Insertional Mutagenesis of the Arginine Cage Domain of the Gonadotropin-Releasing Hormone Receptor
Smiljka Kitanovic,
Tony Yuen,
Colleen A. Flanagan1,
Barbara J. Ebersole and
Stuart C. Sealfon
Fishberg Research Center for Neurobiology (S.K., T.Y., S.C.S.)
and Department of Neurology (C.A.F., B.J.E., S.C.S.) Mount
Sinai School of Medicine New York, New York 10029
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ABSTRACT
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The pattern of side-chain conservation at the
cytoplasmic side of the third transmembrane domain of rhodopsin family
G protein-coupled receptors, Asp/Glu-Arg-Tyr/X-X-X-Ile/Val, defines a
structural "arginine cage" domain. Previous computational and
mutagenesis studies of the GnRH receptor indicated an important
contribution of local interactions to the function of this domain. We
have investigated the functional importance of the intrahelical
position and orientation of the arginine cage using insertional
mutagenesis. Introduction of a single Ala proximal to the conserved
Asp-Arg of this domain caused loss of detectable ligand binding.
Inserting a second Ala, however, restored high-affinity agonist
binding. Further insertion of three or four Ala residues at this site
generated receptors that bound agonist with an affinity 3- to 10-fold
higher than that of the wild-type receptor. Loss of detectable coupling
to inositol phosphate turnover in all these mutant receptors confirms
that the structure required in this region for efficient signaling is
highly constrained. In contrast, the recovery of agonist binding with
the progressive insertion of two to four Ala residues indicates that
specific orientations of this segment can stabilize high-affinity
receptor conformations that are uncoupled from signal transduction.
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INTRODUCTION
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The GnRH receptor (GnRHR) belongs to the large family of
G-protein coupled receptors (GPCRs). Members of this protein group
differ in their activators (neurotransmitter, hormone, light), as well
as in their downstream signaling pathways. However, all rhodopsin-like
GPCRs possess highly conserved residues that are important for
contributing to normal receptor structure and function. Because one
function shared by all GPCRs is the transition between active and
inactive receptor states, it is likely that many of the highly
conserved receptor side chains contribute to the structural mechanisms
that mediate this transition.
The highly conserved Asp/Glu-Arg-Tyr/X motif in helix 3 of
rhodopsin-like GPCRs is a hallmark of this large receptor family.
Studies of this motif in various rhodopsin-like GPCRs support the
hypothesis that conserved side chains contribute to receptor
activation. Mutation of the absolutely conserved Arg 3.50 in many
receptors either diminished or abolished signaling (see Materials
and Methods for consensus numbering scheme) in many receptors
(1, 2, 3, 4, 5). An Arg3.50Ala substitution in the
oxytocin receptor was constitutively active (6). Similarly varying
effects on the function of different GPCRs have been reported with
mutations of Asp 3.49. In several receptors, mutations of Asp 3.49 lead
to increased affinity for agonists, while receptor activation is either
enhanced (constitutively active receptors) (7), unaffected (8), or
undetectable (9, 10). These differing effects on activation suggest
that this domain may contribute to the stabilization of both active and
inactive conformers of these receptors.
We have previously studied the structure and function of this motif
(Asp-Arg-Ser) in the GnRHR using molecular modeling and site-directed
mutagenesis. Our study identified this segment as a component of a
larger, highly conserved "arginine cage domain,"
Asp/Glu-Arg-Tyr/X-X-X-Ile/Val (1). Computational modeling shows that a
clash between the side chains of Arg 3.50 and Ile 3.54 restricts the
orientations of the conserved Arg 3.50 side chain during a
repositioning that occurs with receptor activation (1). The steric
constraint, provided by the ß-branched character of the Ile 3.54 side
chain, supports efficient receptor activation (1). Such an interaction
is feasible, providing that the branched residue at position 3.54
is located one helical turn below Arg 3.50 (1). The recent crystal
structure of rhodopsin in the inactive conformation confirms the
presence of these specific side-chain interactions among conserved
residues that we had identified in the GnRHR (11). The study of the
arginine cage domain demonstrated the importance of the relative
positioning of conserved side chains for normal receptor function.
We have now investigated the functional effects of rotating and
displacing the arginine cage domain in the GnRHR using an insertional
mutagenesis strategy. Ala residues have been sequentially inserted to
displace and rotate the distal segment of the helix, and the effects of
this alteration on receptor function have been investigated.
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RESULTS
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Agonist Binding to Ala Insertional Mutants
GnRH and [D-Ala6,
Pro9-NHEt]-GnRH (GnRH-A) binding to the
wild-type (WT) mouse GnRHR and Ala insertional mutants was
characterized in transfected COS-1 cells. Introduction of one Ala just
proximal to the Asp-Arg-Ser in the GnRHR (mutant ADRS) eliminated
detectable agonist binding in both the whole-cell (Fig. 1
and Table 1
) and membrane binding assays (data not
shown). However, the loss of binding was restored by insertion of
additional alanines in mutants AADRS, AAADRS, and AAAADRS (Fig. 1
and
Table 1
). The affinities of GnRH for the AADRS and WT receptors were
comparable. In contrast, the AAADRS and AAAADRS mutants exhibited
increased affinity for GnRH and GnRH-A. The changes in affinity for
these constructs were greater when assayed at 4 C than at 22 C (Table 1
; see Discussion).
Expression of Epitope-Tagged WT and ADRS Receptors in COS-1
Cells
To determine whether the ADRS mutant, which had no detectable
agonist binding, was expressed in the membranes of transiently
transfected COS-1 cells, an epitope tag attached to a C-terminal tail
segment was incorporated into ADRS and WT mouse GnRHRs to generate the
mutants t-ADRS and t-WT, respectively (see Materials and
Methods). Whole-cell binding and inositol phosphate (IP) assays
confirmed that the t-ADRS has a phenotype similar to the untagged ADRS
mutant, with no detectable binding or activation of the IP signaling
pathway (Tables 1
and 2
). To determine
whether the t-ADRS mutant was synthesized, immunoblot analysis was
performed using both glycosylated and deglycosylated membranes. The
t-ADRS mutant was present at lower intensity than the WT receptor. The
deglycosylated mutant appeared to be full length, and the mutant had a
similar pattern of glycosylation to that of the WT receptor (Fig. 2
).

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Figure 2. Immunoblot of GnRHR Constructs
COS-1 cells were transfected with epitope-tagged WT and mutant GnRHR
constructs. Cell membranes were solubilized, electrophoresed, and
transferred to nitrocellulose. Epitope-tagged receptors were detected
with an antibody generated against tetrahistidine. The four samples on
the right were treated with N-glycosidase F. Equal
amounts of protein (40 µg) were added to each lane.
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Agonist-Stimulated Second Messenger IP Production of Alanine
Insertional Mutants
None of the mutants containing Ala insertions preceding
Asp-Arg-Ser mediated detectable IP production after GnRH stimulation
(Fig. 3
and Table 1
). Basal levels of IP
accumulation in cells expressing the mutant and WT receptors were
comparable (see Fig. 3
). Basal IP accumulation was also equivalent
after transfection of WT receptor and vector alone (data not shown).
Thus, neither the WT receptor nor any of the mutants possesses
detectable constitutive activity.

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Figure 3. GnRH-Stimulated IP Accumulation
COS-1 cells were transfected with WT GnRHR ( ) and ADRS (O), AADRS
( ), AAADRS ( ), AAAADRS ( ), and DRSAAA ( ) mutant receptors.
Data are the mean ± SEM of a representative
experiment performed in triplicate. For these constructs, basal levels
of IP production (cpm per well), are as follows: WT, 1,148; ADRS, 980;
AADRS, 804; AAADRS, 964; AAAADRS, 1,052; and DRSAAA, 978.
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We have previously demonstrated the importance of Ile 3.54 in
restricting the movement of the Arg 3.50 side chain and in contributing
to efficient receptor activation (1). The insertional mutagenesis
alters the position of both Asp-Arg-Ser and distal residues, including
Ile 3.54. To discriminate the contribution of displacement of
Asp-Arg-Ser from that of the distal residues to the phenotype of the
AAADRS mutant, we studied the effect of displacing only the distal
residues by inserting three alanines distal to Asp-Arg-Ser. The DRSAAA
mutant had an affinity for GnRH comparable to that of the WT receptor
(Table 1
). Unlike the AAADRS mutant, the DRSAAA receptor mediated
agonist-stimulated IP production, albeit at a reduced level in
comparison with the WT receptor (Fig. 3
and Table 1
). The coupling of
the DRSAAA mutant was similar to that of the Ile3.54
Ala mutant
previously reported (1).
Effects of GTP Analogs on Agonist Binding
Lack of IP stimulation may be due to the failure of mutant
receptors to interact with Gq/11. It is also
possible that high-affinity agonist binding of these receptors reflects
their interaction with another type of G protein. To study the
potential of insertional mutants to interact with G proteins other than
Gq/11, the effect of guanine nucleotides on
agonist binding was assessed. For these studies, the epitope-tagged WT
and AAADRS mutant receptors containing additional C-terminal sequence
were used because this modification was previously found to enhance
receptor expression (12) and was therefore expected to improve the
signal-to-noise ratio in these assays. The increase in tagged WT and
AAADRS GnRHR protein expression was reflected in elevated maximum
binding (Bmax) values for both constructs (Table 2
). However, the functions of receptors t-WT and t-AAADRS were
unchanged, as assessed by agonist binding affinity and coupling, which
were comparable with the untagged WT and AAADRS constructs,
respectively (Table 2
). Because the modification did not alter receptor
function, these constructs were used to study the effects of GTP
analogs on agonist binding.
In COS-1 membrane preparations, increasing concentrations of GTP
analogs [5'-guanylylimidodiphosphate (GppNHp) and
guanosine-5'-O-(3-thiotriphosphate (GTP
S)] decreased
the specific binding of [125I] GnRH-A to t-WT
GnRHR (Fig. 4
, A and B). This is
consistent with a productive interaction between the receptor and
cytosolic G proteins: the stimulated G protein dissociates from the
receptor, decreases receptor affinity for agonist, and leads to the
loss of detectable high-affinity agonist binding sites, as previously
reported for the bovine pituitary GnRHR at 37 C (17). In contrast,
under the same conditions, the specific binding of
[125I] GnRH-A to t-AAADRS was not affected by
either GppNHp (Fig. 4A
) or GTP
S (Fig. 4B
). In control experiments,
5'-adenylylimidodiphosphate (AppNHp) (Fig. 4C
) and 1 µM
adenosine-5'-O-(3-thiothriphosphate) (ATP
S) (data not
shown) had no effect on the binding of radiolabeled agonist to either
t-WT or t-AAADRS GnRHRs. These data indicate that the effects of GTP
analogs on the WT receptor were specifically mediated through G
proteins and that binding of agonist to the alanine insertional mutant
is not affected by guanine nucleotides.

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Figure 4. Effect of Nucleotide on Agonist Binding
Specific binding of agonist [125I] GnRH-A to t-WT
GnRHR (dark bars) and mutant t-AAADRS (light
bars) in membranes at 37 C in the presence of increasing
concentrations of GppNHp (A), GTP S (B), and AppNHp (C). In these
representative experiments, data obtained from triplicate points
(mean ± SEM) are presented relative to the
specific binding of agonist in the absence of nucleotides. The cpm
values obtained in triplicate ± SEM in this
experiment in the absence of nucleotide were for the GppNHp study,
t-WT: 3,208 ± 68, 288 ± 50; M: 1,962 ± 24, 330
± 15; for the GTP S study, t-WT: 4,446±55, 239 ± 18; M:
1,454 ± 24, 331 ± 30; for the AppNHp study, t-WT:
3,738 ± 72, 351 ± 16; M: 1,805 ± 30, 284 ± 16
with the total and nonspecific values separated by
commas and M representing the t-AAADRS mutant. Each
experiment was replicated at least three times.
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DISCUSSION
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The functional role of the distal segment of helix 3 in the GnRHR
has been studied using insertional mutagenesis, an approach previously
used for studying the muscarinic receptor (13). The periodic nature of
side chain conservation in the arginine cage domain (1) and the results
of spin-labeling studies performed on solubilized rhodopsin (14)
support an
-helical secondary structure of the arginine cage domain,
Asp/Glu-Arg-Tyr/X-X-X-Ile/Val. Assuming an
-helical structure of
this domain, in each of the mutant receptors the inserted alanines
would be expected to progressively rotate the side chains of subsequent
residues of the domain relative to other transmembrane domains (TMDs),
as well as to displace them toward the cytosol (Fig. 5
).

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Figure 5. Proposed Rotation and Extension of Distal Helix 3
with Insertions
The cytoplasmic end of the helix 3 backbone is shown in
yellow. Most side chains are omitted for clarity. The
Ile3.46 side chain (gray) is included to provide a point
of reference. The Ala insertions are shown in green.
Asp3.49 and Arg3.50 are red and blue,
respectively. A, WT. B, ADRS mutant. C, AADRS mutant. D, AAADRS mutant.
E, AAAADRS mutant. With each additional Ala insertion, the position of
Arg 3.50 shifts by 100°.
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Inserting one to four Ala residues proximal to the Asp-Arg-Ser motif
generated the mutants ADRS, AADRS, AAADRS, and AAAADRS. None of these
mutants were found to elicit IP second messenger production, either
constitutively or in response to agonist stimulation (Fig. 3
and Table 1
). These experiments cannot exclude the possibility that the absence
of detectable GnRH-stimulated IP production in these mutants resulted
from the lower levels of receptor expression. However, we believe this
interpretation is unlikely. The mutants with detectable binding express
at 1045% the level of the WT receptor. We have previously reported
that GnRHR mutants that are expressed at levels as low as 5% that of
the WT mediate IP production (12). The results from the guanine
nucleotide radioligand binding experiments also support the lack of G
protein coupling of the insertional mutant tested.
Mutant ADRS, having a single alanine insertion, showed no detectable
agonist binding in either COS-1 whole-cell (Fig. 1
and Table 1
) or
membrane preparations (data not shown). The protein detected on Western
blot analysis is not necessarily plasma membrane protein and could
represent endoplasmic reticulum membrane protein. Nonetheless, the
absence of high-affinity binding to ADRS most likely did not result
from a loss of protein synthesis or stability as indicated by
expression of glycosylated receptor. Progressive rotation of the
Asp-Arg-Ser motif by further insertion of two to four alanines in
mutants AADRS, AAADRS, and AAAADRS partially restored the high affinity
binding of GnRH, which was undetectable in the ADRS mutant.
Function-restoring mutants are an important protein-engineering
tool that can reveal the structural mechanisms contributing to specific
receptor properties. This approach had been used previously in the
GnRHR to elucidate how the interaction between two conserved residues
in helices 2 and 7 contributes to certain receptor functions (15, 16).
In the GnRHR, the single mutation of Asn2.50Asp
eliminates binding, coupling, and receptor expression (16). Introducing
a second mutation to the impaired construct,
Asp7.49Asn in helix 7, restores receptor
expression and allows detection of binding and coupling. In the present
study, the most detrimental mutation, ADRS, disrupts receptor function
without abolishing the expression of the receptor protein. However, in
mutants AADRS, AAADRS, and AAAADRS, additional Ala insertions restore
high-affinity binding of agonists. As the Asp-Arg-Ser motif of helix 3
is located in a region of
-helical secondary structure, each Ala
insertion would be expected to rotate the distal segment of the helix,
including the side chains of the Asp-Arg-Ser motif, by approximately
100o. Thus, recovery of binding in mutants with
two to four alanines inserted suggests that a large range of
Asp-Arg-Ser orientations are permissive for establishing a
high-affinity agonist binding site. The mutants AAADRS and AAAADRS
possess increased affinity for GnRH and GnRH-A relative to the WT
receptor (Table 1
). The increased affinity was specifically found with
displacement of the Asp-Arg-Ser segment; the DRSAAA mutant, which
displaces only the distal portion of the arginine cage motif, had an
affinity comparable to the WT receptor. Contrary to mutants ADRS and
AADRS, insertion of three or four alanines adjacent to the Asp-Arg-Ser
motif rotates and "returns" the residues of this motif to the same
face of the helix as they occupy in the WT receptor (Fig. 5
). Thus, in
the AAADRS and AAAADRS constructs, the residues of the Asp-Arg-Ser
motif may contribute to the formation of intramolecular bonds that
freeze the receptor in a high-affinity state, which is, however, not
able to activate G proteins.
Because assay temperature can influence receptor-G protein
interactions, which in turn regulate binding of agonists, we studied
the effects of altering assay temperature on agonist binding (17). In
addition to the standard binding assays performed at 4 C, ligand
binding was also determined at 22 C. The AAADRS and AAAADRS receptors
showed both an increase in agonist affinity relative to the WT receptor
and a sensitivity of their affinity for agonists to assay temperature
(Table 1
). The affinity of these two mutants for GnRH and GnRH-A
decreased approximately 2-fold with an increase in assay temperature
from 4 to 22 C (Table 1
). However, agonist affinity for the WT GnRHR
remained unchanged when assessed at either 4 or 22 C. At 22 C, the
Bmax values for the WT and all mutant receptors
decreased with respect to those measured at 4 C (Table 1
). The decrease
in Bmax with increasing temperature has been
reported previously for the bovine GnRHR (18). The increased affinity
of two of the insertional mutants suggests that the family of
conformations adopted by the AAADRS and AAAADRS receptors differ from
those of the WT GnRHR.
The phenotypes of mutants AAADRS and AAAADRS are similar to
phenotypes described for two arginine cage domain rhodopsin mutants. In
one mutant, the conserved Glu-Arg-Tyr motif was altered into
Arg-Glu-Tyr by an exchange of residues Glu 3.49 and Arg 3.50 (19). In
the other, Arg 3.50 was mutated into Gly (20). With exposure to light,
the Arg-Glu-Tyr mutant rhodopsin underwent transition into the
activated state, metarhodopsin ll, but failed to stimulate second
messenger production due to a lack of transducin binding (19). In the
second mutant, activated receptor bound transducin but failed to
stimulate GDP dissociation (20). Attempts to distinguish between the
two mechanisms for loss of coupling of the GnRHR insertional mutants,
loss of G protein binding and loss of G protein activation, through
receptor-G protein coimmunoprecipitation experiments were inconclusive.
We propose that the increased affinity GnRHR insertional mutants adopt
a partial active-state receptor conformation, reflected by their
increase in agonist affinity, but fail to stimulate second messenger
due to either impaired G protein binding or activation.
The present data indicate that when the Asp-Arg-Ser segment is
displaced, the receptor conformations that are assumed to form a
high-affinity agonist binding site fail to transmit the signal to the G
protein. This dissociation of high-affinity agonist complexing and
signaling are consistent with the proposed role of the arginine cage
domain in propagating signal transduction through the membrane.
Molecular modeling suggests that the active-state partners for Arg 3.50
are the Asn 2.50 and Asp 7.49 pair, previously identified as forming a
functional microdomain in GPCRs (12, 15, 16). In mutants AAADRS and
AAAADRS, the displacement of the arginine cage apparently disrupts the
formation of these critical interhelical bonds that facilitate receptor
activation, while interhelical bonds that stabilize high-affinity
agonist binding are preserved. Our studies support an emerging model of
sequential side chain rearrangements that underlie signal transduction
through the GnRHR.
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MATERIALS AND METHODS
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Consensus Residue Numbering Scheme
A consensus residue numbering scheme was developed to allow
comparison of corresponding residues among the helices of different
rhodopsin-like GPCRs (21). Each helix contains a highly conserved side
chain, which is used as a reference to identify the remaining residues
of the helix. For example, in helix 3, the most highly conserved
residue corresponds to Arg 139 of the Asp-Arg-Ser motif in GnRHR, which
is designated as Arg3.50(139). The number "3"
denotes helix 3 and "50" indicates the position of Arg 139. The
residues on the C-terminal side of Arg 3.50 are labeled with increasing
numbers, such as Ser3.51(140) in the GnRHR, while
those on the N-terminal side have decreasing numbers, such as
Asp3.49(138).
Mutagenesis
The mouse GnRHR mutants, ADRS, AADRS, AAADRS, AAAADRS, and
DRSAAA, were generated using PCR mutagenesis. In the ADRS mutant, one
alanine residue was introduced proximal to the Asp-Arg-Ser motif, while
in the AADRS, AAADRS, and AAAADRS mutants, two, three, and four
alanines were inserted at the same site, respectively. A silent
BglII restriction site was incorporated in each primer to
facilitate positive identification of mutant receptors. The mutated DNA
fragments were subcloned into ApaI and PflM 1
restriction sites of WT mouse GnRHR in the expression vector pcDNA/Amp
3 (Invitrogen, San Diego, CA), thus replacing the
corresponding WT receptor sequence. Mutations were confirmed by
BglII digestion and sequencing of mutant constructs.
Epitope Tagging of WT and Mutant ADRS and AAADRS GnRHRs
Using PCR and multifragment subcloning into the pcDNA/Amp 3
expression vector, the WT mouse GnRHR was modified by 1) insertion of
the hemagglutinin (HA) tag (YPYDVPDYA) after the initial Met residue,
and 2) addition of a carboxy-terminal domain derived from a putative
type ll human GnRHR, followed by a hexa-histidine tag (12). The
epitope-tagged WT mouse GnRHR was designated t-WT GnRHR. Epitope-tagged
mutants t-ADRS and t-AAADRS were generated by subcloning the mutated
fragments of ADRS and AAADRS into the ApaI and
PflM 1 sites of t-WT GnRHR.
Transfection
COS-1 cells grown in DMEM with 10% FBS were seeded into 100-mm
plates at a density of 3 million cells per plate. These cells were
transfected with DNA plasmids encoding the WT and mutant mouse GnRHR
receptors, using lipofectamine (Life Technologies, Inc.,
Gaithersburg, MD) as previously described (22).
Whole-Cell Agonist Binding Assay
COS-1 cells were assayed for ligand binding 2 days after
transfection with plasmid DNA encoding mouse WT and mutant GnRHRs.
Ligand [D-Ala6,
Pro9-NHEt]-GnRH (GnRH-A, Bachem,
Torrance, CA) radiolabeled with iodogen (Pierce Chemical Co., Rockford, IL) was used in the whole-cell competition
binding assay with increasing concentrations of cold GnRH-A or GnRH
(Bachem) in Ringer-HEPES buffer (pH 7.4) with 0.1% BSA,
at 4 or 22 C as previously described (22). The equilibrium dissociation
constant (Kd) and Bmax
values for GnRH-A were estimated from homologous competition binding
assays, using LIGAND software (23). Nonspecific binding was
subtracted from total binding before data analysis.
IC50 values for GnRH were obtained with
Kaleidagraph software (specific binding =
B0/(1+[GnRH]/IC50); where
B0 is maximum
[125I]GnRH-A bound; and
IC50 is the concentration of GnRH that inhibited
[125I ]GnRH-A binding by
50%), and subsequently converted into Ki
(equilibrium dissociation constant for GnRH) values using the
Cheng-Prusoff equation (24). Protein amounts were determined with the
Bradford reagent (Sigma, St. Louis, MO).
Preparation of Membranes for Western Blotting and Agonist Binding
in the Presence of GTP Analogs
Two days after transfection, COS-1 cells from two 100-mm plates
were each rinsed at room temperature with PBS and subsequently detached
with 3 ml of ice-cold buffer A (50 mM Tris-HCl, 1
mM EGTA, 5 µg/ml leupeptin, and 0.5 mM
phenylmethylsulfonyl fluoride, pH 7.4). Cells remaining on the plates
were collected with an additional 2 ml of buffer and added to the tube.
After centrifugation for 10 min at 500 x g, the cell
pellet was resuspended in buffer A and homogenized by 10 strokes in a
Dounce homogenizer. The suspension was transferred to a microfuge tube
and centrifuged at 500 x g for 10 min. Without
disturbing the pellet, the supernatant was collected and centrifuged at
17,000 x g for 30 min. The resulting membrane pellet
was resuspended in 5 ml of buffer A with 0.1% BSA for use in membrane
binding assays. For Western blotting experiments, membranes were
solubilized in 0.5 ml of buffer A containing CHAPS
([(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, final
concentration 1 mM) at 4 C for 30 min with gentle
rocking, and then centrifuged at 17,000 x g to remove
the insoluble material. Samples to be deglycosylated were treated with
N-glycosidase F (0.2 U per 20 µl of supernatant, Roche Molecular Biochemicals, Indianapolis, IN) for 30 min at 37 C,
before electrophoresis.
Agonist Binding to GnRHR Constructs in the Presence of GTP
Analogs
Cell membranes (5 µg protein) were incubated for 20 min
at 37 C with 200,000 cpm of [125I] GnRH-A,
5 mM MgCl2, and increasing
concentrations of GTP/ATP analogs (GTP
S, GppNHp, ATP
S, AppNHp;
Sigma) in a final volume of 0.4 ml of buffer A with 0.1%
BSA. This assay was performed at 37 C to facilitate guanine nucleotide
exchange and to allow correlation with the signal transduction assay
that is performed at the same temperature. Nonspecific binding was
determined by adding 1 µM GnRH-A. The reaction was
stopped by rapid filtration and washing with 3 x 5 ml of ice-cold
buffer B (50 mM Tris-HCl, 1 mM EDTA, and 0.1%
BSA, pH 7.4) in a cell harvester. Radioactivity bound to GF/C filters
(Brandel Inc., Gaithersburg, MD) was counted in a
-counter.
IP Assay
The IP assay was performed at 37 C, 3 days after transfection of
COS-1 cells with WT and mutant GnRHRs (22). For each concentration of
GnRH, IP production was converted to fold-stimulation of basal IP, then
analyzed and plotted using Kaleidagraph software (Synergy Software, Reading, PA) and the equation E =
Emax/(1 + EC50/[GnRH]),
where E represents the measured IP production,
Emax represents the maximum IP production, and
EC50 is the concentration of agonist that elicits
the half-maximal response.
Immunoblot Analysis
Solubilized COS-1 membranes (10 µl, 5 µg protein)
were mixed with 2x Laemli buffer and incubated for 30 min at 37 C.
Samples were electrophoresed on 12% SDS-PAGE gels, and proteins were
transferred to nitrocellulose membranes using a semidry blotting
apparatus. After blocking for 1 h in 5% milk/Tris-buffered
saline (TBS) at room temperature, the nitrocellulose was incubated
with Tetra-His antibody (QIAGEN, Valencia, CA; dilution
1:1000) in 5% milk/TBS overnight at 4 C. On the next day, the blot was
washed in TBS and incubated with horseradish peroxidase-conjugated
antimouse IgG (Amersham Pharmacia Biotech, Arlington
Heights, IL; dilution 1:1000) in 5% milk/TBS for 1 h at room
temperature, and afterward washed in TBS. The enhanced
chemiluminescence (ECL) kit (Amersham Pharmacia Biotech)
was used to visualize the epitope-tagged WT, ADRS, and AAADRS receptor
proteins adsorbed to the blot.
 |
ACKNOWLEDGMENTS
|
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We thank Dr. Daqun Zhang and Professor Harel Weinstein for
providing the computational graphic shown in Fig. 5
. The
technical assistance of Vladimir Rodic is gratefully appreciated.
 |
FOOTNOTES
|
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Address requests for reprints to: Stuart C. Sealfon M.D., Neurology Box 1137, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029. E-mail: sealfs01{at}doc.mssm.edu
1 Current address: Department of Medical Biochemistry, University of
Cape Town Medical School, Observatory, 7925, South Africa. 
This work was supported by NIH Grant DK-46943.
Received for publication May 25, 2000.
Revision received December 5, 2000.
Accepted for publication December 8, 2000.
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