From the MRC Reproductive Biology Unit, Centre for Reproductive
Biology, 37 Chalmers Street, Edinburgh EH3 9EW, United Kingdom and
Department of Experimental Zoology, Universiteit Utrecht,
Utrecht 3584 CH, The Netherlands
The mammalian gonadotropin-releasing hormone
receptor (GnRH-R) is the only G-protein-coupled receptor (GPCR) in
which the intracellular C-terminal tail is completely absent. In
contrast to other GPCRs, the GnRH-R does not show rapid desensitization of total inositol (IP) production, and the rates of internalization are
exceptionally slow. We investigated whether the incorporation of a
cytoplasmic tail into the C terminus of the GnRH-R affects desensitization events and receptor internalization rates. A
GnRH-R/TRH-R chimera was created where the intracellular tail of the
rat thyrotropin-releasing hormone receptor (TRH-R) was engineered into
the C terminus of the rat GnRH-R. Three different rat GnRH-R cDNA
stop codon mutations (one for each reading frame) were also made. The
GnRH-stimulated IP production of the wild-type rat GnRH-R expressed in
either COS-7 or HEK 293 cells did not desensitize even after prolonged stimulation with GnRH. In contrast, the catfish GnRH-R (which does
possess an intracellular tail) and the TRH-R rapidly (<10 min)
desensitized following agonist stimulation. The
GnRH-R/TRH-R chimera also desensitized following treatment
with GnRH, resembling the pattern shown by the TRH-R and the catfish
GnRH-R. Two of the stop codon mutants did not show desensitization of
IP production, and the third mutant with the longest tail was not
functional. Internalization experiments showed that the rat GnRH-R had
the slowest endocytosis and recycling rates compared with the TRH-R, the catfish GnRH-R, and the chimeric GnRH/TRH-R. This study
demonstrates that the addition of a functional intracellular
C-terminal tail to the GnRH-R produces rapid desensitization of
IP production and significantly increases internalization
rates.
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INTRODUCTION |
Gonadotropin-releasing hormone
(GnRH)1 is a decapeptide
released by the hypothalamus which acts on specific receptors in the anterior pituitary. GnRH and its receptor (GnRH-R) are key mediators of
the regulation of the reproductive axis controlling the synthesis and
release of the gonadotropins, luteinizing hormone, and
follicle-stimulating hormone. The GnRH-R is a member of the large
family of G-protein-coupled receptors (GPCRs) (1) and is preferentially
coupled to phosphoinositidase C via the Gq/G11
family of G-proteins. Following stimulation by GnRH, the production of
inositol phosphates (IPs) and diacylglycerol is induced resulting in
the elevation of cytosolic calcium and the activation of protein kinase
C (2-4). A number of GPCRs exhibit homologous desensitization upon
prolonged stimulation with agonist. This involves uncoupling of
G-proteins, rapid down-regulation of the receptor's effector systems
such as total inositol phosphate (IP) production followed by receptor
internalization, and presumably subsequent proteolytic degradation. In
particular this has been demonstrated for the m3 muscarinic
acetylcholine receptor (5) and the thyrotropin-releasing hormone
receptor (TRH-R) (6-9). The phosphorylation of specific amino acids in
the cytoplasmic part of the C-terminal tail of several GPCRs has been
implicated in desensitization events (3, 10, 11). This phosphorylation event is rapid, occurring within the first few minutes of agonist addition. Of all the GPCRs cloned to date, the mammalian GnRH-R is the
only receptor in which the functionally important intracellular cytoplasmic C-terminal tail is completely absent, as it is truncated at
the cell membrane immediately after the seventh transmembrane domain
(1). Studies by our group and by others (7, 12, 13) have shown that in
contrast to other GPCRs, the GnRH-R does not show rapid desensitization
of IP production. It is possible that during evolution the mammalian
GnRH-R has lost its intracellular cytoplasmic C-terminal tail as a
result of a mutation creating a stop codon, particularly since GnRH
receptors in non-mammalian species such as African catfish (14),
goldfish, frog, and chicken (15) still possess their intracellular
C-terminal tails. Here we have examined the rapid desensitization
events of a non-mammalian (catfish) GnRH receptor. We have also created
a GnRH-R/TRH-R chimera where the cytoplasmic tail of the rat TRH-R has
been engineered into the C-terminal region of the rat GnRH-R.
Furthermore, to investigate the possibility that a functional "ghost
tail" (the amino acids translated from the natural stop codon to the
next stop codon in the untranslated region) exists in mammalian
species, we have created three different stop codon mutations (one for each reading frame) within the rat GnRH-R cDNA. The aim of this study has been to investigate whether the missing cytoplasmic C-terminal tail could be implicated in the inability of the rat's GnRH-R to show rapid desensitization of IP production and its exceptionally slow internalization kinetics.
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EXPERIMENTAL PROCEDURES |
Materials--
Inositol-free Dulbecco's modified Eagle's
medium, penicillin, and streptomycin were obtained from Life
Technologies, Inc., Paisley, UK. Transfectam was obtained from Promega,
Southampton, UK. All other tissue culture reagents and media were
supplied by Sigma Cell Culture, Dorset, UK. Dowex resin was obtained
from Bio-Rad. [3H]Myoinositol was from Amersham Pharmacia
Biotech, Buckinghamshire, UK.
TRH-(3-Me-His2)-[3H] were from NEN Life
Science Products, Hertfordshire; TRH-(3-Me-His2) and
chicken II GnRH were from Peninsula Laboratories, Merseyside, UK. All
other compounds and reagents were obtained from Sigma, Dorset, UK. HEK
293 and COS-7 cells were obtained from the European Collection of
Animal Cell Cultures, Center for Applied Microbiology and Research,
Salisbury, UK.
GnRH Receptor Constructs--
The full-length rat GnRH-R
cDNA in the vector pcDNA3 (Invitrogen) was subjected to
oligonucleotide site-directed mutagenesis using standard methods to
generate three stop codon mutations, one for each reading frame. The
natural stop codon (TAA) was changed to GCAA for the +1 base mutant and
GCA for the in-frame mutant. The
1 base mutant was created by
changing the stop codon to GCA and deleting the naturally occurring T
following the stop codon. In all three cases the stop codon was changed
to a codon coding for alanine (Fig. 1A). To create the
GnRH-R/TRH-R chimera, a ClaI restriction enzyme site was
introduced in the stop codon of the rat GnRH-R cDNA, and
ClaI sites were introduced at the N- and C-terminal ends of
the rat TRH-R cytoplasmic C-terminal tail. The untranslated base
sequence from nucleotides 981 to 990 in the rat GnRH-R was changed from
TAA TTG GGA to CAA ATC GAT introducing the ClaI site and
changing the stop codon to glutamine. The base sequence from
nucleotides 997 to 1003 at the N terminus of the cytoplasmic C-terminal
tail of the TRH-R was changed from AAG CTC to ATC GAT, and the
untranslated base sequence from nucleotides 1238 to 1243 in the TRH-R
was changed from ACT TCC to ATC GAT introducing two ClaI
sites. The mutated C-terminal cytoplasmic tail of the TRH-R was then
amplified using polymerase chain reaction. The polymerase chain
reaction product was first cloned into the TA vector PCR 2.1 (Invitrogen), excised with ClaI enzyme, and then inserted
into the newly created ClaI site of the GnRH-R cDNA in
the vector pcDNA3. The cDNA clones were sequenced several times using an Applied Biosystems (Cheshire, UK) 373A automated sequencer. Sequence analysis was performed by means of the program GeneJockey II
(Biosoft, Cambridge, UK).
Tissue Culture--
Monolayer cultures of COS-7 cells (3 × 106 cells in 75-cm2 flasks) were transiently
transfected with WT rat GnRH-R, rat TRH-R, rat GnRH receptor
constructs, or the catfish GnRH-R in the eukaryotic expression vector
pcDNA3 using a standard DEAE-dextran method. Cells were then grown
for 48 h in Dulbecco's modified Eagle's medium containing 10%
heat-inactivated fetal calf serum, glutamine (0.3 mg/ml), penicillin
(100 units/ml), and streptomycin (100 units/ml) at 37 °C in a
humidified atmosphere of 5% CO2 in air before use. HEK 293 cells were stably transfected with the appropriate DNA using
Transfectam. Receptor expressing clones were selected with Geneticin (1 mg/ml) and identified with a functional receptor binding assay. The
cell lines expanded from these clones were then used for further study.
Iodination of GnRH Agonist--
Iodinated radiolabeled GnRH
analogue was prepared using the glucose oxidase/lactoperoxidase method
and purified by chromatography on a Sephadex G-25 column in 0.01 M acetic acid, 0.1% BSA. The specific activity of the
[125I-des-Gly10,D-Trp6]GnRH
was 56 µCi/µg and was calculated from self-displacement assays
using either rat pituitary homogenates or HEK 293 cells stably
expressing the wild-type GnRH-R. The specific activity of the
125I-chicken II tracer was 90 µCi/µg and was
calculated as described previously (16).
Receptor Internalization Assays--
Cells were plated at a
density of 2 × 105 cells/well in 24-well plates. The
cells were washed once with assay medium (HEPES modified Dulbecco's
modified Eagle's medium with 0.1% BSA, pH 7.4) before being incubated
with 125I-labeled GnRH agonist, 125I-labeled
chicken II GnRH or [3H]TRH-(3-Me-His2) in
0.5-ml assay medium for time intervals ranging from 5 min to 2 h
at 37 °C. At appropriate times, cells were transferred onto ice and
washed twice with ice-cold PBS. Subsequently, the extracellular
receptor-associated ligand was removed by washing once with 1 ml of
acid solution (50 mM acetic acid and 150 mM NaCl, pH 2.8) for 12 min. The acid wash was collected to determine the
surface-bound radioactivity, and the internalized radioactivity was
determined after solubilizing the cells in 0.2 M NaOH and 1% SDS (NaOH/SDS) solution. Nonspecific binding for each time point
was determined under the same conditions in the presence of 1 µM unlabeled agonist. For control experiments,
untransfected HEK 293 cells were assayed under identical conditions.
After subtraction of nonspecific binding, the internalized
radioactivity was expressed as a percentage of the total binding at
that time interval. All time points were performed in triplicate in at
least three separate experiments.
Receptor Binding Assay--
Ligand binding assays were carried
out on cell membranes from receptor expressing cells as described
previously (17). Briefly, purified membranes were incubated for 120 min
in assay buffer (40 mM Tris·HCl, 2 mM
MgCl2, 0.1% BSA, pH 7.4) at 4 °C with
125I-labeled GnRH agonist
([des-Gly10,D-Trp6]GnRH),
125I-labeled chicken II GnRH or
[3H]TRH-(3-Me-His2) with or without
10
6 M unlabeled agonist present. For
dose-displacement assays the unlabeled agonist concentration varied
from 10
10 to 10
6 M. The
membranes were then filtered, and the radioactivity was counted. All
assays were performed in triplicate. Binding parameters were determined
from displacement curves. Protein concentrations were measured using a
Bio-Rad protein assay kit (Bio-Rad) with a BSA standard.
Total Inositol Phosphates--
Total IPs were extracted and
separated as described previously (12, 18). Briefly, 24-well plates
containing 1 × 105 cells per well were incubated with
inositol free Dulbecco's modified Eagle's medium containing dialyzed
heat-inactivated fetal calf serum and [3H]myoinositol (2 µCi/well) for 48 h. Medium was then removed, and cells were
washed with buffer containing 10 mM LiCl followed by
addition of the appropriate ligand at a range of time points. The
reaction was stopped by adding cold perchloric acid or boiling water.
Neutralized supernatants from extracted cells were then transferred to
tubes containing AG 1-X8 anion exchange resin. Total IPs were then
eluted, and the radioactivity was counted.
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RESULTS |
Fig. 1A shows sequence
alignments of the C-terminal end of known sequences of cloned WT GnRH
receptors, whereas Fig. 1B shows the chimeric (GnRH-R/TRH-R)
and stop codon (+1 base,
1 base and in frame) receptor constructs.
Putative phosphorylation sites for protein kinase C and casein kinase
II are highlighted. The +1 base mutant and the
1 base mutant both
exhibited reduced (30-40%) 125I-GnRH agonist binding and
GnRH-stimulated IP production (50-60%) when compared with the WT
GnRH-R (Fig. 2). The in-frame stop codon mutant with the longest extension (52 amino acids) demonstrated no GnRH
agonist binding nor IP production. The GnRH-R/TRH-R chimera was able to
bind GnRH agonist and stimulate IP production even though its tail
extension was the longest (79 amino acids) of all these constructs.

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Fig. 1.
Sequence alignment of GnRH receptors.
A, sequence alignment from amino acid 320 to the stop codon
of the C-terminal region of cloned GnRH-Rs from different species. Note
that the non-mammalian catfish GnRH-R has an extended cytoplasmic
C-terminal tail. B, sequence alignment of the C-terminal
region of rat stop codon mutants and chimera GnRH receptor constructs.
In the rat GnRH-R stop codon mutant constructs, amino acids from the
natural stop codon in the rat GnRH-R have been mutated in each of the
three reading frames thereby extending the C-terminal tail in the
3'-untranslated region to the next occurring stop codon. Three
different rat GnRH-R stop codon mutant constructs, rat GnRH-R + 1 base,
1 base, and in frame, were created. The GnRH-R chimera was
constructed by inserting the rat TRH-R cytoplasmic tail in frame into
the rat GnRH-R stop codon. Putative phosphorylation sites for protein
kinase C (PKC) are shown in bold, and casein kinase II
phosphorylation sites are in bold and
underlined.
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Fig. 2.
Receptor binding and IP production of rat
GnRH receptor constructs. 125I-Labeled GnRH agonist
([des-Gly10,D-Trp6]GnRH) binding
to cell membranes prepared from COS-7 cells transiently expressing WT
rat GnRH-R and rat GnRH-R stop codon mutant and chimeric constructs.
Receptor binding assays were performed in triplicate ± S.E.
(n = 3) in the presence or absence of 10 6
M unlabeled GnRH agonist, and the binding is shown as a
percentage of agonist binding to the WT rat GnRH-R (solid
bars). GnRH-induced total IP accumulation in COS-7 cells
transiently expressing WT rat GnRH-R and rat GnRH-R stop codon mutant
and chimeric constructs was measured after 45 min stimulation with
10 6 M GnRH as described under "Experimental
Procedures." IP production is expressed as a percentage of total IP
production of the WT GnRH-R (open bars). Results shown are
the mean ± S.E. of triplicate observations from a single
representative experiment.
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In COS-7 cells transiently expressing the rat WT GnRH-R, we observed
that the GnRH-stimulated IP production did not desensitize even after
stimulation with GnRH for up to 45 min (the rate of IP production did
not slow down) (Fig. 3A). In
contrast, the IP production of the WT rat TRH-R did undergo
desensitization after the first 5 min of TRH stimulation (Fig.
3B) following which a decrease in IP production was
observed. The GnRH-R/TRH-R chimera was also able to desensitize after
10 min stimulation with GnRH (Fig. 3C). Desensitization of
IP production seen for the chimera resembled that observed for the
TRH-R, demonstrating the importance of the functional C-terminal tail.
Unlike the mammalian GnRH-R, the catfish GnRH-R desensitized rapidly
(within the first 5 min) following stimulation with GnRH (in this case,
chicken II GnRH, the preferred endogenous ligand for the catfish GnRH-R
(14)) (Fig. 3D). From the data shown in Fig. 3 the initial
rate of IP production (the first 10 min of stimulation) and the rate
after 10 min was calculated as described by Perlman and Gershengorn (8). As seen in Table I the rat WT GnRH-R
did not show significant desensitization in contrast to the rat WT
TRH-R, the GnRH-R/TRH-R chimera, and the catfish GnRH-R. The +1 base
and
1 base GnRH-R stop codon mutants stimulated IP production;
however, they did not undergo IP desensitization thereby resembling the
WT GnRH-R (data not shown).

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Fig. 3.
Desensitization of total IP production in
COS-7 cells. GnRH-induced total IP accumulation in COS-7 cells
transiently expressing WT rat GnRH-R (A), TRH-induced total
IP accumulation of WT rat TRH-R (B), GnRH-induced total IP
accumulation of the GnRH-R/TRH-R chimera (C), and chicken II
GnRH-induced total IP accumulation of WT catfish GnRH-R (D).
Cells in 24-well plates were stimulated with the appropriate agonist
(10 6 M) for the indicated time before total
IP was measured as described under "Experimental Procedures." IP
production is expressed as a percentage of total cell incorporated
radioactivity. Results shown are the mean ± S.E. of triplicate
observations from a single representative experiment.
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Table I
Rates of agonist-stimulated total IP production in COS-7 cells
Experiments were performed as described in the legend to Fig. 3. Rates
are given as percent of total IP produced per minute.
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In order to establish whether desensitization of IP production was cell
type-specific, we carried out similar experiments with HEK 293 cells
stably expressing WT rat GnRH-R, WT catfish GnRH-R, WT TRH-R, and the
GnRH-R/TRH-R chimera. We again observed that the TRH-R, catfish GnRH-R,
and the chimera desensitized, whereas the rat GnRH-R did not (Fig.
4). We did, however, observe a difference
between the two sets of experiments. In the 293 cells expressing the
TRH-R, catfish GnRH-R, and the chimera, the IP response desensitized
after approximately 100 s which is considerably faster than the
COS-7 cells that desensitized after 5-10 min. From the data shown in
Fig. 4 the initial rate of IP production (the first 120 s of
stimulation) and the rate after 120 s was calculated as described
by Perlman and Gershengorn (8). As seen in Table
II the rat WT GnRH-R did not show
significant desensitization in contrast to the rat WT TRH-R, the
GnRH-R/TRH-R chimera, and the catfish GnRH-R when stably expressed in
HEK 293 cells. Equilibrium dissociation constants
(Kd) and receptor numbers
(Bmax) were 0.2 nM and 3.1 pmol/mg
protein for the rat GnRH-R expressing HEK 293 cells,
Kd = 0.7 nM and
Bmax = 3.7 pmol/mg protein for the catfish
expressing HEK 293 cells, and Kd = 1.6 nM and Bmax = 9.5 pmol/mg protein
for the GnRH-R/TRH-R expressing HEK 293 cells. We have previously
published Kd = 4 nM and
Bmax = 13.5 pmol/mg protein values for the TRH-R
expressing HEK 293 cells (13). In GH3 cells which
endogenously express TRH-Rs, we observed that the desensitization of IP
production occurred after 10 min stimulation (data not shown), and we
have previously shown that pituitary gonadotrope alpha T3-1 cells
endogenously expressing GnRH-Rs do not undergo rapid desensitization of
IP production (19). Untransfected cells and cells transfected with receptors not matching the stimulating agonist did not respond to
stimulation (data not shown).

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Fig. 4.
Desensitization of total IP production in HEK
293 cells. GnRH-induced total IP accumulation in HEK 293 cells
stably expressing WT rat GnRH-R (A), TRH-induced total IP
accumulation of WT rat TRH-R (B), GnRH-induced total IP
accumulation of the GnRH-R/TRH-R chimera (C), and chicken II
GnRH-induced total IP accumulation of WT catfish GnRH-R (D).
Cells in 24-well plates were stimulated with the appropriate agonist
(10 6 M) for the indicated time before total
IP was measured as described under "Experimental Procedures." IP
production is expressed as a percentage of total cell incorporated
radioactivity. Results shown are the mean ± S.E. of triplicate
observations from a single representative experiment.
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Table II
Rates of agonist-stimulated total IP production in HEK 293 cells
Experiments were performed as described in the legend to Fig. 4. Rates
are given as percent of total IP produced per second.
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To investigate the possible role of the added cytoplasmic tail on the
ability of the rat GnRH-R to internalize, we performed a series of
internalization experiments using the four stably transfected HEK 293 cell lines described above. Approximately 70% of the rat TRH-Rs were
internalized after 15 min stimulation with TRH, whereas approximately
10% of the rat GnRH-Rs were internalized after 15 min stimulation with
GnRH (Fig. 5). The rat GnRH-R/TRH-R chimera and the catfish GnRH-R internalized considerably faster than
the rat GnRH-R but slower than the TRH-R. More than 20% of the
GnRH-R/TRH-R chimera receptors were internalized after 15 min
stimulation with GnRH, and approximately 50% of the catfish GnRH-Rs
were internalized after 15 min stimulation with chicken II GnRH (Fig.
5). By using a two-component model described by Koenig and Edwardson
(20), we calculated the rate constant for endocytosis
(ke) and the rate constant for recycling
(kr) (Table III).
The rate of endocytosis for the TRH-R was faster than for the other
receptors, and the rate of recycling was similar to that of the chimera
and the catfish GnRH-R but twice that of the rat GnRH-R. The rate
constants for the GnRH-R/TRH-R chimera were approximately double the
rate constants for the rat GnRH-R. Internalization experiments were
also performed with GH3 and alpha T3-1 cells, and
internalization rates were comparable to those observed for the HEK 293 cells expressing TRH or GnRH receptors (data not shown).

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Fig. 5.
Internalization of receptors in HEK 293 cells. Stably transfected HEK 293 cells were incubated with the
appropriately labeled agonist for the indicated time. Surface-bound
radioactivity was then determined as the radioactivity that could be
removed by acid wash. Internalized radioactivity was determined after
solubilization of the cells as described under "Experimental
Procedures." A curve has been fitted to the data points as described
by Koenig and Edwardson (20). The results shown are the mean ± S.E. of triplicate observations from a single representative
experiment.
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Table III
Rate constants of receptor endocytosis and recycling in HEK 293 cells
By using a two-compartment model described by Koenig and Edwardson
(20), the rate constant for endocytosis (ke) and the
rate constant for recycling (kr) were calculated for
WT rat GnRH-R, WT rat TRH-R, GnRH-R/TRH-R chimera, and WT catfish
GnRH-Rs stably expressed in HEK 293 cells.
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DISCUSSION |
The aim of this study was to investigate the desensitization and
internalization events of the WT rat GnRH-R and compare these findings
with results obtained from GnRH-Rs with naturally occurring or
artificially added C-terminal tails. In this paper we describe functional characteristics of the WT rat TRH-R, WT catfish GnRH-R, WT
rat GnRH-R, three rat GnRH-R stop codon mutants, and the rat GnRH-R/TRH-R chimera where the cytoplasmic tail of the TRH-R has been
engineered into the stop codon of the rat GnRH-R.
We found no sequence homology between the C-terminal region of the
three stop codon mutants and the catfish GnRH-R. This result is perhaps
not surprising as there has been no evolutionary pressure on the ghost
tails (the amino acids translated in each reading frame beyond the
natural stop codon to the next stop codon in the 3'-untranslated
region) for millions of years. We also demonstrated that cells
transfected with the stop codon mutants bound GnRH agonist less well
than cells transfected with WT GnRH-R, possibly due to reduced
expression. The binding was observed to decrease with increasing length
of the tail, and the stop codon mutant with the longest tail (52 amino
acids) showed no binding at all, indicating that the cells might not be
able to process a GnRH-R with a long unnatural C-terminal tail. In
support of this, COS-7 cells transfected with the GnRH-R/TRH-R chimera
bound GnRH agonist about 40% as well as cells transfected with WT
GnRH-R, even though the added TRH-R C-terminal tail (79 amino acids) is
longer than any of the ghost tails.
To investigate whether the addition of a known C-terminal tail
belonging to another GPCR would affect the function of the rat GnRH-R,
we created a GnRH-R/TRH-R chimera where the cytoplasmic tail of the rat
TRH-R was engineered into the C terminus of the rat GnRH-R. It could be
argued that the creation of a rat GnRH-R/catfish GnRH-R chimera where
the C-terminal tail of the catfish GnRH-R had been added to the
C-terminal of the rat GnRH-R would be more relevant. However, we chose
to use the C-terminal tail of the rat TRH-R as this receptor is also a
pituitary GPCR and is from the same species as the GnRH-R. Moreover,
the C-terminal tail of the TRH-R is of functional importance as has
been shown in several studies where mutated TRH receptors with
increasingly truncated C-terminal tails were studied (6, 10). The TRH-R has also been shown by several groups to undergo desensitization at the
level of IP production (7, 8). Similar data were not available for the
catfish GnRH-R before this study. In addition the amino acid sequence
homology between the mammalian and the catfish GnRH-R is only 38% (14)
and also the catfish GnRH-R does not recognize mammalian GnRH as its
primary agonist.
We observed that GnRH stimulated total IP production in cells
expressing WT rat GnRH-R did not desensitize even after prolonged stimulation with GnRH. This is in agreement with several other reports
(7, 12, 19) describing the lack of desensitization of GnRH induced IP
production in various cell lines (HEK 293, GH3, and alpha
T3-1 cells) expressing the receptor. The rapid desensitization observed
for the TRH-R is also supported by the findings of other groups (7, 8)
who studied the rapid desensitization events of TRH-R-stimulated IP
production in GH3 cells. The onset of desensitization in
these cells varied considerably (from approximately 1 min to 20 min) in
these studies, perhaps a result of different methods used.
The GnRH-R/TRH-R chimera desensitized in a manner similar to that
observed for WT TRH-R and catfish GnRH-R, indicating that the inability
of the rat's GnRH-R to desensitize is probably caused by the
receptor's lack of a cytoplasmic C-terminal tail. It has been
established that upon agonist stimulation of GPCRs the receptors become
phosphorylated on different intracellular amino acids, many of which
are situated on the C-terminal tail. This phosphorylation is mediated
by protein kinases such as protein kinase A, protein kinase C,
-adrenoreceptor kinase, or other members of the family of
G-protein receptor kinases (3, 5, 20-22). The fact that the GnRH-R
lacks a cytoplasmic C-terminal tail and thus important phosphorylation
sites may explain its inability to undergo rapid desensitization. The
time course of desensitization of total IP production was cell
line-dependent as desensitization was faster in HEK 293 cells (100 s) than in COS-7 cells (5-10 min). The reason for this is
not clear but could possibly be due to differences in the intracellular
mechanisms of the two cell types.
Phosphorylation of receptors has also been reported to be important for
endocytosis, for example via phosphorylation by
-adrenoreceptor kinase followed by recruitment of the cytosolic protein
-arrestin (20, 23) or by phosphorylation of amino acids on the C-terminal tail
(11). Further studies will investigate possible phosphorylation and the
functional importance of specific amino acids in the C-terminal tails
of the GnRH-R/TRH-R chimera and the catfish GnRH-R. Because truncation
of the C-terminal tail of the TRH-R impairs endocytosis (6), it is
perhaps not surprising that the rat GnRH-R internalizes with a lower
rate of endocytosis than the catfish GnRH-R and the TRH-R. The finding
that the GnRH-R/TRH-R chimera also internalizes faster than the WT
GnRH-R suggests that a cytoplasmic C-terminal tail is necessary for
rapid internalization. We used a two-component model to calculate
endocytosis and recycling rate constants ke and
kr, respectively. This model does not take into
account synthesis of new receptors or receptor degradation. However,
the model was found to be acceptable as receptor degradation is not
believed to take place during the time course of an internalization
experiment (21). Furthermore, we have found that internalization
experiments performed in the presence of cycloheximide (10 µg/ml), a
protein synthesis inhibitor, did not affect the results (data not
shown). All the receptors gave similar kr
values, with the exception of the rat GnRH-R which recycled at about
half the rate of the others. The ke for the
GnRH-R/TRH-R chimera was approximately 2-fold higher than the
corresponding value for the WT GnRH-R but still lower than the values
for the catfish GnRH-R and the TRH-R. Internalization rates of the
GnRH-R/TRH-R are not the same as the internalization rates for the
TRH-R, thus demonstrating that factors other than the C-terminal tail
might also be involved in the rate of internalization. In support of
this, recent evidence has suggested that sites within the third
intracellular loop of the m2 muscarinic acetylcholine receptor and in
the second intracellular loop of the GnRH-R are involved in receptor
internalization (24, 25).
The physiological relevance of the slow internalization and lack of
rapid desensitization of the mammalian GnRH-R are not understood. It
is, however, possible that the receptor's slow internalization and
lack of rapid desensitization is important for keeping a large number
of active receptors at the gonadotrope cell surface, in particular
during the GnRH surge that precedes the preovulatory luteinizing
hormone surge (26). It can be speculated that in more primitive
ancestral species the GnRH surge is not as profound as in mammals. In
summary, we have found evidence that shows the missing cytoplasmic
C-terminal tail of the rat GnRH-R to be at least partly responsible for
the inability of this receptor to desensitize at the level of total IP
production. We also found that the introduction of the cytoplasmic
C-terminal tail of the rat TRH-R into the C terminus of the rat GnRH-R
increased the internalization rates.
We are grateful to Dr. T. A. Bramley for
the 125I-chicken II tracer; to Dr. B. Byrne, Dr. E. Faccenda, and Dr. H. Rahe for critical evaluation of the manuscript;
and to Dr. G. B. Willars for helpful advice.