Department of Cancer Research (S.H.H., T.B.) ASTA Medica
AG D-60314 Frankfurt/Main, Germany
Department of
Molecular Membrane Biology (S.H.H., H.R.) Max-Planck-Institute of
Biophysics D-60528 Frankfurt/Main, Germany
Institute of
Molecular Pharmacology (T.t.L., R.K.) D-10315 Berlin,
Germany
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ABSTRACT |
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The substitution of residues K36, Q204, W205, H207, Q208, F210, F213, F216, and S217 for alanine had no or only a marginal effect on ligand binding and signaling. In contrast, substitution of N87, E90, D98, R179, W206, Y211, F214, and T215 for alanine resulted in receptor proteins neither capable of ligand binding nor signal transduction. Within those mutants affecting ligand binding and signaling to various degrees, W101A, N102A, and N212Q differentiate between agonists and antagonists. Thus, in addition to N102 already described, the residues W101 in TMH2 and N212 in TMH5 are important for the architecture of the ligand-binding pocket. Based on the experimental data, three-dimensional models for binding of the superagonist D-Trp6-GnRH (Triptorelin) and the antagonist Cetrorelix to the hGnRH-R are proposed. Both decapeptidic ligands are bound to the receptor in a bent conformation with distinct interactions within the binding pocket formed by all TMHs, E2, and E3. The antagonist Cetrorelix with bulky hydrophobic N-terminal amino acids interacts with quite different receptor residues, a hint at the failure to induce an active, G protein-coupling receptor conformation.
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INTRODUCTION |
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The GnRH receptor (GnRH-R) expressed on gonadotropic cells of the anterior pituitary gland belongs to the family of rhodopsin-like G protein-coupled receptors (GPCRs), characterized by seven putative transmembrane helices (TMH 17) (reviewed in Refs. 12 13 ). The GnRH-R was cloned from several mammalian (human, marmoset monkey, mice, rat, sheep, bovine) and nonmammalian species (catfish, goldfish, chicken) (reviewed in Ref. 13 ). The mammalian GnRH-R shares several unique features including the absence of a cytoplasmic C-terminal tail, replacement of Y by S140 in the conserved DRY sequence in TMH3 and the reciprocal interchange of two residues highly conserved among GPCRs, N87 in TMH2, and D319 in TMH71. Studies on GnRH-R signal transduction revealed a versatile and dynamic network of temporally segregated responses (reviewed in Refs. 14 15 ). The GnRH-R couples to Gq/G11 but may as well interact with Gi and Gs in rat pituitary cells (16 17 ). Heterologously expressed GnRH-R also couples to adenylate cyclase in GH3 rat pituitary tumor cells (18 19 ) or insect cells (20 ), emphasizing the promiscuity of the GnRH-R as a function of the availability of G proteins in the microenvironment of the test cell.
Caused by the poor solubility of GPCRs and difficulties in obtaining crystals suitable for x-ray structural analysis, existing structural information of G protein-coupled receptors is limited to rhodopsin (21 22 23 ). Thus, to date, generating site-specific mutants of GPCRs is the only experimental approach to study structure-function relationships (24 ). Concurring knowledge for the GnRH-R has been accumulated during the last years (13 25 26 ). The relevance of N87 (TMH2) and D318 (TMH7) for the murine and rat GnRH-R structure has been shown by several groups (27 28 29 ). The N87D mutant was unable to bind agonists, whereas the D318N mutant showed ligand binding but signaling was severely impaired (27 29 ). Reciprocal interchange in the N87D/D318N mutant restored ligand binding but not signal transduction. A recent study by Mitchell et al. (30 ) revealed that D318N, as well as the N87D/D318N mutant of murine GnRH-R, was unable to activate phospholipase D in an ADP-ribosylation factor (ARF) independent way (30 ). These results indicate that N87 and D319 and therefore TMH2 and 7 are located in close proximity and are important for receptor signaling.
With L58, L73, S74, and L80 as part of intracellular loop I (I1) (31 ), L147 within the conserved DRYXXV/ISSPL motif of GPCRs I2 (32 ), R262 in I3 (33 ), and Y322 from the DPLIY motif in TMH7 (34 ), several other residues affecting signal transduction have been identified. Mutants in I1 abolished or severely affected signaling by cAMP, but not inositol phosphate (IP) responses (31 ). Thus the I1 loop might be important for binding and activating Gs proteins. The R262Q mutant, which was characterized by wild-type (wt) agonist binding affinity but a 10-fold reduced stimulation of IP production in heterologous expression experiments, was detected as a GnRH-R gene mutant in a family with idiopathic hypogonadotropic hypogonadotropism (IHH) (33 36 ). The DRS138140 motif at the cytoplasmic border of TMH3 was also subject to mutagenesis. The D138A mutant abolished ligand binding and signal transduction, whereas the D138N and D138E mutants were characterized by almost wt affinity for agonists, yet signaling was affected (37 38 ). The I143A mutant showed impaired receptor activation, presumably by solvation of R139 in the cytoplasmic aqueous environment. Proper positioning of R139 achieved by I143 allows interaction with, e.g. D138 and is supposed to be a prerequisite for receptor activation. Therefore, in the proposed model, R139 is involved in the transition between the inactive and active receptor conformation (37 ).
Until now, only N102 (TMH2), K121 (TMH3), and D302 (E3/TMH7) have been identified as being important for ligand binding. The interaction of Arg8 of mammalian GnRH with E301 of the murine GnRH-R (D302 in human) was proposed by Flanagan et al. (39 ). The mutants K121L, D, or Q abolished ligand binding, but antagonist binding to the K121Q mutant was unaffected and therefore an interaction of K121 with amino-terminal residues was proposed (40 ). The N102A mutant described by Davidson et al. (41 ) differentiates between agonists with glycineamide and ethylamide C termini, suggesting that N102 forms a hydrogen bond with the C-terminal amide moiety. The authors assumed that N102 is located proximal to D302 and K121, which are part of the ligand-binding pocket formed by TMH 1, 2, 3, and 7.
To facilitate the functional characterization of hGnRH-R mutants and for having a reasonable throughput of ligands, we have established a modified reporter gene assay based on GnRH-R signaling via adenylate cyclase (42 ). Here we applied this methodology for the functional characterization of 24 site-specific mutants of the human GnRH-R. Receptor residues in TMH1, TMH2 with focus to the E2-loop, and TMH5 were selected to detect those residues involved in ligand binding. In most cases, residues were subjected to alanine substitution (Ala-scan mutagenesis). After selecting cell lines with stable expression of hGnRH-R mutants, functional properties as well as binding affinities for the antagonist Cetrorelix were analyzed in detail. In this report we present two models of the human GnRH-R with the agonistic and antagonistic ligands D-Trp6-GnRH and Cetrorelix, respectively, docked into the GnRH binding pocket. A deep hydrophobic binding pocket is formed by TMHs 1, 2, 3, and 7 as described recently (41 ), but also with a major contribution of residues within TMH 5 and 6. The residue N212 in TMH 5 was found to be important for agonist binding and for the stabilization of the antagonist binding pocket. By comparing the functional and binding activity of agonists and antagonists, W101 and N102 in TMH2 were found to be important for agonist, but much less so for antagonist binding.
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RESULTS |
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The A mutants of residues N87 and D98 showed neither receptor signaling nor ligand binding. Data published by several groups for the N87D and D98N mutants described no or decreased activity, respectively (27 28 30 39 ). Our finding that D98A is completely inactive stresses the importance of residue D98 for GnRH-R function. Contrary to a normal activity for the E90Q mutant claimed by Flanagan et al. (39 ), our result revealed that the mutant E90A was inactive.
The N102A mutant was capable of coupling to G
protein but with significant differences between the various agonists
(Table 1 and Fig. 5
, AC). With an EC50 of
530 ± 100 nM for GnRH with the glycineamide C
terminus the activity index was 240 (Fig. 5A
). In contrast, the
activity index of the agonist
des-Gly10-Pro9-NHEt-GnRH
showing an EC50 of 29 ± 4 nM
was calculated to 62 (Fig. 5B
). Similar results were obtained
with D-Ala6-GnRH and
des-Gly10-D-Ala6-Pro-NHEt9-GnRH
and to some extent also with
D-Trp6-GnRH (Fig. 5C
) and
des-Gly10-D-Trp6-Pro-NHEt9-GnRH.
A new feature of the N102 residue is that it
proved to be of minor importance for antagonist binding. The activities
of Cetrorelix and Antarelix with an IC50 of
5.4 ± 0.9 nM (activity index 4.9) and 8.2 ± 0.8
nM (activity index 5.9), respectively, were close to that
of the wt receptor. For comparison, the activities of the
conformationally constrained agonists were significantly more affected
(Table 1
and Fig. 5
, C and D).
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The amino acid residue N212 located in TMH5 was
mutated to A and Q. Both mutations and here especially the
N212A mutant displayed a marked increase of the
EC50 values for agonists (Table 1 and Fig. 5G
for
GnRH). This result suggested that N212 is located
within the binding pocket and contributes to agonist binding, which is
further supported by the receptor model (Fig. 6C
and Discussion). Yet a
surprising difference between the N212A and
N212Q mutants was detected during testing with
antagonist (Table 1
and Fig. 5H
for Cetrorelix). The
N212A mutant exhibited a significant effect on
the antagonistic potency of Antarelix and Cetrorelix (activity indices
of 40 and 48, respectively), which also correlated with the decreased
binding affinity determined for Cetrorelix. Thus, the
N212A mutant exhibited no discrimination between
agonists and antagonists. However, the N212Q
mutant displayed almost wt behavior in respect to Cetrorelix binding
and signal inhibition.
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DISCUSSION |
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Various mutants of the GnRH-R affecting ligand binding or G protein
coupling and signal transduction have been described (for review see
Refs. 13 25 ). The labor-intensive functional assays based on IP
production and detection limits with standard radioligand assays kept
mutants or ligands to be tested to small numbers. Therefore, no
detailed model for ligand binding and almost no data concerning the
architecture of the antagonist binding pocket are currently available.
In the present study multiple receptor mutants were characterized by
using a panel of peptidic agonistic and antagonistic GnRH derivatives.
A high-throughput functional assay was applied to generate a
comprehensive set of data needed to propose and validate computational
models for D-Trp6-GnRH and Cetrorelix
binding to the human GnRH-R. With focus on TMH5 and E2, 20 residues of
the hGnRH-R were exchanged by site-directed mutagenesis to A and in a
few cases to closely related residues (NQ, F
Y). The
functional assay as described (42 ) was adopted for
characterization of the mutant hGnRH-Rs in stably transfected cells. In
the antagonist experiments,
D-Trp6-GnRH was used for receptor
activation in a concentration of 2x EC50 of the
respective receptor mutant. By doing so, comparable and practicable
experimental conditions were generated to compensate for the very
different activities of receptor mutants. In addition, all mutants were
characterized by displacement binding experiments with intact cells and
[125I] Cetrorelix (44 ). This radioligand is
superior to agonists like [125I]-Triptorelin in
terms of high binding affinity and
80% of tracer capable for
specific receptor association. Thus, it was possible to determine
dissociation constants for receptor mutants with decreased expression
or binding affinity, a major problem in many studies (58 ). There was a
very good correlation of the binding affinity KD
and the antagonistic potency IC50 of Cetrorelix
(Fig. 4
), and those mutants without signaling activity also did not
bind the antagonist. Therefore, we assume that the mutations affected
ligand binding, while signal transduction is only affected as a
consequence. From the displacement binding assays
Bmax values for the different mutant receptor
proteins exposed on the cell surface were calculated (Table 1
, last
column). Relative to wt hGnRH-R production, the mutant receptor
proteins were produced between 150% (F210A) and
17% (W101A). This can be explained either by
differences in transfection efficiency and/or mutant receptor protein
synthesis, processing, or transport. As has been discussed recently,
the EC50 could depend on the number of receptors
expressed on the cell surface (40 59 ). From our data we conclude that
this is unlikely, because 1) weakly or strongly expressed mutants like
H207A, W205A, or
F210A had wt activity, 2) the shape of all
dose-response curves was identical, and 3) receptor affinity and
antagonistic potency did correlate. In addition the maximal effect
observed ín model experiments to decrease transiently
overexpressed GnRH-R number artificially was 10-fold (40 ).
One can deduce from the mutational analysis that the residues
K36 (TMH1), Q204,
W205, H207
Q208, F210 (E2), and
F213 and S217 (TMH5) played
a minor role in binding of the peptidic ligands. In accordance to the
receptor model, their activity and affinity were identical or close to
that of the wt receptor (Fig. 6 and Table 1
). The
S217R mutant has been described recently in a
family with IHH together with mutations in E1
(Q106R) and I3 (R262Q)
(36 ). The S217R receptor mutant alone was
incapable of ligand binding and signaling. Since
S217 is conserved in all mammalian GnRH-Rs except
mouse (G216), it is likely that only small,
uncharged residues are tolerated at this position. In the receptor
model, S217 is pointing toward the lipid
membrane; thus, the positively charged guanidinium group of the
S217R mutant most probably hinders a proper
membrane integration whereas the alanine exchange is tolerated.
Mutations of residues N87, E90, D98, R179, W206, Y211, F214, and T215 to A resulted in a complete loss of ligand binding as well as signal transduction. One has to keep in mind that this can be explained by 1) destruction of the overall receptor structure and/or ligand binding pocket, 2) failure of proper integration into the membrane and therefore loss of surface expression, or 3) synthesis of a misfolded protein targeted to the proteasome. Mutants at the loci N87, E90, and D98 have already been published for the murine (27 28 30 ), rat (29 ), and catfish GnRH-R (61 ). For mammalian GnRH-R, the N87D mutation was inactive, and N87 was considered to form a salt bridge with D319 in TMH7, which is important for the structural integrity of the receptor. Therefore, it was not surprising to find that the N87A mutant of hGnRH-R neither binds ligand nor undergoes agonist-induced receptor activation and signaling.
The E90Q mutant of the murine GnRH-R showed wt
behavior considering agonist binding and signal transduction (39 ). In
the receptor model E90 forms a salt bridge with
K121 in TMH3, which probably is essential for
receptor structure and explains the loss of function due to the alanine
mutation (Fig. 6). Interaction with K121 via
hydrogen bonds is also possible for a glutamine side chain, which
explains the wt behavior of the E90Q mutant. In
our modeling studies we have experienced that
K121 was able to interact with
pGlu1 or His2 of agonists
without interfering with residual receptor-ligand interactions. Taking
into consideration the calculated interaction energies, the
pGlu1-K121 interaction was
slightly favored (data not shown).
In comparison to the wt hGnRH-R and depending on the ligands
studied, signaling and ligand binding capacity of the
W101A, N102A (TMH2),
N212A/Q, and F216Y (TMH5)
mutants was affected quite differently. The W101A
mutant displayed the most pronounced shift in agonist-induced signal
transduction published so far (Fig. 5E). In the model,
W101 is involved in binding via a hydrogen bond
with the Leu7 backbone oxygen of
D-Trp6-GnRH (Fig. 6C
). In contrast,
this interaction was not observed in the Cetrorelix receptor binding
model which is in agreement with the smaller effect in case of
antagonists (Fig. 5F
). Therefore, we assume that
W101 might be important for the proper formation
of the binding pocket around the C-termini in case of antagonists.
Mutants of N102 were already described recently:
the N102Q mutation neither affected neither
ligand binding, signaling, nor receptor production (26 41 ). In
contrast, the N102A mutation caused a marked bias
between the C termini of agonists. The activity of GnRH and the
agonists with a glycineamide C terminus was reduced about 27- to
220-fold, whereas the activity of agonists with an ethylamide C
terminus was much less affected (Table 1 and Ref. 41 ). The authors
assumed that N102 was important for binding of
the C terminus of agonists. In our experiments, we were able to confirm
this assumption for agonists. Nevertheless, in contrast to agonists,
the antagonistic potency and binding affinity of Cetrorelix was only
slightly reduced. According to our receptor models,
N102 most probably is directly involved in ligand
binding of the glycineamide C terminus of
D-Trp6-GnRH and the
D-alanylamide C terminus of Cetrorelix via a hydrogen bond.
The D-Ala side chain stabilizes the orientation of the C
terminus of Cetrorelix and, therefore, antagonist binding is less
affected by this mutation.
As was deduced from the data of the N212A/Q mutants that exhibited a different behavior for agonists and antagonists, the residue N212 in TMH5 directly contributes to the architecture of the ligand binding pocket. In our model, N212 is involved in the binding of the D-Trp6-GnRH backbone (His2 = O) via a hydrogen bond. In the case of Cetrorelix, the N212 residue appears not to be directly involved in binding of the antagonist, but seems to be more important for stabilizing the binding pocket through hydrogen bonds with W206. These differences in agonist/antagonist binding are supported by the result that the N212Q mutant did not affect antagonist binding, whereas agonist binding was severely reduced most probably by steric hindrance.
Although the F216A mutant showed wt activity, the F216Y mutant affected agonist as well as antagonist binding. In our model, the side chain of F216 is near the ligand binding pocket and not directly involved in ligand interaction. It is likely that at this position a hydrophobic side chain is needed and that the hydrophilic phenolic side chain of tyrosine distorts the receptor conformation due to an interaction with the Y283.
By comparing the EC50 values of agonistic ligands of the W101A, N102A, N212A/Q, and F216Y receptor mutants, a common theme became apparent. In contrast to GnRH, signaling of these GnRH-R mutants after induction with the structurally constrained analog D-Trp6-GnRH and to some extent D-Ala6-GnRH was significantly less affected. For example, D-Trp6-GnRH and GnRH have an activity index of 27 and 240, respectively, for the N102A mutant and of 3.2 and 19, respectively, for the F216Y mutant. Similar results have been published for the N102A mutant by Davidson et al. (41 ) and the D301Q murine receptor mutant by Flanagan et al. (39 ). The antagonistic potency and binding affinity of Cetrorelix or Antarelix is also much less affected by these receptor mutants. Therefore, it is reasonable to postulate that the initial steps of ligand binding, i.e. the docking of superagonistic and antagonistic GnRH analogs might be quite similar. In contrast to GnRH, these ligands are conformationally constrained due to the incorporation of bulky and D-configurated amino acid side chains (13 ). GnRH has more conformational freedom and, therefore, needs an induced fit for receptor binding and activation. In the case of agonists, receptor-ligand interactions are required to induce an active receptor complex. Such interactions are of less importance for the constrained analogs. Although there are similarities between D-Trp6-GnRH and Cetrorelix, the functional data and the receptor models highlight the differences. Many interactions of the N-terminal residues of D-Trp6-GnRH (pGlu1-His2-Trp3) are not present in the hydrophobic and bulky N-terminal sequence D-Nal1-D-pClPh-Ala2-D-Pal3 of Cetrorelix. In addition, the Cetrorelix model lacks the K121-pGlu1 or alternatively the K121-His2 interaction. Here K121 interacts only with E90 in TMH2. Since the N-terminal parts of the agonistic GnRH analogs are predominantly responsible for receptor activation (13 ), this gives a hint to the antagonistic function of analogs like Cetrorelix. Certainly, the models as proposed have to be refined and confirmed by additional receptor mutants, especially to gain a comprehensive understanding of antagonist binding and function on a molecular level. Nevertheless, the receptor mutants and the computational models as presented are very useful tools for defining putative binding sites for small, peptidomimetic antagonistic ligands like T-98475 (56 ) and, subsequently, the design of derivatives with a higher potency or specificity. Based on cell lines with stable expression of hGnRH-R receptor mutants and a sensitive reporter system for quantification of receptor activation at hand, it is feasible to comprehensively characterize new receptor mutants and peptidomimetic compounds.
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MATERIALS AND METHODS |
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K36A: 5'-CTTGTCTGGAGCGATCCGAGTG-3', N87A: 5'-GACCTTAGCCGCCCTGTTG GAG-3', D98A: 5'-CATGCCACTGGCTGGGATGTGG-3', W101A: 5'- GGATGGGATGGC CAACATTACAGTC-3', N102A: 5'-GGGATGTGGGCCATTACAGTCC-3', R179A: 5'-CAGTTATACA TCTTCGCGATGATTCATCTAG-3', Q204A: 5'-CTGCAGTTTTTCAGCCTGGTGG-CATC-3', W205A: 5'-CAGTTTTTCACAAGCTTGGCATCAAG-C-3', W206A: 5'-GTTTTTCACAATGGGCCCATC AAGCA-TTTTATAAC-3', W206Y: 5'-GTTTTTCACAATGGTACCATCA-AGCATTTTATAAC-3', W205A/W206A: 5'-CAGTTTTTCACAA-GCTGCGCATCAAGCATTTTATAAC-3', H207A: 5'-CACAAT-GGTGGGCCCAAGCATTTTATAAC-3', Q208A: 5'-CAATGGT-GGCATGCAGCATTTTATAAC-3', F210A: 5'-GCATCAAG-CGGCCTATAACTTTTTCACC-3', Y211A: 5'-GCA TCAAG-CATTCGCGAACTTTTTCACC-3', N212A: 5'-CATCAAGCAT-TTTACGCGTTTTTCACCTTCAGC-3', N212Q: 5'-CATCA-AGCATTTTATCAATTTTT-CACCTTCAG-3', F213A: 5'-CAAG-CATTTTATAACGCGTTCACCTTCAG-3', F214A: 5'-GCATTT-TATAACTTCGCG ACCTTCAGCTG-3', T215A: 5'-CTAGGA-ATTTGGGCCTGGTTTGATCC 3'; F216A: 5'-CTTT TTCAC-CGCGAGCTGCCTCTTC-3', F216Y: 5'-CTTTTTCACCTACAG-CTGCCTC-3', S217A: 5'-CTTTTTCACCTTCGCATGCCTC-TTCATC-3', C218A: 5'-ACCTTCAGCCTCTCTTCATCATC 3'.
By AseI/NotI fragment shuffling between the pSBC1-hGnRHR derivatives and pSBC2-SEAP, dicistronic expression vectors pSBC mt hGnRHR/IRES/SEAP were generated (44 ). Mutations were verified by restriction analysis and DNA sequencing, using the dideoxy chain termination method according to Sanger.
Mammalian Cell Culture, Stable Transfection, and Selection of
Cell Clones
Murine thymidine kinase-deficient L cells
(LTK-) and transfectants thereof were cultured
at 37 C, 5% CO2 in DMEM supplemented with 10%
inactivated FCS (FCSi), penicillin/streptomycin,
and glutamine. LTK- cells were transfected by
calcium phosphate/DNA coprecipitation (45 ) with plasmids
pSBC(CRE3)CMVmin-Luc
containing the firefly luciferase cDNA under control of the
cAMP-response element (CRE) fused to the cytomegalovirus (CMV) minimal
promoter (our unpublished data) and pSV2 PAC containing the
puromycin-N-acetyl-transferase gene (46 ). After selection
(culture medium supplemented with 5 µg/ml puromycin), single-cell
clones were isolated by limited dilution and about 200 clones were
analyzed for expression of the Luc reporter gene after stimulation with
forskolin. Four clones with optimal signal-noise ratio and intermediate
or high signal after forskolin stimulation were selected and stably
supertransfected with the pSBC-wt hGnRHR/IRES/SEAP plasmid and pAG60
containing the amino-glycoside phosphotransferase gene (47 ). After
selection (culture medium supplemented with 800 µg/ml G418/Life Technologies, Inc., Gaithersburg, MD), cell pools were analyzed
for luciferase expression after stimulation with
D-Trp6-GnRH. Clonal cell
line 122 showed superior stimulation characteristics (75,000
vs. 6,000 relative light units of unstimulated cells) and,
therefore, was chosen as the reporter cell line for stable
supertransfection with the hGnRH-R mutants. The cell pools obtained
after selection with G418 were used for analysis of signal transduction
and ligand binding.
Isolation of RNA and Northern Blot Analysis
Subconfluent cultures of cells were stimulated with 1
nM D-Trp6-GnRH and 500
µM 3-isobutyl-1-methylxanthine (IBMX) and harvested after
0, 30, 60, 120, 240, and 360 min by adding lysis buffer (4
M guanidinium thiocyanate, 0.5% (wt/vol) sodium lauryl
sarcosinate, 285 mM ß-mercaptoethanol, 25 mM
sodium citrate, pH 7) directly to the culture dishes. Preparation of
total RNA, electrophoretic separation, transfer to the Hybond
N+ nylon membrane, hybridization with
[32P]dCTP random labeled probes, and finally
stringent washing was performed as has been described previously (48 ).
For experiments with the antagonist, 0.11000 nM
Cetrorelix was added 1 h before stimulation with 1 nM
D-Trp6-GnRH/500 µM
IBMX, and cells were lysed 6 h after stimulation. For
hybridization, a cDNA probe of Photinus pyralis luciferase
(EcoRI/HindIII fragment from pSBC2 Luc (49 ) was
used.
Agonistic and Antagonistic GnRH Analogs, Cell Treatment, and
Reporter Gene Assay
Cetrorelix/D-20761
(Ac-D-Nap-Ala1-D-ClPh-Ala2-D-Pyr-Ala3-Ser4-Tyr5-D-Cit6-Leu7-Arg8-Pro9-D-Ala10-NH2)
and Antarelix/D-23234
(Ac-D-Nap-Ala1-D-ClPh-Ala2-D-Pyr-Ala3-Ser4-Tyr5-D-Hci6-Leu7-Lys(iPr)8-Pro9-D-Ala10-NH2)
were synthesized at ASTA Medica AG as described previously (6 ). The
agonistic peptides GnRH,
des-Gly10-Pro-NHEt9-GnRH,
D-Trp6-GnRH,
Des-Gly10-D-Trp6-Pro-NHEt9-GnRH,
D-Ala6-GnRH,
des-Gly10-D-Ala6-Pro-NHEt9-GnRH
were purchased from Bachem Biochemica GmbH
(Heidelberg, Germany). All peptides with exception of Cetrorelix were
dissolved in water at 1 mM final concentration and stored
in siliconized polypropylene tubes at -20 C. Cetrorelix was dissolved
in 0.01 N CH3COOH.
For functional analysis of the receptor mutants 1 x
104 cells per well were cultivated for 24 h
in 96-well microtiter plates using DMEM with supplements and 10%
(vol/vol) FCSi. Subsequently cells were
stimulated for 6 h with the respective agonist. For signal
enhancement, a phosphodiesterase (PDE) inhibitor (IBMX or the PDE4
specific inhibitor Rolipram) was added. Cells were lysed for
quantification of cellular Luc activity. Cetrorelix and Antarelix were
added 15 min before the stimulation of cells with
D-Trp6-GnRH. For stimulation a
variable D-Trp6-GnRH concentration of
2x EC50 was used, dependent on the GnRH-R mutant
tested. The luciferase reporter gene assay was performed as described
(42 ). Calculation of the EC50 and
IC50 values was done by nonlinear regression
analysis using the Hill model (program EDX 2.0, our unpublished
data) and mean values ± SD (sample standard
deviation n-1) from at
least two independent experiments are shown.
Radioligand Binding Assay
For receptor binding studies,
[125I]Cetrorelix was prepared and used as a
tracer that exhibited about 80% of peptide capable for specific
receptor association. The binding assay was performed on intact cells
under physiological conditions essentially as described (44 ). For
displacement binding assays, 1 x 106 cells
in 100 µl were incubated with 225 pM
[125I]Cetrorelix (specific activity 510
x 105 dpm/pmol) and different concentrations of
unlabeled Cetrorelix as a competitor for 1 h at 37 C. Binding
affinity (KD) and cell surface receptor
expression (receptor concentration Bmax) were
calculated from the displacement binding data by using the EBDA/Ligand
analysis program (Biosoft V3.0) (50 ) and mean values ±
SD (sample SD
n-1) from independent
experiments are shown.
Molecular Modeling Experiments
The starting structure for the model of the GnRH-R transmembrane
domains was built from the sequence of the hGnRH-R (Swiss-Prot P30968)
(51 52 ) using the -carbon template of the transmembrane helices of
rhodopsin (53 ). To obtain a complete model, the extra- and
intracellular loops were added using the loop search option in
SYBYL6.4. Two disulfide bridges
(C14-C200 and
C114-C196) reported by
Davidson et al. (54 ) were included in the model. According
to earlier studies on the stability of transmembrane bundles in
molecular dynamics simulations (55 ), we used positively charged
arginine and lysine residues and negatively charged glutamate and
aspartate residues in all calculations. To obtain better
three-dimensional structures for the intra- and extracellular loop
regions of the hGnRH-R, a simulated annealing (SA) method was performed
to sample a large number of different conformations using the AMBER 4.1
force field. In 25 SA runs the hGnRH-R was solvated with
explicit water molecules and the TMH domains were restrained to
maintain the template (55 ). The hGnRH-R model with intra- and
extracellular loop structures having the lowest potential energy was
used for further modeling studies. In comparison to the rhodopsin
template structure some of the TMHs were extended as a result of the SA
calculations (TMH boundaries:
Lys36-Lys62,
Leu73-Ile103,
Leu112-Arg145,
Val155-Ile181,
Asn212-Val241,
Arg260-Trp291,
His306-Leu328). The
rhodopsin template represents an arbitrary transmembrane bundle of a
GPCR in the inactive state and does not reflect receptor-specific
conformational changes such as proline kinks. To simulate such effects,
an AMBER4.1 molecular dynamics simulation in vacuo was
performed in a next step, fixing the arrangement of the seven-helix
bundle by a set of distance restraints between the centers of three
C
-atoms in each TMH (TMH1: Thr51,
Phe52, Asn53; TMH2:
Val94, Met95,
Pro96; TMH3: Tyr126,
Ala127, Pro128; TMH4:
Ala171, Gly172,
Pro173; TMH5: Ile221,
Ile222, Pro223; TMH6:
Trp280, Thr281,
Pro282; TMH7: Phe318,
Asp319, Pro320). This
allows rotations of the TMHs around their own axis and enables helix
kinks near prolines.
D-Trp6-GnRH was docked into the
putative agonist binding site during 150 psec of AMBER4.1
molecular dynamics simulation in vacuo starting from about
3040 Å outside the hGnRH-R pocket (55 ). The mass centers of
interacting amino acid residues (pGlu1 with
K121, Arg8 with
D302, and
Gly10-NH2 with
N102) were restrained to gradually approach a
distance smaller than 35Å or a distance smaller than 8Å
(Trp6 with C14). During the
simulation the TMHs were stabilized by restraints on all helix hydrogen
bonds and the center of mass restraints described previously. The
GnRH-R model as obtained was used to generate different
D-Trp6-GnRH binding modes
within the receptor binding site with SA method (40 runs, AMBER4.1,
in vacuo, heating up to 1500 K in 5 psec and then slowly
cooling down to 0 K between 25 psec and 50 psec). All SA runs were
performed using the same set of restraints (distance restraints between
pGlu1 with K121,
Arg8 with D302, and
Gly10-NH2 with
N102; harmonic restraints of 1 kcal
mol-1 Å-2 on all C-atoms of the TMHs to
fix the TM regions; distance restraints between the mass centers of the
extra- and intracellular loops to avoid unfolding of loop regions
during SA; torsional restraints to fix chirality of all
C
-atoms).
In the case of Cetrorelix, SA runs were performed starting with the docked D-Trp6-GnRH-hGnRH-R complex in which the five distinct residues were mutated to those of Cetrorelix (pGlu1 to D-Nal1; His2 to D-pClPhe2; Trp3 to D-Pal3; D-Trp6 to D-Cit6; Gly10-NH2 to D-Ala10-NH2) using the SYBYL mutate option and only two biases for receptor-ligand interaction during the 40 SA runs (Arg8 with D302 and D-Ala10-NH2 with N102). The restraints to fix the receptor structure were identical to these described for the D-Trp6-GnRH-binding study.
After AMBER 4.1 force field minimization, the obtained receptor binding models for both D-Trp6-GnRH and Cetrorelix were ranked according to the calculated ligand binding energies. Models having the highest ligand binding energies were selected, and the influence of the mutations presented in this study on the ligand binding energy was calculated for each of these models. The binding models showing the best agreement between experimental data and calculated ligand binding energies were selected.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by the Federal Ministry of Research, Education, Science and Technology (BMBF), Grant 0310697A.
1 Present address: Vrije Universiteit Amsterdam, Division of Chemistry,
De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Received for publication January 18, 2000. Revision received March 6, 2000. Accepted for publication March 21, 2000.
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REFERENCES |
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