability to inhibit Ca2+-induced accumulation of [3H]inositol phosphates. Three

Mapping the binding pocket of a calcilytic

1

Modeling and mutagenesis of the binding site of Calex 231, a novel negative allosteric

modulator of the extracellular Ca2+ sensing receptor

by

Christophe Petrel*, Albane Kessler&, Fouzia Maslah#, Philippe Dauban&, Robert H.

Dodd&, Didier Rognan# and Martial Ruat*†

Running title: Mapping the binding pocket of a calcilytic

*Institut de Neurobiologie Alfred Fessard, IFR 2118 CNRSLaboratoire de Neurobiologie Cellulaire et Moléculaire, UPR 9040 CNRSBâtiment 33, 1 avenue de la terrasse, 91198 Gif sur Yvette, France.

&Institut de Chimie des Substances Naturelles, UPR 2301 CNRS, 1 avenue de la terrasse, 91198 Gifsur Yvette, France.

#Laboratoire de Pharmacochimie de la Communication Cellulaire, UMR 7081 CNRS, 74 route duRhin, B.P. 24, 67401 Illkirch, France.

† Corresponding author: [email protected];phone: 33 1 69 82 36 41; fax: 33 1 63 82 36 39

Abbreviations: CaSR, Calcium sensing receptor; Calex 231, (1S,2S,1'R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane; ECL2, extracellular loop 2; GABAB, gamma aminobutyric acid; GST, Glutathion S-transferase; GPCR, G-protein coupled receptor; HEK, humanembryonic kidney; PTH, parathyroid hormone; TM, transmembrane; mGlu, metabotropic glutamate;2-D, two dimensional; 3-D, three dimensional; [3H]IP, tritiated inositol phosphates.

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ABSTRACT

A model of the calcium sensing receptor (CaSR) seven transmembrane domains was

constructed based on the crystal structure of bovine rhodopsin. This model was used for

docking (1S,2S,1'R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane

(Calex 231), a novel potent negative allosteric modulator which blocks (IC50 of 0.39 µM)

increases of [3H]inositol phosphates elicited by activating the human wild type CaSR

transiently expressed in HEK293 cells. In this model, E8377.39 plays a pivotal role in

anchoring the two nitrogen atoms of Calex 231 and locating the aromatic moieties in two

adjacent hydrophobic pockets delineated by transmembrane domains 3, 5 and 6, and

transmembrane domains 1, 2, 3 and 7, respectively. To demonstrate its validity, we have

mutated selected residues and analyzed biochemical and pharmacological properties of the

mutated receptors transfected in HEK293 cells. Two receptor mutations, F684A3.32 and

E837A7.39, caused a loss of Calex 231’s ability to inhibit Ca2+-induced accumulation of

[3H]inositol phosphates. Three other mutations, F688A3.36, W818A6.48, and I841A7.43,

produced a marked increase of the IC50 of Calex 231 for the Ca2+ response whereas L776A5.42

and F821A6.51 mutations led to a decrease of the IC50. Our data validate the proposed model

for the allosteric interaction of Calex 231 with the seven transmembrane domains of the

CaSR. Interestingly, the residues at the same positions have been shown to delimit the

antagonist-binding cavity of many diverse GPCRs. Our present study furthermore suggests

that the crystal structure of bovine rhodopsin exhibits sufficient mimicry with the ground state

of a very divergent class 3 receptor to predict the interaction of antagonists with the

heptahelical bundle of diverse GPCRs.


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INTRODUCTION

The extracellular Ca2+ sensing receptor (CaSR) plays an essential role in the regulation

of Ca2+ homeostasis. Located at the cell surface of the parathyroid cell, the CaSR is stimulated

by serum Ca2+ and controls PTH release (1). Initially cloned from bovine parathyroid (2), the

CaSR has been isolated from various species and tissues (3-6). CaSR activation results in

calcitonin secretion in the thyroid and Ca2+ reabsorption in the kidney. CaSR on nerve

terminals may regulate neurotransmitter release (3,7) and its presence on oligodendrocyte

cells suggests that it participates in the complex processes of myelination (8,9). Its

physiological importance is further illustrated in several disorders linked to Ca2+ homeostasis

resulting in gain or loss of function mutations (10).

The CaSR belongs to family 3 of G-protein coupled receptors (GPCRs) which

comprises eight mGlu receptors, GABAB, vomeronasal, pheromone and taste receptors. These

GPCRs possess an unusual long bilobed aminoterminal extracellular domain resembling

bacteria periplasmic binding protein implicated in nutrients transport and postulated to contain

the ligand binding sites of these receptors (11,12). The CaSR is activated by Ca2+ and Mg2+

present in the extracellular fluids, by charged molecules including spermine, spermidine, β-

amyloid peptides and several antibiotics (2,4,8,13-15). Recently, low molecular-weight

synthetic molecules activating the CaSR have been identified and their pharmacological

properties with respect to cloned CaSR reported (16-19). It has been proposed that these

molecules, named calcimimetics, interact allosterically within the 7 transmembrane (TM)

domains to potentiate the effect of Ca2+ (20-22). On the other hand, compounds that inhibit

the effect of Ca2+ toward the CaSR are called calcilytics (23). Controlling transient PTH

release by blocking the parathyroid CaSR with such molecules has been hypothesized to

produce anabolic effects in bone and represents a major therapeutic interest in the treatment of

osteoporosis (23). Moreover, such compounds might be useful for studying the roles played


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by the CaSR in tissues under physiological and pathological states. NPS 2143 was the first

negative allosteric modulator acting on the CaSR whose properties have been investigated

both in vitro and in vivo (24,25). We have recently synthesized and evaluated the in vitro

pharmacological properties of a novel structurally different series of calcilytics acting on the

cloned rat CaSR (26). We now report the calcilytic properties of (1S,2S,1'R)-N1-(4-

chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane (Calex 231, Fig. 1) which

belongs to this family of molecules, and the characterization of its potency towards the human

CaSR transiently expressed in HEK293 cells. Calex 231 shows in vitro potency comparable to

NPS 2143 in inhibiting Ca2+-induced activation of the human CaSR (25). We have developed

a 3-D model of the TM domains of the human CaSR based on the crystal structure of bovine

rhodopsin (27). This model has allowed us to dock Calex 231 into a hydrophobic cavity

centered on Glu837 (7.39 in Ballesteros numbering) (28) having two adjacent hydrophobic

pockets. We used site directed mutagenesis of amino acid residues likely involved in the

recognition of Calex 231 to demonstrate the validity of this model, and to propose a possible

binding mode of this negative allosteric CaSR modulator within the 7TM domains.

EXPERIMENTAL PROCEDURES

Material

Preparation of Calex 231

(1S,2S,1'R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane

(Calex 231) was prepared as its hydrochloride salt. Briefly, cyclohexene in acetonitrile was

treated with [(N-p-nitrobenzenesulfonyl)imino]phenyliodane in the presence of a catalytic

quantity of copper (II) triflate to give the aziridine (±)-7-(4-nitrobenzenesulfonyl)-7-

azabicyclo[4.1.0]heptane. The latter was reacted in triethylamine-containing THF with (R)-1-


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(1-naphthyl)ethylamine to afford the aziridine ring-opened product N1-(4-

nitrobenzenesulfonyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane. Removal of the p-

nitrobenzenesulfonyl group by the action of thiophenol/potassium carbonate in

acetonitrile/DMSO followed by acylation of the resulting free amine with 4-chlorobenzoyl

chloride provided (1S,2S,1’R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-

diaminocyclohexane, i.e., Calex 231, as the slower moving of two components on silica gel.

The absolute configuration of Calex 231 was deduced by X-ray crystallography. Details of the

synthesis and structural characterization of Calex 231 will be published elsewhere.

Site-directed mutagenesis

In order to mutate amino acids possibly involved in the binding site of Calex 231, the

coding region of the human wild type (WT) CaSR, kindly provided by Pr. M. Freichel (6),

was first cloned in HindIII/XbaI sites in a modified pUC18 plasmid where SacI and SmaI

restriction sites were removed (pUCmCaSR). A SacI-BamHI insert encompassing the coding

region corresponding to TM domain 2 to 7 was then cloned in pBluescript SK+ plasmid

(Stratagene) and site directed mutagenesis was performed. A SacI-SmaI fragment containing

the mutation was then replaced in the pUCmCaSR plasmid to obtain the final mutated CaSR.

Finally, WT and mutant CaSR coding region were subcloned into HindIII-XbaI sites of

pcDNA3 expression vector (Invitrogen). All point mutants were constructed using the

QuickChange site-directed mutagenesis kit (Stratagene), performed with specific

oligonucleotides (Eurobio, Les Ulis, France) to convert residues to alanine (sequences of

oligonucleotides available on request). Sequencing was performed on both strands on

pBluescript SK+ plasmid containing the mutated fragment and on the final pcDNA3 vector

(Eurogentec, Ivoz-Ramet, Belgium).


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Cell culture and transfection

HEK293 cells (Eurobio, Les Ulis, France) were cultured in Dulbecco’s modified

Eagle’s medium (DMEM) supplemented with 10 % dialyzed fetal calf serum (Life

Technologies) and were transiently transfected using gene pulser apparatus (Bio-Rad) by

electroporation (270 Volts, 975 microfarads). Briefly, 4 µg of pcDNA3 plasmids containing

WT or mutated human CaSR DNA were supplemented with 6 µg of pRK5 plasmid and were

used to transfect 106 cells in a total volume of 300 µL of electroporation buffer (K2HPO4, 50

mM; CH3COOK, 20 mM; KOH, 20 mM; MgSO4, 26.6 mM; pH 7.4). After electroporation,

cells were resuspended in culture medium and distributed on a 24 well plate coated with 100

µg/ml rat tail collagen (Becton Dickinson, Meylan, France) for [3H]inositol phosphates

([3H]IP) analysis or plated on a 75 cm2 tissue culture flask for Western blot analysis,

respectively.

[3H]IP formation

Cells were labeled by 0.5 µCi/well of myo-[3H]inositol (Amersham) for 20 h in their

growth medium and measurement of [3H]IP accumulation was performed as described (13).

Data are expressed as mean ± S.E.M of triplicate determinations and are representative of one

out of three to ten independent experiments. The activities of WT and mutant CaSRs were

determined in response to increasing extracellular Ca2+ concentration, or to Calex 231 in the

presence of 10 mM Ca2+. EC50 values for Ca2+ and IC50 values for Calex 231 were calculated

using GraphPad prism 2.01 (GraphPad Prism Software Inc., San Diego, USA) and

significance was assayed by the Excel 98 Student T test (Microsoft, Seattle, USA).

Generation of polyclonal antiserum to human CaSR

A DNA encoding part of the carboxy-terminal domain of the human CaSR (residues

747-1077) (6) was amplified by PCR, inserted into a glutathion S-transferase (GST) fusion


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pGEX-4T-1 vector (Amersham Bioscience, Saclay, France) and sequenced (Eurogentec, Ivoz-

Ramet, Belgium). The production of a GST fusion protein in the bacteria strain Escherichia

coli BL21 was then purified (29) and 100 µg were injected into a rabbit to generate 141Ab

antiserum.

Western blot analysis

Cells were homogenized in ice cold 50 mM Tris-HCl, pH 7.4, containing 1 mM

EDTA, aprotinin (10 µg/ml), benzamidine (10 µg/ml), phenylmethylsulfonyl fluoride (10

µg/ml) and leupeptin (10 µg/ml). Proteins from whole cell lysate (30) were diluted into lysis

buffer (25 mM Tris pH 6.8; 6 % glycerol; 0.005 % bromophenol blue; 2.5 % SDS; 2.5 % β-

mercaptoethanol). 4 µg of proteins were separated on 8 % SDS-polyacrylamide gel then

transferred to nitrocellulose membranes that were probed for 2 h at room temperature with

141Ab antiserum (1/3000). The immunoreactivity was then revealed as described (16).

Alignment of amino acid sequences

The amino acid sequences of four human GPCRs were retrieved from the Swiss-Prot

database (accession numbers: CaSR receptor, P41180; mGluR type 1 receptor, Q13255;

subunit 1 of the GABAB receptor, Q9UBS5; beta2 adrenergic receptor, P07550). Their TM

domains were extracted and aligned with those of bovine rhodopsin (accession number:

P02699) using the in-house developed GPCRmod program (31). A ClustalW multiple

alignment (32) was then performed on extra- and intracellular loops while maintaining the

relative alignment of the 7TM domains fixed. A slow pairwise alignment using BLOSUM

matrix series (33) and a gap opening penalty of 15.0 were chosen for aligning the amino acid

sequences to the sequence of bovine rhodopsin.


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Preparation of starting protein coordinates

The 3-D model of the human CaSR was constructed using a previously described

procedure (34). Briefly, starting from the X-ray structure of bovine rhodopsin (pdb entry:

1f88), a first model of the 7TM domains was obtained by mutating the side chains of the

amino acids in rhodopsin. Standard geometries for the mutated side chains were given by the

BIOPOLYMER module of SYBYL (Tripos Assoc., Inc, St. Louis, U.S.A.). Whenever

possible, the side chain torsional angles were kept to the values occurring in bovine

rhodopsin. Otherwise, a short scanning of side chain angles was performed to remove steric

clashes between the mutated side chain and the other amino acids. The third intracellular loop

between TM domain 5 and 6, which shows a high degree of variability, was not included in

any of the models. This loop is believed for most GPCRs to be far away from the TM binding

cavity (35). We therefore assume that omitting this loop should not influence our docking

results. The observed insertions/deletions in the loops of the CaSR were achieved through a

simple knowledge-based loop search procedure as previously reported (34). Special caution

had to be given to the second extracellular (ECL2) loop, which has been described in bovine

rhodopsin to fold back over the heptahelical bundle (27), and therefore limits the size of the

active site. Two models of this loop were proposed. A first one assumes a rhodopsin-

dependent folding and was then obtained by direct threading to the rhodopsin template. In this

model, one residue was inserted 8 positions before Cys765 and 9 amino acids were deleted

after His766. A second model assuming a rhodopsin-independent fold was obtained by

searching for two loops of 12 and 11 residues, respectively, linking Ser750 to Thr764 and

His766 to Ser769. After the heavy atoms were modeled, all hydrogen atoms were added, and

the protein coordinates were then minimized with AMBER6 using the AMBER95 force field

as previously described (34).


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Modeling the Calex 231-bound CaSR

To obtain a ligand-bound model of the CaSR, the above-described coordinates were

refined as previously described (34) to enlarge the binding cavity. Briefly, Calex 231 was first

manually docked into the TM cavity by anchoring its protonable nitrogen atom to the only

negatively-charged residue of the binding site (Glu837) and fitting the shape of the two

aromatic moieties into the two proximal hydrophobic pockets. The bulky naphthalene group

was docked into the bigger of the two pockets (pocket A: Pro682, Phe688, Val689, Tyr744,

Pro748, Leu776, Trp818, Phe821, Ala840) while the substituted phenyl moiety was located

into the smaller of the two pockets (pocket B: Phe612, Ala615, Leu616, Ser665, Phe668,

Ile669, Phe684, Val838, Ile841). After parameterization of Calex 231 for the AMBER6 force-

field using a previously-reported protocol (36), the resulting protein-ligand complex was then

refined by minimization using the above-described AMBER parameters. Removing the ligand

atoms from the minimized complex finally yielded one set of coordinates for the Calex 231-

bound receptor model.

Automated docking of Calex 231

To verify that the above-described coordinates were not biased by the manual docking

procedure, the Surflex docking program (37) was used to automatically dock Calex 231. An

idealized active site ligand or protomol (38) was first generated from 33 consensus positions

(34) supposed to map the TM cavity of most GPCRs. This protomol consists of the preferred

locations of various molecular probes (CH4, C=O, N-H) that are then used by the docking

engine to search for the best 3-D morphological similarity between the protomol and the

ligand to dock. A proto_thresh value of 0.5 and a proto_bloat value of 0 were used to generate

a compact protomol. A TRIPOS mol2file of Calex 231, obtained from a 2-D sketch as

previously reported (34) was docked into the TM cavity using standard parameters of Surflex


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used in the "whole" docking approach (37). The best 30 solutions were finally stored in mol2

format.

RESULTS

Potency of Calex 231

In a recent preliminary report, we described the synthesis and the characterization of a

novel series of molecules displaying calcilytic properties towards rat CaSR (26). We have

now synthesized Calex 231, which belongs to this family of molecules, and we have

investigated its potency towards the human CaSR by measuring its effects on Ca2+-induced

accumulation of [3H]IP, a well characterized response linked to CaSR activation (2,13,39).

Increasing the concentration of extracellular Ca2+ from 0.3 to 10 mM caused a 10 fold

increase of [3H]IP accumulation in HEK293 cells transiently transfected with a plasmid

containing the human WT CaSR whereas we did not detect a significant increase of [3H]IP

accumulation in cells transiently transfected with an empty control plasmid (Fig. 2A and data

not shown). Analysis of the dose response curve led to an EC50 for Ca2+ of 3.4 ± 0.1 mM

(mean ± S.E.M., n = 10). These data fit well with the affinity for Ca2+-mediated increase of IP

accumulation previously determined for the human CaSR (39). Preincubation of HEK293

cells expressing the human WT CaSR with Calex 231 caused a concentration-dependent

inhibition of the IP response to 10 mM Ca2+ (Fig. 2B). Analysis of the dose response curve

led to an IC50 for Calex 231 of 0.39 ± 0.08 µM (mean ± S.E.M., n = 7). These data indicate

that Calex 231 is a potent calcilytic of the human CaSR transiently expressed in HEK293

cells.


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Molecular modeling of the 7TM domains of the human CaSR

To elucidate the binding mode of Calex 231, we postulated that the ligand binding

pocket could be localized within the 7TM domains of the CaSR. First, we developed a model

of the human CaSR based on the X-ray structure of bovine rhodopsin (27) which was used as

a template to model the 7TM domains of the CaSR. Using the in-house developed GPCRmod

program (31), we could unambiguously assign the position of the seven helices by the use of

GPCR-dependent TM-specific amino acid fingerprints (Fig. 3A), and thread CaSR 3-D

coordinates onto that of bovine rhodopsin. Two models were generated differing only in the

fold of the ECL2 loop (Figs. 3B, C). A first one assumes a conserved folding of the ECL2

loop over the 7TM bundle as in bovine rhodopsin. A rationale for this first choice was the

presence of two conserved cystein residues at positions 677 in TM domain 3 and 765 in ECL2

of the CaSR which are also present in bovine rhodopsin to form a disulfide bridge. A second

model of the ECL2 loop was derived independently from the rhodopsin structure as it is

questionable whether the particular fold of the ECL2 loop in rhodopsin is a common feature

of most GPCRs (Fig. 3C).

As previously reported (34), the TM cavity was enlarged in order to accommodate a

ligand for automated docking. This procedure requires the manual positioning of Calex 231.

As there is only a single accessible negatively-charged residue in the TM cavity (Glu8377.39)

available to neutralize the positively-charged secondary amine of the ligand, anchoring Calex

231 into the TM cavity was straightforward (see experimental procedures). After energy

refinement of the receptor-ligand complex and subsequent expansion of the binding cavity,

the CaSR forms a well-defined hydrophobic cavity centered on Glu837 located in the 7TM

domain with two adjacent hydrophobic pockets A and B. The bigger pocket A is delineated

by hydrophobic side chains between TM domains 3, 5 and 6 whereas the smaller pocket B is

located between TM domains 1, 2, 3 and 7.


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Hypothesized binding mode of Calex 231

Automated docking of Calex 231 with the recently-described Surflex docking program

(37) disclosed a preferred binding mode (Figs. 4A, B) in which both nitrogen atoms are H-

bonded to Glu837. The close proximity of the protonated secondary amine to the negatively-

charged Glu837 side chain indicates a likely ionic interaction between both moieties. The

naphthalene moiety is embedded in pocket A and interacts with neighboring hydrophobic side

chains (Pro682, Phe688, Val689, Tyr744, Pro748, Leu776, Trp818, Phe821). The para-

chlorophenylgroup is buried in the additional pocket B (Phe612, Ala615, Leu616, Phe668,

Ile669, Phe684, Val838, Ile841). Interestingly, the cyclohexyl scaffold is proposed to be

located in a small hydrophobic niche delimited by Pro682, Phe684 and Gly685. The important

methyl group at the chiral carbon atom is directly facing the Phe821 aromatic ring. The

proposed interaction model suggests a tight binding of Calex 231 as 85 % of it overall surface

(573 out of 674 Å2) is buried upon binding to the TM cavity.

Generation of point mutations and characterization of the mutated CaSR

We have mutated into alanine T764 and H766 located in ECL2 as well as 7 other

amino acid residues located in TM domains 3, 5, 6 and 7 by site-directed mutagenesis to

investigate their possible interactions with Calex 231. These mutants as well as the WT

receptor were transiently transfected into HEK293 cells. We then analyzed their ability to

respond to Ca2+ by measuring [3H]IP accumulation and their expression by Western blots

using a specific rabbit antiserum (141Ab) developed against the carboxyl-terminal tail of the

human CaSR.

This antiserum was generated against a 330 amino acid polypeptide starting from

amino acid 747 of the CaSR and fused to GST. The 141Ab antiserum was first evaluated by

Western blot analysis against the human WT receptor. Under reducing conditions, two


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polypeptides migrating with a mobility corresponding to relative molecular masses of 150 and

130 kDa were identified in membrane preparations from cells transfected with the WT

receptor whereas these signals were absent from mock cell preparations thereby indicating

their specificity (Fig. 5). Our results are in agreement with previous carbohydrate analysis of

the human receptor expressed in HEK293 cells showing that the polypeptides of higher

molecular weight correspond to N-linked glycosylated receptors expressed at the cell surface

whereas the polypeptides of lower molecular weight represent intracellular mannose-modified

receptors (40). A polypeptide complex migrating above 200 kDa was also identified and

might correspond to intermolecular linked dimers as previously observed (40). Importantly,

the expression pattern of the different mutant receptors was comparable to that of the WT

receptor as accessed by immunoblot (Fig. 5) indicating that the alanine substitution at the

various positions analyzed did not abolish the expression of the CaSR.

All tested mutants responded to Ca2+ (Figs. 6A, B and Table 1). The mutants harboring

F684A or F688A mutations showed a rightward shift in sensitivity with an EC50 of 5.9 ± 0.4

mM and 5.9 ± 0.2 mM (mean ± S.E.M., n = 3, p<0.001), respectively, whereas three mutants

with L776A, F821A or I841A mutations, showed a moderate but significant increase in Ca2+

sensitivity with EC50's below 3 mM (p<0.05, n = 3). The other mutants studied did not differ

in Ca2+ sensitivity compared to the WT receptor. The maximal response to Ca2+ was reduced

by 2 fold for mutants harboring the F684A or F688A mutation compared to the WT receptor

and we observed only a moderate decrease of the maximal response (30 % reduction) for

mutants with W818A or E837A mutations (Table 1). These data demonstrate that the mutant

and WT receptors are functional after transfection in HEK293 cells.


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Functional analysis of ECL2 mutants for Calex 231 inhibition of Ca2+-promoted increases

of IP response

Since all the mutants examined display a maximal IP response at 10 mM Ca2+ (Figs.

6A, B and Table 1), we chose to analyze the effects of Calex 231 in inhibiting this maximal

response for each mutant. We first transfected in HEK293 cells the mutants harboring T764A

or H766A mutations located in the ECL2, and we constructed the dose response curves for

Calex 231 inhibition of Ca2+-induced accumulation of IP. Calex 231 dose-dependently

inhibited the IP response induced by 10 mM Ca2+ with a potency in T764A (IC50 = 0.28 ±

0.05 µM) and H766A (IC50 = 0.64 ± 0.03 µM) (mean ± S.E.M., n = 3) mutant receptors

similar to the WT receptor. These data indicate that these amino acid residues do not

participate significantly in the binding of Calex 231 to the receptor.

Functional analysis for Calex 231 inhibition of mutants located in TM domains

We then analyzed the effect of mutations located in TM domains 3, 5, 6 and 7 on

Calex 231 inhibition of Ca2+-induced increases of IP response (Fig. 7 and Table 1). The dose

response curves of the ligand for the two mutants with the F684A or E837A mutations located

in TM domains 3 and 7, respectively, were profoundly affected and are shown in Figs. 7A and

7B, respectively. Calex 231 lost its ability to block the Ca2+-induced IP response in CaSR

having the point mutation F684A or E837A (<30% and <20% inhibition by 10 µM Calex 231,

respectively). These data indicate that these two amino acid residues are crucial for Calex 231

recognition. Its dose response curve for each of three other mutants with F688A, W818A or

I841A mutations, was right-shifted, indicating a marked increase (~10 fold) of the IC50 value.

It is worth noting that F688 and I841 are located near the two crucial residues F684 and E837,

respectively, confirming the importance of these two regions for Calex 231 recognition. The

two last mutations studied, L776A and F821A, led to significant decrease of the IC50 of Calex


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231 in inhibiting Ca2+-induced IP response for the mutants (IC50 = 0.07 ± 0.03 µM and IC50 =

0.06 ± 0.01 µM, respectively) compared to the WT receptor (IC50 = 0.39 ± 0.08 µM, p<0.01)

DISCUSSION

In this study, we report the characterization of Calex 231, a novel negative allosteric

modulator of the human CaSR. We used molecular modeling approaches, mutagenesis and

functional activity (PLC) in order to identify for the first time residues involved in the binding

pocket of a negative modulator of the CaSR. The aminoterminal domain of the CaSR is

thought to contain the Ca2+ binding sites and has been submitted to extensive mutation and

deletion studies that have given insight on the mechanism of CaSR activation (41-43).

However, little is known about the binding sites of positive or negative allosteric modulators

of the CaSR. An amino acid residue (E837) located in TM7 has been reported to interact with

the calcimimetic NPS R-568 (21), whose pharmacological properties with respect to cloned

CaSR have been previously reported (16,18). At the present time, the sites of interaction with

the CaSR of NPS 2143, the first and sole calcilytic whose pharmacokinetic properties have

been reported in vitro and in vivo (24,25), have not yet been described. We have recently

identified a novel class of molecules inhibiting the effect of Ca2+ on the cloned rat CaSR

expressed in Chinese ovary cells (26). We have now synthesized Calex 231 which belongs to

this family of molecules and shown that it behaves as a potent and high affinity negative

allosteric modulator of the human CaSR. Although the CaSR and rhodopsin display little

amino acid identity, we have generated a 3-D model of the 7TM domains of the CaSR which

has allowed identification of putative residues implicated in the recognition of Calex 231. We

have submitted nine of these residues to mutations and found that seven of them affect the

binding affinity of Calex 231 as measured by inhibition of Ca2+-induced IP accumulation, a


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well characterized functional response linked to CaSR activation in these cells (39), thus

confirming that Calex 231 is a negative allosteric modulator of the CaSR.

Indeed, the binding cavity of Calex 231, as disclosed by the present study, shares

numerous similarities with antagonist-binding cavities of other GPCRs (Table 2 and

Supplementary information). Our current data demonstrate that three residues, Phe6843.32,

Phe6883.36, and Glu8377.39, occupying central positions in the 7TM bundle (Fig. 3A) are in

direct contact with Calex 231. These three positions are well known to map the competitive

antagonist-binding cavity of many GPCRs (Table 2). For example, position 3.32 is the

principal anchoring residue (Asp3.32) of all monoamine receptors (44). Position 3.36 has also

been shown to play a pivotal role in antagonist binding to several different GPCRs. Lastly,

Glu8377.39 is a key residue of various unrelated GPCRs for anchoring competitive antagonists

and is implicated in the recognition of a reference calcimimetic in the CaSR (21). Our results

concerning the E837A mutant are in agreement with those obtained by Hu and collaborators

showing that this mutant is expressed at comparable levels as the WT when transfected in

HEK293 cells, and its sensitivity to Ca2+ is not altered despite a lower maximal response (21).

Interestingly, mutating a glutamic acid at this position in the few GPCRs where it is

conserved (most chemokine receptors, interleukine 8 receptors) leads to the same detrimental

effect on ligand binding (45-47). The fourth important CaSR residue (Trp8186.48) delineated

by the present study is conserved in ~70% of all GPCRs of class I, II and III. Its hydrophobic

side chain is believed to lock the GPCR in a ground state and to interact with most antagonists

(Table 2). In the hypothesized model, Trp8186.48 is surrounded by two other aromatic residues

(Phe8216.51, Phe6883.36) forming an aromatic cluster around Glu8377.39 and preventing

rotation of TM domain 6 which seems mandatory for activation of many GPCRs (48).

A significant decrease of Calex 231’s effect is observed after mutation of Ile8417.43. In

our model, Ile8417.43 interacts with the para-chlorophenyl moiety of Calex 231. This position


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has already been shown to directly contact adenosine as well as peptide receptor antagonists

(Table 2). Surprisingly, the mutation of two positions (Leu7765.42, Phe8216.51) leads to

receptor mutants with significantly enhanced Calex 231 antagonist activities. In many

monoamine receptors, position 5.42 is a serine that has been shown to directly interact with

the catechol group of many GPCR ligands (49). Phe8216.51 is involved in the conformational

switch triggering of GPCR activation (48). The consequence of these particular mutations is

difficult to explain at the molecular level. Both contribute to enlarge pocket A and perhaps

induce slight conformational changes allowing better accommodation of the bulky

naphthalene group of Calex 231.

Altogether, the present data suggest that the bovine rhodopsin X-ray structure exhibits

a significant molecular mimicry with the ground state of not only class 1 monoamine

receptors (28) but also with the TM domains of the very divergent class 3 GPCRs, as very

recently proposed for the metabotropic type 1 receptor (50). Interestingly, the central 6.48

(Trp818) and 7.39 (Glu837) positions also play a key role in recognizing a non competitive

mGluR1 antagonist (50) suggesting that the TM binding cavities of class III GPCRs largely

overlap.

However, some discrepancies with the bovine rhodopsin structure remain. By

opposition to retinal, the two residues (Thr764, His766) adjacent to a conserved cystein

(Cys765) involved in disulfide bridging between ECL2 and TM3 (Cys677) do not contribute

to the binding site of Calex 231. Thus it is likely that the overall folding of the ECL2 loop

differs significantly from that of bovine rhodopsin bound to retinal.

In summary, we have to our knowledge identified for the first time the amino acids

involved in defining the ligand binding pocket of a negative allosteric modulator of the CaSR.

Since calcilytics are proposed to represent a novel therapeutic approach for treating

osteoporosis (23), it is of major interest to delineate the residues involved in their recognition.


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Our study should facilitate the understanding of how calcilytics interact with the CaSR and

the development of molecules with increased affinity and selectivity. Moreover, Calex 231

represents a novel calcilytic which should be highly useful for studying the role of CaSR in

tissues such as bone, kidney and brain under physiological and pathological conditions.

Acknowledgment: We thank H. Faure and E. Traiffort for advice concerning transfection and

mutagenesis.


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LEGEND OF THE FIGURES AND TABLES

Fig. 1. Chemical structure of Calex 231

Fig. 2. Potency of Calex 231 in inhibiting Ca2+-induced accumulation of [3H]IP by the

human CaSR

(A) Concentration-response curve of Ca2+-induced IP stimulation (expressed as

cpm/well x 1000) in HEK293 cells transiently transfected with WT human CaSR. Cells were

prelabeled overnight with 0.5 µCi/ml myo-[3H]inositol, washed twice with basal Ham’s F12

medium supplemented with 10 mM of LiCl, and incubated with increasing concentrations of

CaCl2 for 30 min. (B) Concentration-dependent inhibition of Ca2+-stimulated (10 mM)

increases of IP response by Calex 231 in HEK293 cells expressing the WT human CaSR.

After the prelabeling and washing procedures, cells were incubated with 10 mM Ca2+ alone or

in the presence of increasing concentrations of Calex 231 for 30 min. Results are expressed as

% of maximal response observed with 10 mM Ca2+. IP in (A) and (B) were measured as

described in “Experimental procedures” and data are means ± S.E.M. of triplicates from a

typical experiment representative of seven to ten experiments.

Fig. 3. Amino acid sequence alignment of the seven TM domains of five GPCRs and

close-up of the ECL2 alignment of the human CaSR to bovine rhodopsin

(A) Alignment of the human CaSR, MGR1, GBR1, B2AR, OPSD was performed by

GPCRalign (31). Residues in bold face are typical fingerprints (51) from either class I (B2AR,

OPSD) or class III GPCRs (CaSR, MGR1, GBR1). Boxed positions correspond to herein

described mutation effects. The Ballesteros residue numbering (28) is indicated above the

proposed sequence alignment. CaSR, calcium sensing receptor; MGR1, metabotropic

glutamate receptor type 1; GBR1, gamma amino butyric receptor type 1, B2AR, beta


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adrenoreceptor type 2; OPSD, bovine rhodopsin. (B) Close-up of the sequence alignment of

the ECL2 of the human CaSR to bovine rhodopsin. Residues neighboring the conserved

cystein residue involved in a disulfide bridge with the third transmembrane domain are boxed.

(C) Two putative models of the ECL2 loop of the CaSR obtained either by direct threading to

the rhodopsin X-ray structure (in green) or by a loop search procedure (in cyan). The SCG

residues of bovine rhodopsin (in white) facing retinal (27) are displayed as sticks with the

following color coding: carbon atom, white; oxygen atom, red; nitrogen atom, blue; sulfur

atom, yellow. Retinal structure and location of TM domains 4 and 5 (TM4, TM5) are shown.

Arrows indicate the path of the main chain.

Fig. 4. Molecular modeling of the human CaSR complexed with Calex 231

Proposed interaction model between Calex 231 and the CaSR binding cavity. TM

helices are displayed as yellow ribbons. Calex 231 as well as important CaSR heavy atoms

are indicated by sticks using the following color coding: carbon atom of Calex 231, cyan;

carbon atom of the CaSR, white; oxygen atom, red; nitrogen atom, blue; chloride atom, green.

Important side chain positions of the CaSR are labeled at the Cα atom. Intermolecular

hydrogen bonds between Glu837 and the two nitrogen atoms of Calex 231 are represented by

dotted yellow lines. Mutations discussed herein are displayed by yellow labels. (A) Front

view. (B) Top view from the extracellular side. Calex 231 is embedded in pockets A and B.

Fig. 5. Expression of the CaSR mutants

Immunoblot analysis of whole cell lysates (4 µg proteins) from HEK293 cells

transiently transfected with an empty vector (MOCK), or a vector containing the wild type

CaSR (WT) or the indicated mutant CaSRs, was performed by SDS polyacrylamide gel

electrophoresis as described in “Experimental procedures”. CaSR proteins were detected


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using the specific rabbit 141Ab serum directed against the carboxyl terminal region of the

human CaSR. The position of the molecular mass markers is shown on the left. Arrow heads

on the right indicate the molecular weight (kDa) of two major bands corresponding to the WT

and mutant receptors.

Fig. 6. Effect of some CaSR mutations on Ca2+-induced accumulation of [3H]IP after

transient transfection in HEK293 cells

Concentration-response curves of Ca2+ induced IP stimulation (expressed as % of

maximal response observed with 10 mM Ca2+) in HEK293 cells transfected with WT or

mutant receptors as indicated in A and B. The cells were transfected with the adequate vector

and the IP response to Ca2+ performed as described in “Experimental procedures”. Data are

means ± S.E.M. of triplicates from a typical experiment representative of 3-5 experiments.

Fig. 7. Effect of CaSR mutations on inhibition of Ca2+-stimulated increases of IP by

Calex 231

Concentration-dependent inhibition of Ca2+-stimulated (10 mM) increases of IP

response by Calex 231 in HEK293 cells expressing the WT or mutated receptors as indicated

in A and B. The cells were transfected with the adequate vector and the IP response to Ca2+

performed as described in “Experimental procedures”. After prelabeling and washing

procedures, cells were incubated with 10 mM Ca2+ alone or in the presence of increasing

concentrations of Calex 231 for 30 min. Data are expressed as % of maximal IP response

observed with 10 mM Ca2+ and are means ± S.E.M. of triplicates from a typical experiment

representative of 3-5 experiments.


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Table 1: Summary of the effects of various CaSR mutations on the properties of Ca2+

and Calex 231 on the IP response

Receptor Position % of maximal Ca2+ Calex 231 WT response EC50 ± S.E.M., mM IC50 ± S.E.M., µM

WT 100 ± 4 3.4 ± 0.1 0.39 ± 0.08F684A TM3 50 ± 4** 5.9 ± 0.4** >10 -5

F688A TM3 50 ± 3** 5.9 ± 0.2** 3.20 ± 0.98**T764A ECL2 120 ± 9 3.0 ± 0.3 0.28 ± 0.05H766A ECL2 102 ± 8 3.2 ± 0.3 0.64 ± 0.03L776A TM5 121 ± 9 2.2 ± 0.3 0.07 ± 0.03*W818A TM6 72 ± 4** 3.4 ± 0.2 3.30 ± 0.50**F821A TM6 112 ± 10 2.6 ± 0.2 0.06 ± 0.01*E837A TM7 72 ± 5** 3.8 ± 0.2 >10 -5

I841A TM7 98 ± 6 2.9 ± 0.2 2.71 ± 0.10**

Concentration-response curves for Ca2+ and for Calex 231 were generated as described

in the legend of figures 6 and 7. EC50’s and maximal stimulation for Ca2+ compared to a

maximal Ca2+ response at WT CaSR, and IC50’s for Calex 231 were calculated. Data shown

are means ± S.E.M. from three to ten independent experiments. Level of significance

compared to wild type receptor: * p<0.01; **. p<0.001


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Table 2. GPCRs sharing with the CaSR the same TM positions for delimiting the

antagonist-binding cavity.

Positiona Receptorsb

3.32 monoamine receptors, OPSD

3.36 A1AR, CKR5, D2DR, D3DR, MGR5, NK1R, OPSD, OXYR, TSHR

5.42 AG2R, CKR5, monoamine receptors, OPSD

6.48 AA3R, ACM1, ACM2, AG2R, BRB2, D2DR, GASR, GRHR, OPRD,

OPRX OPSD, MGR1, TRFR, V1AR

6.51 5H4, A1AB , ACM1, AG2R, BRB2, D2DR, GASR, GRHR, NK1R, NK2R,

NTR1, NY2R, OPSD, V1AR

7.39 5H1D, AA1R, AA2A, ACM1, B2AR, C5a, CCKR, CKR2, CKR5, D2DR,

D3DR, GASR, MGR1, OPRD, OPRX, OPSD, P2YR, PI2R, US28, V1AR

7.43 AA1R, AA2A, AA3R, ACM1, AG2R, BRB2, GRPR, NTR1, OPRD,

OPRM, OPSD

a Ballesteros numbering

b Swiss-Prot identification: OPSD, bovine rhodopsin; monoamine receptors (acetylcholine,

adrenergic, dopamine, histamine, serotonin, octopamine and trace amine receptors); A1AR,

alpha adrenoreceptor type 1a; CKR5, C-C chemokine type 5; D2DR, dopamine D2; D3DR,

dopamine D3; MGR5, metabotropic glutamate type 5; NK1R, neurokinin receptor type 1;

OXYR, oxytocin receptor; TSHR, thyrotropin stimulating hormone receptor; AG2R,

angiotensin receptor type 1; AA3R, adenosine type 3; ACM1, muscarinic M1; ACM2,

muscarinic M2; BRB2, bradykinine type 2; GASR, cholecystokinin type B; GRHR,

gonadotropin-releasing hormone receptor; OPRD, opioid delta receptor; OPRX, nociceptin

receptor; MGR1, metabotropic glutamate receptor type 1; TRFR, thyrotropin releasing


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hormone receptor; V1AR, vasopressin receptor type 1a; 5H4, serotonin 5-HT4; A1AB,

adrenoreceptor type 1B; NK2R, neurokinin receptor type 2; NTR1, neurotensin receptor type

1; NY2R, neuropeptide Y receptor type 2; 5H1D, serotonin 5-HT1D; AA2A, adenosine type

2; B2AR, beta adrenoreceptor type 2; C5a, C5a anaphylatoxin chemotactic receptor; CCKR,

cholecystokinin receptor type A, CKR2, C-C chemokine receptor type 2; P2YR, purinergic

P2Y1 receptor; PI2R, prostacyclin receptor, AA1R; adenosine type 1; US28, human

cytomegalovirus US28 receptor; GRPR, bombesin receptor; OPRM, opioid mu receptor


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FIGURES

Fig. 1

Cl

O

NHNH


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Fig. 2

-8 -7 -6 -50

50

100

Log[Calex 231], (M)

[3 H]IP

s,%

of m

axim

al re

spon

se

0 2 4 6 8 100

1

2

[Calcium], mM

[3 H]IP

s, CP

M/WE

LL x

1000

A

B


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Fig. 3

TM1 TM2 30 40 50 40 50 60 | | | | | | CaSR 607 SWTEPFGIALTLFAVLGIFLTAFVLGVFIK 642 IVKATNRELSYLLLFSLLCCFSSSLFFIGE MGR1 587 EWSNIEPIIAIAFSCLGILVTLFVTLIFVL 622 VVKSSSRELCYIILAGIFLGYVCPFTLIAK GBR1 586 FLSQKLFISVSVLSSLGIVLAVVCLSFNIY 621 YIQNSQPNLNNLTAVGCSLALAAVFPLGLD B2AR 31 VWVVGMGIVMSLIVLAIVFGNVLVITAIAK 67 VTNYFITSLACADLVMGLAVVPFGAAHILM OPSD 35 WQFSMLAAYMFLLIMLGFPINFLTLYVTVQ 71 PLNYILLNLAVADLFMVFGGFTTTLYTSLH

TM3 TM4 30 40 50 30 40 50 | | | | | | CaSR 674 DWTCRLRQPAFGISFVLCISCILVKTNRVLLVF 728 VFLCTFMQIVICVIWLYTAPPSS MGR1 654 TTSCYLQRLLVGLSSAMCYSALVTKTNRIARIL 710 ASILISVQLTLVVTLIIMEPPMP GBR1 660 PFVCQARLWLLGLGFSLGYGSMFTKIWWVHTVF 714 VGLLVGMDVLTLAIWQIVDPLHR B2AR 103 NFWCEFWTSIDVLCVTASIETLCVIAVDRYFAI 148 NKARVIILMVWIVSGLTSFLPIQ OPSD 107 PTGCNLEGFFATLGGEIALWSLVVLAIERYVVV 151 NHAIMGVAFTWVMALACAAPPLV

TM5 TM6 40 50 60 30 40 50 | | | | | | CaSR 769 SLMALGFLIGYTCLLAAICFFFAFKS 800 NFNEAKFITFSMLIFFIVWISFIPAY MGR1 749 SNLGVVAPLGYNGLLIMSCTYYAFKT 780 NFNEAKYIAFTMYTTCIIWLAFVPIY GBR1 766 MNTWLGIFYGYKGLLLLLGIFLAYET 798 KINDHRAVGMAIYNVAVLCLITAPVT B2AR 196 NQAYAIASSIVSFYVPLVIMVFVYSR 268 EHKALKTLGIIMGTFTLCWLPFFIVN OPSD 200 NESFVIYMFVVHFIIPLIVIFFCYGQ 247 EKEVTRMVIIMVIAFLICWLPYAGVA

TM7 40 50 | | CaSR 831 KFVSAVEVIAILAASFGLLAC MGR1 809 NYKIITTCFAVSLSVTVALGC GBR1 831 DAAFAFASLAIVFSSYITLVV B2AR 306 EVYILLNWIGYVNSGFNPLIY OPSD 286 IFMTIPAFFAKTSAVYNPVIY

TM4 TM5CaSR APPSSYRNQELEDEIIFITCH--------EG-SLMAMOPSD APPLVGWSRYI-PEGMQCSCGIDYYTPHEETNNESVF

S186 C187 G188 TM5

TM4

retinal

A

B

C


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Fig. 4

Phe612

Ala615

Leu616

Val838

Phe684

Glu837

Phe821

Phe688

Val689

Tyr744

Pro748Leu776

Trp818Pro682

TM1

TM3

TM4

TM5TM7 TM6

TM2

Ala840

Ser665

Phe668Ile669

Pocket A

Pocket B

Ile841

Ile841

Phe684

Glu837

Phe821

Phe688

Leu776

Trp818

TM1

TM2

TM3TM4

TM5

TM6TM7

Ala840

Pro682Pro748

Tyr744

Phe612Ala615

Leu616

Ser665Phe668

Val838

Ile669Val689

Pocket APocket B

A

B


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34

Fig. 5

W81

8AE8

37A

MO

CK

F684

AF6

88A

T764

AH

766A

L776

A

F821

A

I841

A

200

97

66

116

55

kDa WT

150130


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Fig. 6

0 2 4 6 8 100

25

50

75

100

WTL776AW818AF821AI841AE837A

[Calcium], mM

[3 H]IPs

, %

of m

axim

al res

pons

e

0 2 4 6 8 100

25

50

75

100

T764AH766A

WT

F688AF684A

[Calcium], mM

[3 H]IPs

,%

of ma

ximal

respo

nse

A

B


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Fig. 7

-8 -7 -6 -50

25

50

75

100

T764AH766A

F684AF688A

WT

Log[Calex 231], (M)

[3 H]IPs

,%

of ma

ximal

respo

nse

-8 -7 -6 -50

25

50

75

100

L776AW818AF821AI841A

WT

E837A

Log[Calex 231], (M)

[3 H]IPs

,%

of ma

ximal

respo

nse

A

B


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Didier Rognan and Martial RuatChristophe Petrel, Albane Kessler, Fouzia Maslah, Philippe Dauban, Robert H. Dodd,

allosteric modulator of the extracellular Ca2+ sensing receptorModeling and mutagenesis of the binding site of Calex 231, a novel negative

published online September 23, 2003J. Biol. Chem.

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Documents

Preparation of Calex 231 - Journal of Biological Chemistry · E837A7.39, caused a loss of Calex 231’s ability to inhibit Ca2+-induced accumulation of [3H]inositol phosphates. Three