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Medical Research Council Research Unit for Molecular Reproductive Endocrinology, Department of Chemical Pathology (C.A.F., B.J.F., J.S.D., R.P.M.), and Endocrine Laboratory, Department of Medicine (C.A.F., R.P.M.), University of Cape Town Medical School, Observatory 7925, South Africa
Address all correspondence and requests for reprints to: Robert P. Millar, Director, Medical Research Council Reproductive Biology Unit, 37 Chalmers Street, Edinburgh EH, United Kingdom. E-mail: Bob{at}ed-rbu.mrc.ac.uk
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Abstract |
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In competition binding assays, total binding of 125I-[His5,D-Tyr6]GnRH was higher than binding of a conventional tracer ligand, 125I-[D-Ala6,N-MeLeu7,Pro9NHEt]GnRH. The bindable fractions and specific activities of both peptides were similar, and the receptor binding affinities of the unlabeled peptides were indistinguishable. However, comparison of the radiolabeled peptides in saturation binding assays showed that the affinity of the peptide,125I-[His5,D-Tyr6]GnRH, (Kd, 0.19 nM), was approximately 2-fold higher than that of the conventional tracer. The increased binding of 125I-[His5,D-Tyr6]GnRH has allowed the development of a sensitive GnRH receptor binding assay for analysis of mutant GnRH receptors that exhibit decreased ligand binding.
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Introduction |
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Radioiodinated GnRH agonists are used as tracers in the competition
binding assays that are used to determine expression levels and
affinity of GnRH receptors (4, 5). A tracer with increased affinity for
the GnRH receptor would be expected to facilitate binding assays for
some of the mutant GnRH receptors that cannot be measured using
currently available technology. The GnRH agonists that are used as
tracers contain a Tyr residue in position 5, to which an atom of
125I is attached by oxidative radioiodination reactions.
However, the Tyr5 residue is believed to have a role in
maintaining the active conformation of the GnRH peptide for interaction
with its receptor (6) and may also interact directly with the receptor.
Iodination of GnRH has been reported to decrease its biological
activity by a small amount (7). Although it has been reported that the
GnRH receptor binding affinities of iodinated peptides are the same as
those for the corresponding noniodinated peptides (4, 8), differences
in the methods used to determine affinities and the log-normal
distribution of ligand-receptor interactions (9) make it difficult to
detect small (
2-fold) differences in affinity. Nevertheless, small
differences in affinity have a significant effect on the amount of
tracer bound under the nonsaturating conditions of competition binding
assays.
Replacement of the achiral Gly residue in position 6 of GnRH with D-amino acids increases GnRH potency (reviewed in Ref. 10) and increases affinity for the GnRH receptor (11). D-Amino acids with large hydrophobic side-chains are not only tolerated in position 6, but improve GnRH activity (10). Our studies using chimeric analogs of naturally occurring forms of GnRH showed that substitution of Tyr5 with His slightly increases the affinity of GnRH agonists for GnRH receptors (12). We have therefore designed and synthesized a peptide, [His5,D-Tyr6]GnRH, that is expected to bind to the GnRH receptor with high affinity when it is radioiodinated in position 6 rather than the usual position 5.
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Materials and Methods |
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Radioiodination
Peptides were radioiodinated by a variation of the chloramine-T
method. Five micrograms of peptide in 20 µl 0.5 M
phosphate buffer (pH 7.4) were reacted for about 10 sec with
Na125I (1 mCi; Amersham, Aylesbury, UK) and chloramine-T
(10 µl of 3 mg/ml in phosphate buffer). The reaction was terminated
by the addition of sodium metabisulfite (50 µl of 1.2 mg/ml in
phosphate buffer). Iodinated peptides were purified on C18
reverse phase HPLC with a gradient of 0–60% or 80% acetonitrile/0.01
M ammonium acetate (pH 4.6) at a flow rate of 1.5 ml/min.
Specific activity was determined by the method of Hulme and Birdsall
(13)
Cell culture, transfection, and receptor binding assays
The 
T3 gonadotrope cell line, which contains the mouse GnRH
receptor (14), and COS-1 cells (American Type Culture Collection) were
maintained in DMEM (Life Technologies, Paisley, UK) containing 10% FBS
(Highveld Biological, Kelvin, South Africa). Complementary DNA for the
wild-type human GnRH receptor (15) and mutant GnRH receptors containing
a second N-glycosylation site (16) or an Asn substitution
for Asp302 were subcloned into the pcDNA I/Amp expression
vector and transfected into COS-1 cells using the
diethylaminoethyl-dextran method (17) as previously described (5).
Binding assays were performed as previously described with minor
alterations. Cells (T3 or transfected COS-1) were detached from
culture dishes in binding buffer [10 mM HEPES (pH 7.4), 1
mM EDTA and 0.1% wt/vol BSA], homogenized in a Dounce
homogenizer (Kontes, Vineland, NJ), and centrifuged (10,000 x
g, 4 C, 40 min). The crude membrane pellet was resuspended
in binding buffer and incubated at 4 C, for the times indicated in the
figure legends, in the presence of labeled peptide at the indicated
concentrations and varying concentrations of unlabeled peptides in a
final volume of 0.5 ml. Incubations were terminated by the addition of
3 ml cold polyethylenimine solution (0.01%; Sigma, St. Louis, MO) and
were filtered through GF/C filters (Whatman, Clifton, NJ) presoaked in
1% polyethylenimine. Nonspecific binding was estimated in the presence
of 10-6 M antagonist 26, which does not
displace label bound by membranes prepared from untransfected COS-1
cells.
Data analysis
Experiments were performed in triplicate. Four-parameter
nonlinear curve fitting (Sigmaplot, Jandel Scientific, Corte Madera,
CA) was used to calculate the maximum bindable fraction of tracer and
to draw the curves presented in figures. Equilibrium dissociation
constants (Kd) were estimated using the Ligand data
analysis program (18).
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Results |
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T3 mouse gonadotrope cells, specific binding of
125I-[His5,D-Tyr6]GnRH
was 30.9% of the total radioactivity (70,000 cpm) compared with 13.3%
for
125I-[D-Ala6,N-MeLeu7,Pro9NHEt]GnRH.
This result confirmed that increased binding could be obtained with the
new tracer. Although the
125I-[His5,D-Tyr6]GnRH
peptide was designed to have increased affinity for the receptor, its
higher binding could also be due to trivial causes, such as higher
specific activity or a higher bindable fraction of the
[His5,D-Tyr6]GnRH tracer
preparation. The cloned human GnRH receptor was used for subsequent
experiments because the mutant receptors for which we had been unable
to measure ligand binding were derived from the human GnRH
receptor.
Tracer preparations frequently contain radioactive impurities that do
not bind to the receptor of interest. These impurities may include
diiodinated peptides, iodinated peptide fragments, and peptides that
have been oxidized during the iodination reaction. As the increased
binding of
125I-[His5,D-Tyr6]GnRH
could be due to a lower proportion of unbindable radioactive
impurities, we determined what fraction of the tracer preparation could
be bound by a GnRH receptor. A human GnRH receptor mutant that contains
an extra N-glycosylation site was used in experiments to
determine the bindable fraction of
125I-[His5,D-Tyr6]GnRH.
This mutation increases the number of receptors present on the cell
membrane (16) and therefore makes it possible to achieve receptor
concentrations greater than the Kd, which is necessary for
determination of the bindable fraction of a radioligand (13).
Incubation of a limiting concentration of
125I-[His5,D-Tyr6]GnRH
tracer (22 pM) with increasing concentrations of
receptor-containing membranes showed that 69.3% of the radioactivity
in the tracer could be bound by the glycosylated GnRH receptor (Fig. 1
), similar to the 74.7% bindable
fraction of the
125I-[D-Ala6,N-MeLeu7,Pro9NHEt]GnRH
tracer. These results show that unbindable radiolabeled material made
up 30.7% of the
125I-[His5,D-Tyr6]GnRH
preparation and 25.3% of the
125I-[D-Ala6,N-MeLeu7,Pro9NHEt]GnRH
preparation, and we conclude, therefore, that the increased binding of
the
125I-[His5,D-Tyr6]GnRH
tracer could not be accounted for by higher purity of the tracer
preparation. The specific activity of the
125I-[His5,D-Tyr6]GnRH
tracer ranged between 900-1800 µCi/µg, which was similar to what we
have previously found for
125I-[D-Ala6,N-MeLeu7,Pro9NHEt]GnRH
(12). The similar specific activities of the two tracer preparations
indicate that both peptides are iodinated to a similar degree, and as
GnRH peptides that contain diiodotyrosine in position 5 do not bind to
the GnRH receptor, it is likely that the bindable fractions of both
tracers consist of peptides that contain a single atom of radioiodine.
125I-[His5,D-Tyr6]GnRH
probably was not radioiodinated on His residues because attempts to
radioiodinate the homologous peptide,
[His5,D-Trp6]GnRH, which contains
His residues in positions 2 and 5, but D-Trp instead of
D-Tyr in position 6, yielded only a small amount of
radiolabeled peptide that did not bind to GnRH receptors (not shown).
This result shows that modification of residues other than
Tyr5 or D-Tyr6 yields peptides that
do not exhibit high affinity binding to GnRH receptors.
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). In subsequent
binding experiments, incubations were performed overnight (18–24
h).
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).
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). Thus, the new tracer,
125I-[His5,D-Tyr6]GnRH,
binds to the human GnRH receptor with higher affinity than the
conventional GnRH analog.
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).
In contrast, sufficient binding of
125I-[His5,D-Tyr6]GnRH
was achieved to allow competition binding curves and, thus, estimation
of the Kd for the mutant receptor (Fig. 5). In addition,
nonspecific binding and the associated noise were lower (Fig. 5)
because less radioactivity and fewer cell membranes were required to
achieve measurable binding.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, the binding affinities of the unlabeled peptides [His5,D-Tyr6]GnRH and [D-Ala6,N-MeLeu7,Pro9NHEt]GnRH were indistinguishable. Thus, any difference in the receptor binding affinities of the unlabeled peptides is smaller than the experimental error in determining them. There remained the possibility, however, that radioiodination affects the two peptides differently and that the labeled peptides might therefore have distinct affinities for the recombinant human GnRH receptor. This possibility was tested using saturation binding experiments. Although the Kd of 0.19 nM determined for 125I-[His5,D-Tyr6]GnRH is slightly lower than the Kd determined for the unlabeled peptide in competition binding experiments, indicating a higher affinity of the labeled peptide, the difference is too small to draw any firm conclusion as to whether iodination of the D-Tyr6 residue increases binding affinity because of the different methods used to determine these values. Nevertheless, it is clear from our experiments that the affinity of 125I-[His5,D-Tyr6]GnRH is higher than that of the more conventional tracer, 125I-[D-Ala6,N-MeLeu7,Pro9NHEt]GnRH at the human GnRH receptor.
Previous studies showed that the affinities of iodinated GnRH analogs
were the same as those for equivalent unmodified peptides (4, 8) in rat
pituitary membranes, even though iodinated GnRH had exhibited decreased
potency in vivo (7). The Tyr5 residue, which is
modified in the iodination reaction, has been proposed to participate
in intramolecular interactions that stabilize the active conformation
of the GnRH peptide (6). Most substitutions for Tyr5 caused
decreases (40–95%) in GnRH activity (7, 19). However, substitution of
a His residue for Tyr5 slightly increased affinity for
mammalian GnRH receptors (12). This makes it possible to minimize
potential deleterious effects of modifying Tyr5 by
replacing it with His. Substitution of the achiral Gly residue, in
position 6 of GnRH, with D-isomers of natural or unnatural
amino acids is well known to increase affinity for GnRH receptors (10, 12, 20). Thus, a D-Tyr substitution is expected to be well
tolerated in position 6. In addition, GnRH activity increases with
increasing hydrophobicity of the side-chain of the residue in position
6 (21). Thus, radioiodination of a D-Tyr residue in
position 6 is likely to result in a tracer with increased affinity for
GnRH receptors. Therefore, the
[His5,D-Tyr6]GnRH peptide was
designed to obviate any deleterious effects of iodination at position 5
while capitalizing on the increased affinity associated with increased
hydrophobicity at position 6. The present study has not established
whether iodination of Tyr in either position 5 or 6 modifies the
affinity of GnRH agonists for the human GnRH receptor, but it is clear
that the
125I-[His5,D-Tyr6]GnRH
tracer has higher affinity for the GnRH receptor than does the
conventional tracer. The increased affinity for the GnRH receptor
accounts for the higher total binding that was obtained under the
nonsaturating conditions (tracer concentration, 50 pM)
of the preliminary competition binding assay.
We have previously described a mutation of the mouse GnRH receptor
(Glu301
Gln) that exhibited decreased affinity for GnRH,
but not for certain GnRH analogs (20). The equivalent mutation of the
human GnRH receptor (Asp302Asn) caused a decrease in
total binding of
125I-[D-Ala6,N-MeLeu7,Pro9NHEt]GnRH
such that we were unable to characterize the binding of the mutant
receptor. The
125I-[His5,D-Tyr6]GnRH
tracer allowed characterization of the mutant receptor. The high
affinity of the new tracer makes it possible to perform competition
binding assays with lower receptor concentrations and thus allows
analysis of mutant receptors with significantly decreased expression.
In addition,
125I-[His5,D-Tyr6]GnRH
should be useful for analysis of receptor mutations that cause small
decreases (<10-fold) in affinity if expression levels are not
compromised.
In conclusion, we have designed and synthesized a high affinity tracer
for GnRH receptor binding assays that is radioiodinated at position 6.
125I-[His5,D-Tyr6]GnRH
has allowed us to establish a more sensitive GnRH receptor binding
assay that requires fewer cells (5-fold) for assays using the
wild-type GnRH receptor and allows ligand binding analysis of some
mutant GnRH receptors that exhibit decreased expression or decreased
affinity for GnRH.
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Acknowledgments |
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Footnotes |
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2 Current address: Fishberg Research Center in Neurobiology, Box 1137,
Mount Sinai School of Medicine, 1 Gustav L. Levy Place, New York, New
York 10029.
3 Current address: Medical Research Council Reproductive Biology
Unit, 37 Chalmers Street, Edinburgh EH, United Kingdom.
Received January 29, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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