(Received for publication, May 2, 1995)
From the
The decapeptide gonadotropin-releasing hormone controls
reproductive function via interaction with a heptahelical G
protein-coupled receptor. Because a molecular model of the receptor
predicts that Lys in the third transmembrane helix
contributes to the binding pocket, the function of this side chain was
studied by site-directed mutagenesis. Substitution of Arg at this
position preserved high affinity agonist binding, whereas Gln at this
position reduced binding below the limits of detection. Leu and Asp at
this locus abolished both binding and detectable signal transduction.
The EC
of concentration-response curves for coupling to
phosphatidyl inositol hydrolysis obtained with the Gln
receptor was more than 3 orders of magnitude higher than that
obtained for the wild-type receptor. In order to determine whether the
increased EC
obtained with this mutant reflects an altered
receptor affinity, the effect of decreases in wild-type receptor
density on concentration-response curves was determined by irreversible
antagonism. Progressively decreasing the concentration of the wild-type
receptor increased the EC
values obtained to a maximal
level of 2.4 ± 0.2 nM. Comparison of this value with
the EC
of 282 ± 52 nM observed with the
Gln
receptor mutant indicates that the agonist affinity
for this mutant is reduced more than 100-fold. In contrast, antagonist
had comparable high affinities for the wild-type, Arg
,
and Gln
mutants. The results indicate that a
charge-strengthened hydrogen bond donor is required at this locus for
high affinity agonist binding but not for high affinity antagonist
binding.
GnRH, ()a decapeptide secreted from neurons in the
medial-basal hypothalamus, has a central role in regulating the
mammalian reproductive system. GnRH induces its biological effect by
interacting with high affinity pituitary receptors. cDNA cloning of the
GnRH receptor (GnRHR) from five mammalian
species(1, 2, 3, 4, 5, 6, 7, 8, 9) has
revealed that the receptor is a member of the large family of
homologous seven-transmembrane helix (TMH) G protein-coupled receptors,
which includes receptors for neurotransmitters and
peptides(10, 11, 12) .
Modulation of the pituitary-gonadal axis via the GnRHR has proven to be therapeutically important, and extensive research has led to the development of several thousand peptide analogs(13) . In contrast to some other peptides such as the tachykinins and cholecystokinin, for which small nonpeptide analogs have been identified, all GnRHR ligands reported thus far are peptides. Delineation of the precise contact sites between GnRH and its receptor is critical for developing an understanding of the relationship of the GnRHR binding pocket to that of other neurotransmitter and peptide receptors and for determining the molecular mechanisms underlying receptor activation. Ultimately this insight may lead to the design of novel GnRHR ligands.
We have
previously reported that a mutation in TMH 7 of the GnRHR restored
binding, which had been eliminated by a mutation in TMH 2 (14) . The revertant character of mutations at these loci
suggested that these two sites are in spatial proximity, a hypothesis
that facilitated the refinement of a preliminary three-dimensional
model of the receptor helix bundle constructed according to a set of
integrated methods (15) . This receptor model predicts that
Lys, located in the third TMH, is positioned in the
ligand binding pocket of the receptor and would be accessible to GnRH.
Lys
is found in all six cloned mammalian GnRH receptors
at a locus that corresponds to the position of the conserved Asp of the
cationic amine receptors (Asp
in the
-adrenergic
receptor), a residue required for high affinity neurotransmitter
binding(12, 16, 17) . In order to investigate
the role of Lys
in ligand binding and activation of the
GnRHR, a series of mutations was introduced at this position, and the
resulting receptors were expressed and characterized in COS-1 cells.
The results identify the role of charge-augmented hydrogen bonding at
this position for the affinity of agonists but not antagonists.
Expression of the wild-type receptor in COS-1
cells generated high affinity binding of the radiolabeled agonist
[I]GnRH-A (K
of
GnRH-A = 1.8 ± 0.3 nM). When Arg was substituted
for Lys
, affinity was comparable with that of the
wild-type receptor (K
= 3.3
± 1.0 nM, Table1, Fig. 1). However, with
the substitution of Gln, Leu, and Asp at this position, agonist binding
was reduced below detectable limits. In order to provide further
insight into the function of the mutant receptors, agonist-stimulated
PI hydrolysis was also evaluated. The wild-type, Arg
, and
Gln
receptors were all able to mediate phosphatidyl
inositol hydrolysis. No stimulation was detected in cells transfected
with the Leu
or Asp
receptor mutants (Fig.2). The EC
obtained with the Arg
construct was comparable with that found with the expression of
the wild-type receptor. The EC
obtained with the
Gln
receptor, however, was more than 3 orders of
magnitude higher ( Fig.2and Table 1). The EC
values obtained for stimulation with GnRH-A of the wild-type,
Arg
, and Gln
mutants showed the same trend,
including the large relative increase for the Gln
mutant (Table1).
Figure 1:
Agonist binding to the wild-type
() and Arg
mutant (
) GnRHRs expressed in COS-1
cells. Data represent mean and standard error of triplicate
determinations from one competition binding experiment, as described
under ``Experimental Procedures.'' The data are
representative of five replicate
experiments.
Figure 2:
GnRH-stimulated PI hydrolysis in COS-1
cells expressing mutant GnRHRs. The cells were transfected with the
expression vector pcDNAI/Amp (), Leu
(
),
Gln
(
), Arg
(
), or wild-type
(
) GnRHRs, and the resulting increase in intracellular inositol
phosphates was measured. The response was normalized to that obtained
in the wild-type receptor. Data shown are the mean ± S.E. Each curve is representative of four to five replicate
experiments.
To evaluate the cause of the increased EC values obtained with the Gln
receptor, the
relationship between receptor expression and EC
was
examined. In several receptor expression systems, the EC
has been found to depend on the number of receptors
expressed(22, 23, 24) . Such findings are
compatible with the presence of ``spare receptors'' in these
systems (25) . If the wild-type GnRHR were expressed in COS-1
cells with a high proportion of spare receptors, the increased
EC
observed with the Gln
receptor could, in
principle, be due to a marked decline in the level of mutant receptor
expression. To evaluate this possibility, the effect of decreasing
levels of receptor expression was studied using partial chemical
modification to irreversibly antagonize an increasing proportion of
receptors(26) . Cells previously transfected with the wild-type
GnRHR were exposed to varying concentrations of TNBS, which eliminates
binding sites by reacting with free amino
groups(27, 28) . Concentration-response curves were
then obtained in cells containing varying GnRHR concentrations (Fig.3). TNBS did not interfere with the capacity for the cells
to mediate signal transduction, as demonstrated by the lack of effect
of this treatment on the capacity of aluminum fluoride to stimulate PI
hydrolysis (data not shown). Thus the increasing EC
values
and decreasing E
observed with TNBS treatment
reflects a decrease in GnRHR receptors/cell and allows a precise
assessment of the effect of decreasing receptor expression on
EC
. Prior exposure to TNBS at concentrations that
decreased the maximal level of PI hydrolysis led to EC
values of 2.4 ± 0.2 nM. Since this value is more
than 100-fold lower than the EC
obtained for the
Gln
receptor, the difference in the
concentration-response curves observed cannot be attributed to altered
receptor expression. These results demonstrate, therefore, that the
affinity of agonist for the Gln
mutant receptor is
significantly reduced.
Figure 3: Effect of varying receptor expression on PI hydrolysis dose-response relationships. COS-1 cells were transfected with the wild-type receptor DNA and exposed to TNBS at the concentrations indicated prior to obtaining the concentration-response curves for GnRH.
Figure 4:
Blockade by antagonist of GnRH stimulation
of PI hydrolysis by the wild-type GnRHR (panels A and C) and Gln mutant receptor (panels B and D). A and B, GnRH stimulation of PI
hydrolysis was performed in the absence of antagonist 27 (
) or in
the presence of 3
10
M (
),
10
M (
), 3
10
M (
), 10
M (
),
or 3
10
M (
) of antagonist
27. C, Schild regression from one representative experiment
with the wild-type receptor. D, Schild regression representing
data obtained in four experiments with the Gln
receptor.
Antagonist 27 alone did not cause any PI stimulation at concentrations
up to 10
M (data not
shown).
Mammalian GnRH
contains an Arg in position 8, whereas chicken I GnRH, which has low
affinity for the mammalian receptor, has a Gln in this
position(30) . We have previously found that an acidic residue
in the third extracellular loop of the GnRH receptor is required for
this selectivity for mammalian Arg-GnRH(31) . The
effect of having an acidic residue at this position, however, is
smaller for constrained GnRH agonist analogs like GnRH-A. Thus,
substitution of Gln for Glu
in the mouse GnRH receptor
induces a 50-fold decrease in the affinity for GnRH but only a
4-5 fold change in the affinity for GnRH-A. These results are
consistent with the presence of different, although presumably
overlapping, binding sites for different classes of agonists. The
binding site of GnRH requires an acidic residue in the third
extracellular loop, whereas the binding site of GnRH-A has minimal
involvement of this locus. Agonist interaction sites that play a
fundamental role in positioning the pharmacophore for receptor
activation or in transmitting the activation itself are likely to be
conserved among all agonist classes. Because both GnRH activity and
GnRH-A activity require a positive charge at Lys
,
interaction with this site is likely to play a crucial role in receptor
activation. Taken together, these considerations identify at least
three types of determinants for ligand-receptor interaction: residues
required for antagonist binding, residues required for binding of
certain agonists, and residues involved in binding of all agonists.
Clarification of the role of various receptor loci in ligand
interaction should facilitate the understanding of the mechanism of
agonist activation of the receptor.
The two potential contact sites between GnRH and the
receptor identified to date, Lys and Glu
,
have similarities in their tolerance for variability at each position.
In both cases little alteration in affinity is induced by substitution
with a similarly charged residue. The Glu in extracellular loop 3 of
the mouse GnRHR, which is involved in high affinity GnRH binding, can
be replaced by an Asp with little effect on receptor
affinity(31) . In the present study, we found that the
Arg
substitution, presenting the hydrogen bond donor
group at either
or
positions that can mimic the
position of the group in Lys, gives comparable affinity. In either
locus of the GnRHR, substitution by Gln significantly decreases GnRH
affinity. Thus alterations in the length of the side chain of charged
residues in TMH 3 and in the third extracellular loop are both well
tolerated, suggesting flexibility in the spatial constraints for the
binding interaction.
In contrast to affinity, the receptor
activation mechanism may be sensitive to the length of the side chain
at position 121. Thus, the maximal level of GnRH-stimulated inositol
phosphate accumulation obtained with expression of the Arg receptor was significantly higher than that generated by the
wild-type receptor in all experiments (paired two-tailed t test, p < .05, n = 5 experiments).
Expression of the Gln
receptor led to a lower maximal
stimulation. Because the Gln
receptor was undetectable in
radioligand binding, the possibility that the reduction in E
was due to a lower level of expression of this
receptor cannot be definitively excluded. In the case of the
Arg
construct, however, the higher level of stimulation
was accompanied by a reduction in the level of receptor expression,
suggesting that the activated state of the Arg
receptor
is more efficient at G protein coupling than the activated wild-type
receptor. A qualitatively similar effect of side chain length on
efficacy was observed in the
-adrenergic receptor.
Asp
, which is located at a position homologous to that of
Lys
in the GnRHR, serves as the counterion for binding of
the catecholamine head group of the ligand. Substitution of Glu at this
position led to the development of partial agonist activity from
antagonists for the wild-type
-adrenergic receptor(34) .
In the GnRHR, replacing Lys
with Arg leads to an
augmentation of the efficiency of signal transduction by GnRH. In both
receptors, altering the length of this charged side chain leads to an
alteration of measured drug efficacy. This similarity between a
neurotransmitter and a peptide receptor suggests that the interaction
with this locus can contribute to activation, possibly by serving to
position the component of the ligand that triggers the receptor.
We have identified a transmembrane domain site in the GnRHR that is specifically involved in docking GnRH agonists. We have previously provided evidence for the proximity of specific side chains in helix 2 and helix 7 (14) and have proposed another site of GnRH docking in the third extracellular domain(31) . This study provides insight into the structure of the receptor and of the GnRH binding site. The results obtained constitute a useful basis for extending and refining an experimentally testable model of the structure of the receptor-hormone complex that will make possible the elucidation of the molecular dynamics of receptor activation.