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Endocrinology and Reproduction Research Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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K191 human receptor was similar to that of the wild-type receptor,
but the EC50 was decreased by about 5-fold. In
addition, the rate of internalization of the K191 human receptor was
significantly reduced and was similar to that of the mouse receptor. In
contrast to these effects of deletion of Lys191, its replacement by
Arg, Glu, Gln, or Ala caused no significant change in receptor
expression or function. These findings demonstrate that a specific
residue in the extracellular region of the human GnRH receptor is
a significant determinant of receptor expression, agonist-induced
activation, and internalization. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In functional studies, the human receptor has been found to be more rapidly internalized than the mouse receptor and is less abundantly expressed in transfected mammalian cells. Among the structural differences between the two species is the slightly larger size of the human GnRH receptor. Whereas the mouse and rat GnRH receptors contain 327 amino acids, the human, cow, and sheep receptors contain 328 amino acids, due to the presence of an additional residue in the second extracellular loop. This residue is Lys191 in the human GnRH receptor and Glu191 in the ungulate receptors (4). The present study was performed to examine the role of the additional Lys191 residue in the function of the human GnRH receptor. For this purpose, the Lys191 residue was deleted or substituted by other amino acids. The mutant GnRH receptors were transiently expressed in COS-1 cells and analyzed for ligand binding, agonist-stimulated inositol phosphate (InsP) production, and agonist-induced internalization of the hormone-receptor complex. The results of this study have demonstrated that deletion of Lys191 from the human GnRH receptor significantly increases its expression at the cell surface and alters its signaling properties and internalization kinetics. However, receptors in which Lys191 was substituted with arginine, glutamine, glutamic acid, or alanine behaved in the same manner as the wild-type receptor. These findings indicate that the Lys191 residue in the second extracellular loop of the human GnRH receptor has an unexpectedly prominent role in several aspects of receptor function.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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K191 human GnRH receptor to compare the binding,
signaling, and internalization properties of the two receptors. As
shown in Fig. 1
, cells expressing the
K191 human GnRH receptor bound more than 4 times as much
[125I]GnRH-Ag as the wild-type (WT) receptor at 37 C. To
determine the surface expression level and structural integrity of
these receptors, radioligand binding to COS-1 cells expressing WT or
mutant receptors was measured at 4 C. Scatchard analysis of the binding
data revealed that the dissociation constant (Kd) of the WT
receptor for the GnRH agonist was 10.5 ± 0.45 nM
(n = 3), whereas that of the K191 receptor was slightly but
consistently lower (7.6 ± 0.40 nM, n = 3). The
4-fold greater degree of GnRH-Ag binding to cells expressing the
K191 receptor vs. the WT receptor was due to its higher
expression level. The Bmax values were 72 ± 8 and
307 ± 30 fmol/well for the WT and K191 receptors,
respectively. The increase in expression level observed for the K191
receptor was also evident when enzyme-linked immunosorbent assay
(ELISA) measurements of receptor protein levels were performed (Fig. 2). Furthermore, when HEK 293 cells were
used to transiently express human GnRH receptors, the K191 receptors
were nearly 8-fold better expressed than the WT receptor (data not
shown). However, when Lys191 was replaced by Arg, Gln, Glu, or Ala, the
mutant receptors were expressed at almost the same level as the WT
human receptor (Fig. 1B). These results indicate that deletion of
Lys191 from the human GnRH receptor significantly increases the
expression level of the receptor, whereas its substitution by other
residues maintains the basal level of receptor expression.
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K191 human receptors is similar to that
of the WT mouse receptors, which are well expressed in this transient
cell expression system (see Fig. 1
). Interestingly, the insertion of a
Lys residue at position 191 in the mouse receptor did not have a
deleterious effect on its expression. In fact, the +K191 mouse receptor
is equally well expressed and has the same binding affinity as the WT
mouse receptor. The Kd values of the WT and +K191 mouse
receptors were 2.4 ± 0.22 (n = 3) and 2.0 ± 0.13
nM (n = 3), respectively.
In addition to the presence of an extra amino acid (Lys191) in
the second extracellular loop of the human receptor, this region has at
least four other differences from the sequence of the mouse receptor.
The human receptor contains His, Ser, Gln, and Ser at positions 182,
186, 189 and 203, whereas the mouse receptor has Tyr, Gly, Pro, and Pro
as the analogous residues. When these residues in the human receptor
were changed individually to the corresponding amino acids in the mouse
receptor, radioligand binding to the mutant receptors
(H182Y, S186G, Q 189P or S
203P) was similar to that of the WT human receptor (Fig. 3). These findings suggest that the other
regional amino acid differences between two species do not account for
the observed differences in receptor expression. As observed in Fig. 1, deletion of Lys191 caused a marked increase in the expression of the
human receptor.
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K191 human receptors were similar to those of the WT receptors, but
the EC50 for agonist stimulation was decreased by about
5-fold (Fig. 4A); EC50 values
were 75.0 ± 10.0 pM (n = 3) and 350 ± 70
pM (n = 3) for the K191 and WT human receptors,
respectively. The InsP response profiles for the mouse, +K191 mouse,
and K191R, K191E, K191Q, and
K191A human GnRH receptors were virtually identical to that
of the WT human receptor (Fig. 4, A and B) and had similar
EC50 values. These results indicate that overexpressed
K191 receptors are functionally active and show significantly
enhanced ability to elicit phosphoinositide hydrolysis.
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T3–1 cells (7, 8, 9, 10). A comparison of
internalization kinetics for the WT mouse and human GnRH receptors is
shown in Fig. 5A. It is evident that
human receptors have at least 2-fold higher internalization kinetics
than the mouse receptor. The endocytotic rate constant, which defines
the probability of an occupied receptor being internalized in 1 min at
37 C, was calculated from these data as described previously (8). These
values were 0.007 for the mouse and 0.022 for the human receptor.
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, A and B).
When Lys191 was deleted from the human receptor, the kinetics of
internalization were much slower than those of the native human
receptor (Fig. 5A), and the percent of radioligand sequestered at 60
min was similar to that of the mouse WT or +K191 receptors (Fig. 5C).
However, the internalization rates of the four Lys191-substituted
receptors were similar to that of the WT human receptor (Fig. 5B), with
the same amounts of radioligand sequestered at 60 min (Fig.
5C). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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K191 receptors bind GnRH agonist in a manner similar to the native
WT receptor. The major functional difference between the two receptors
is that the K191 receptors internalize slowly, similar to the mouse
receptor, whereas native human receptors have comparatively rapid
internalization kinetics with an almost 3-fold higher endocytotic rate
constant. The reason(s) for the low rate of K191 receptor
internalization are not apparent. The finding is intriguing in view of
the extracellular location of the Lys191 residue, at a site distant
from the transmembrane and intracellular regions that are believed to
engage in interactions with the signaling and internalization
machineries of the cell. It is remotely possible that the greater level
of expression of the mouse and the K191 receptors could saturate the
endocytic machinery, leading to the relatively decreased
internalization relative to the human receptor, which has lower
expression but exhibits 2-fold more rapid internalization. However,
this possibility is unlikely in view of our previous observations that
several mutations in the mouse receptor caused increased
internalization without any significant effect on receptor expression
(7, 8). The nature of the amino acid at position 191 does not appear to be critical, since its substitution by residues with different side-chain properties (Arg, Gln, Glu, or Ala) had no effect on receptor expression or function. It is possible that a motif containing Lys191 in the human receptor is not favorable to its folding and/or helices packing. Another possibility is that its presence in the human receptor augments the turnover rate, thus causing enhanced internalization of the receptor. If this is the case, then the mouse receptor bearing lysine at position 191 should have exhibited properties similar to that of the WT human receptor, but this was not observed. In the present study, no effect on receptor expression and internalization was seen after replacement of Lys191 with Glu, the amino acid residue that is usually present in the ungulate receptors. This implies that the expression and internalization patterns of ungulate receptors would resemble those of human rather than mouse receptors. These findings demonstrate that the presence of an additional residue at position 191 in the human receptor, and possibly in ungulates, is a critical determinant of receptor expression and internalization and that its deletion imparts the mouse phenotype.
The structural basis for the observation made in the present study is
not yet known. As a first approximation, examination of the primary
sequence in the second extracellular loop of the GnRH receptor revealed
that, although this protein is highly homologous in human and mouse,
there are a few differences upstream of residue Lys191. For example,
the human receptor has His, Ser, and Gln at positions 182, 186, and
189, whereas the mouse receptor has Tyr, Gly, and Pro as the analogous
residues. Downstream of Lys191, the human receptor contains Ser at
position 203, and the corresponding residue in the mouse receptor is
Pro. Individual mutations of these amino acids in the human receptor to
the corresponding residues in the mouse had no significant effect on
receptor expression, in contrast to the effect of deletion of Lys191
(Fig. 3). These amino acid differences presumably give rise to
different local secondary structures in the second extracellular loop
that are related to the observed interspecies differences in GnRH
receptor expression and function. The secondary structure predicted by
the algorithms of Chou and Fasman (11) in the region surrounding Lys191
of the human GnRH receptor indicates that there is no defined
structure, whereas its deletion confers a higher order structure that
could presumably stabilize the receptor protein (Fig. 6). The physiological relevance of these
results is not clear at this time. One possibility is that the
molecular factors governing the reproductive process at the level of
the GnRH receptor differ in humans and mice. This could, in part, be
related to the presence of the additional residue in the second
extracellular loop, which influences its expression and internalization
and possibly its desensitization mechanisms.
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K191 human receptor in transfected mammalian cells could be of
value in screening therapeutically useful GnRH analogs. The most
interesting aspect of this finding, the mechanism whereby this single
amino acid in the extracellular domain influences receptor expression
and function, remains to be clarified. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of WT and Mutant GnRH Receptors
The 1550-bp human GnRH receptor cDNA (kindly provided by Dr.
Sham S. Kakar, University of Alabama at Birmingham, Birmingham,
AL) was subcloned into pcDNA1/Amp at the EcoRI and
XbaI sites, and a hemagglutinin (HA) epitope
(YPYDVPDYA) was introduced at its amino terminus after the first
methionine residue. The HA-tagged human GnRH receptor was used as a
template for creating site-directed mutations according to the method
of Kunkel et al. (12) using a Muta-Gene phagemid in
vitro mutagenesis kit. Mutations were identified using the Thermo
Sequenase radiolabeled terminator cycle sequencing kit. The
construction of the plasmid expressing the mouse GnRH receptor has been
described previously (7).
Receptor Expression in COS-1 Cells
WT and mutant GnRH receptors were transiently expressed in COS-1
cells. To measure InsP responses, [125I]GnRH-Ag binding
to intact cells, and internalization kinetics, cultures were seeded in
24-well plates (Costar, Cambridge, MA) at a density of
0.15 x 106 cells per well 1 day before transfection.
The plated cells were cultured in DMEM supplemented with 10%
heat-inactivated FBS containing 100 U/ml of penicillin and 100 µg/ml
streptomycin (Pen-Strep) at 37 C in an atmosphere consisting of 5%
CO2/95% humidified air. Next day, the cells were
transfected with WT or mutant plasmid DNA (1 µg/well) using Fugene 6
transfection reagent. The cultures were maintained for 48 h before
analysis of receptor function by ligand binding and other assays.
Receptor Binding and Internalization Assays
The binding affinity and abundance of the mutant receptors were
determined in transfected COS-1 cells incubated with 2 nM
[125I]GnRH-Ag in binding medium (M199 containing 25
mM HEPES and 0.1% BSA) in the absence or presence of
increasing concentrations of unlabeled peptide for 3–4 h at 4 C. The
cells were then rapidly washed twice with ice-cold PBS (pH 7.4) and
solubilized in 0.5 M NaOH/1% SDS solution. The
cell-associated radioactivity was measured by 
-spectrometry. All
time studies were performed in duplicate on at least three occasions,
and displacement curves were analyzed for binding affinity and capacity
by the LIGAND program using a one-site model (13). The
nonspecific binding of [125I]GnRH-Ag determined in the
presence of unlabeled agonist (1 µM) for WT or mutant
receptors was always less than 5% of the respective total binding.
For internalization studies, transfected COS-1 cells were washed once
with binding medium before the addition of 2 nM
125I-labeled GnRH-Ag. After incubation at 37 C for the
indicated times, the cells were washed twice with ice-cold PBS (pH 7.4)
and incubated with 1 ml of 50 mM acetic acid/150
mM NaCl (pH 2.8) for 12 min to remove surface-bound tracer.
The acid-released radioactivity was collected to determine the
receptor-bound radioactivity, and the internalized (acid-resistant)
radioactivity was quantitated after the cells were solubilized in
NaOH/SDS solution. Radioactivities were measured by -spectrometry,
and the internalized radioligand at each time-point was expressed as a
percent of the total (acid-resistant + acid-released) binding.
InsP Production
COS-1 cells were labeled 24 h after transfection by
incubation in inositol-free DMEM containing 20 µCi/ml
[3H]inositol as described previously (7). After 24
h, cells were washed with inositol-free M199 medium and preincubated in
the same medium containing 10 mM LiCl for 30 min at 37 C,
and then stimulated with 10-10 to 10-6
M GnRH for 20 min. Incubations were terminated by the
addition of ice-cold perchloric acid [5% (vol/vol) final
concentration]. The InsPs were extracted and separated by anion
exchange chromatography as described previously (8), and their
radioactivities were measured by liquid scintillation
ß-spectrometry.
ELISA
An indirect ELISA protocol was used to quantify the expression
of epitope-tagged WT or mutant GnRH receptors in the plasma membrane.
COS-1 cells were seeded at a density of 90,000 cells per well in
48-well plates and transfected after 24 h with WT or mutant
receptor cDNA. After 48 h, the cells were fixed with 4%
paraformaldehyde in PBS for 30 min at room temperature. After washing
with PBS three times, treatment with DMEM containing 10% FBS to block
nonspecific binding sites, the cells were incubated at 37 C for 2
h with a monoclonal antibody directed against the HA-epitope tag
(Babco, Richmond, CA) at a dilution of 1:500 in DMEM. Plates
were then washed in DMEM and incubated with a 1:2000 dilution (in DMEM)
of peroxidase-conjugated goat antimouse IgG antibody (Sigma Chemical Co., St. Louis, MO) for 1 h at room temperature.
Hydrogen peroxide (0.03%) and o-phenylenediamine (5
mM) in 0.1 M phosphate-citrate buffer (pH 5.0),
serving as substrate and chromogen, respectively, were then added, and
the plates were kept in the dark for 30 min. The enzymatic reaction was
terminated with 1 M H2SO4
containing 0.05 M Na2SO3, and the
color development was measured at 495 nm using a Titertek Multiskan
plate reader (EFLABOY, Helsinki, Finland).
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FOOTNOTES |
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1 Both of these authors contributed equally to this work. 
Received for publication November 30, 1998. Revision received March 8, 1999. Accepted for publication March 10, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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T3–1 cells. Endocrinology 135:1165–1170[Abstract]
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