Addition of Catfish Gonadotropin-Releasing Hormone (GnRH) Receptor Intracellular Carboxyl-Terminal Tail to Rat GnRH Receptor Alters Receptor Expression and Regulation
Xinwei Lin,
Jo Ann Janovick,
Shaun Brothers,
Marion Blömenrohr,
Jan Bogerd and
P. Michael Conn
Oregon Regional Primate Research Center (X.L., J.A.J., S.B.,
P.M.C.) Beaverton, Oregon 97006
Department of Physiology
and Pharmacology (P.M.C.) Oregon Health Sciences University
Portland, Oregon 97201
Research Group for Comparative
Endocrinology (M.B., J.B.) University of Utrecht 3584 CH
Utrecht, The Netherlands
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ABSTRACT
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Mammalian GnRH receptor (GnRHR) is unique among G
protein-coupled seven-transmembrane segment receptors due to the
absence of an intracellular C-terminal tail frequently important for
internalization and/or desensitization of other G protein-coupled
receptors. The recent cloning of nonmammalian (i.e.
catfish, goldfish, frog, and chicken) GnRHRs shows that these contain
an intracellular C terminus. Addition of the 51-amino acid
intracellular C terminus from catfish GnRHR (cfGnRHR) to rat GnRHR
(rGnRHR) did not affect rGnRHR binding affinity but elevated receptor
expression by about 5-fold. Truncation of the added C terminus impaired
the elevated receptor-binding sites by 3- to 8-fold, depending on the
truncation site. In addition, introducing the C terminus to rGnRHR
altered the pattern of receptor regulation from biphasic
down-regulation and recovery to monophasic down-regulation. The extent
of down-regulation was also enhanced. The alteration in receptor
regulation due to the addition of a C terminus was reversed by
truncation of the added C terminus. Furthermore, addition of the
cfGnRHR C terminus to rGnRHR significantly augmented the inositol
phospholipid (IP) response of transfected cells to Buserelin, but this
did not result from the elevation of receptor-binding sites. Addition
of the C terminus did not affect Buserelin-stimulated cAMP and PRL
release. GH3 cells transfected with
wild-type cfGnRHR did not show measurable Buserelin binding
or significant stimulation of IP, cAMP, or PRL in response to Buserelin
(10-13-10-9
M). GH3 cells
transfected with C terminus-truncated cfGnRHR showed no IP response to
Buserelin (10-13-10-7
M). These results suggest that addition of the
cfGnRHR intracellular C terminus to rGnRHR has a significant impact on
rGnRHR expression and regulation and efficiency of differential
receptor coupling to G proteins.
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INTRODUCTION
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Pituitary gonadotropes respond to GnRH with the synthesis and
release of gonadotropins (LH and FSH), development of desensitization,
and regulation of GnRH receptors (GnRHR). The first step in GnRH action
is its recognition by the specific high-affinity GnRHR at the surface
of gonadotrope cells (1). The mammalian GnRHR has been cloned from at
least six species (2); the amino acid sequences from these sources are
substantially homologous and contain seven putative transmembrane
domains and many of the conserved residues and sequences,
characteristic of other members of the rhodopsin-like G protein-coupled
receptor (GPCR) family (2), consistent with a role for multiple G
proteins in GnRH action (3, 4). GnRHR is coupled to
Gq/11
in
T31 gonadotrope cells (5, 6). In
GGH3 cells (GH3 cells expressing rat GnRHR),
GnRHR is coupled to Gq/11
, resulting in activation of
phospholipase C and inositol phospholipid (IP) turnover (7, 8). In
addition, GnRHR appears to be coupled to adenylate cyclase-mediated PRL
release through Gs
in GGH3 cells (9, 10),
further emphasizing the promiscuity of GnRHR as a function of the
availability of G protein in the microenvironment of the target cells
(11). More recent studies using G protein knockout mice and confocal
microscopy showed that GnRHR in the primary pituitary cell is coupled
to Gq/11
(7, 12, 13).
Mammalian GnRHR has several unique features that distinguish
it from other GPCRs. Most striking is the absence of the intracellular
carboxyl-terminal tail (2, 14). The intracellular C terminus of many
GPCRs has been shown to be functionally important for G protein
coupling (15, 16, 17), agonist-induced receptor internalization (17, 18, 19, 20, 21, 22, 23, 24, 25),
and/or Ser/Thr phosphorylation-mediated desensitization (17, 26, 27, 28, 29, 30).
The intracellular C terminus of most GPCRs also contains a highly
conserved Cys that may be palmitoylated and form a fourth intracellular
loop (31, 32, 33, 34). However, the function of the intracellular C terminus
appears to be different among GPCRs. For example, in some GPCRs
agonist-stimulated Ser/Thr phosphorylation of the C terminus has been
implicated in receptor desensitization (26, 27, 28), while the C terminus
of others is involved in agonist-stimulated internalization, but not in
desensitization (21, 25, 35). Truncation of the CCK-A and
ß-adrenergic receptor did not result in altered internalization (20, 36), and truncation of the LH and FSH receptor did not affect
desensitization (37, 38).
Recently, a GnRHR cDNA was cloned from a teleost, the African catfish,
with only 38% amino acid sequence identity with mammalian
GnRHR (39). Catfish GnRHR (cfGnRHR) expressed in HEK 293 cells was
shown to mediate the native cfGnRH-stimulated phosphatidylinositol
hydrolysis and production of cAMP (39, 40), suggesting G protein
coupling for cfGnRHR similar to that observed in the mammalian GnRHR.
Another recent report of cloning of goldfish, frog, and chicken GnRHR
cDNAs showed that these nonmammalian GnRHRs have a high overall
homology (5867%) with each other, but only 4247% homology with
mammalian GnRHR (41). The surprising feature of these nonmammalian
GnRHRs is that they all contain an intracellular C terminus with
phosphorylation consensus sites and Cys residues. The presence of
intracellular C terminus in nonmammalian GnRHRs and in other GPCRs
raises the question of the evolutionary significance and physiological
implication of the absence of the intracellular C-tail in mammalian
GnRHR.
To elucidate the structural determinants and
structure/function evolution of GnRHR, a chimeric receptor was
constructed by addition of cfGnRHR intracellular C terminus to rat
GnRHR (rGnRHR). The chimera was truncated in some instances to create
mutant receptors containing different lengths of the intracellular C
terminus. The wild-type (wt) and mutant receptor cDNAs were
transiently expressed in GH3 cells, and the receptor
binding, homologous regulation, and receptor-mediated signal
transduction pathways were examined.
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RESULTS
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Addition of an Intracellular C Terminus Does Not Affect rGnRHR
Binding Affinity but Significantly Elevates the Number of rGnRHR
Binding Sites
A chimeric receptor (rGnRHR-Ctail, Fig. 1
) was constructed by addition of cfGnRHR
intracellular C terminus to rGnRHR. The intracellular C terminus of
cfGnRHR contains 51 amino acids, including two consensus
phosphorylation sites for protein kinase C and two Cys residues (Fig. 1
). Thus, the chimeric rGnRHR-Ctail is comprised of the 327 amino acids
of wt rGnRHR and 51 amino acid of cfGnRHR, forming a chimera of 378
amino acids with a presumptive intracellular C terminus of 53 amino
acids (Fig. 1
).

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Figure 1. Schematic Model of the rGnRHR Showing Addition of
the Intracellular Carboxyl-Terminal Tail of cfGnRHR
The putative structure of rGnRHR is shown by open
circles; the portion of intracellular carboxyl-terminal tail of
the cfGnRHR is represented by solid circles. The amino
acid sequences of the cfGnRHR intracellular carboxyl terminus and three
truncated C termini are indicated. Two consensus phosphorylation sites
for protein kinase C are underlined.
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wt rGnRHR, wt cfGnRHR, and chimeric rGnRHR-Ctail were transiently
expressed in GH3 cells. To compare the receptor expression
and binding characteristics of wt rGnRHR with cfGnRHR and chimeric
rGnRHR-Ctail, receptor binding assays were performed using a
metabolically stable agonist of GnRH, [125I]Buserelin.
Scatchard analysis of the binding of [125I]Buserelin
(Fig. 2
) showed that rGnRHR and
rGnRHR-Ctail have similar binding affinity for Buserelin, with
dissociation constant (Kd) values of 1.52 nM
(rGnRHR) and 1.46 nM (rGnRHR-Ctail), while wt cfGnRHR
showed no measurable binding of Buserelin. In contrast to the
Kd, the number of binding sites of chimeric rGnRHR-Ctail
receptor was about 5-fold higher than that of wt rGnRHR, with
Bmax of 17,087 sites per cell (assuming similar
transfection efficiency) for wt rGnRHR and Bmax of 92,046
sites per cell (assuming similar transfection efficiency) for
rGnRHR-Ctail, indicating a significantly increased receptor expression
at the cell surface due to the addition of the intracellular C terminus
of cfGnRHR. RT-PCR showed no difference between the mRNA levels for wt
rGnRHR and rGnRHR-Ctail (data not shown).

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Figure 2. Scatchard Plots for Binding of
[125I]Buserelin to GH3 Cells Expressed wt
rGnRHR, wt cfGnRHR, or Chimeric rGnRHR-Ctail
Seventy two hours after transfection of GH3 cells, the cell
suspension (106 cells) was incubated with increasing
concentrations of [125I]Buserelin, as indicated, for
3 h at 4 C. Cell-associated specific activity was measured (see
Materials and Methods).
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Addition of an Intracellular C Terminus Changes the Pattern of
Homologous Regulation of rGnRHR and Enhances the Extent of
Down-Regulation
To study the homologous regulation of wt and chimeric receptor,
GH3 cells were transiently transfected with either wt
rGnRHR or rGnRHR-Ctail and incubated with 10 nM GnRH for
the indicated times (Fig. 3
). Consistent
with results of the binding study (Fig. 2
), the number of binding sites
of rGnRHR-Ctail was about 5-fold higher than that of wt rGnRH (Fig. 3
, upper panel). In addition, the receptor-binding assay showed
that the receptor number of wt rGnRHR and chimeric rGnRHR-Ctail was
regulated differentially (Fig. 3
, lower panel). The wt
rGnRHR number was regulated in a biphasic fashion. The receptor was
initially down-regulated, reaching its nadir at 2 h, with
approximately 25% reduction of specific binding compared with control
cells at initial time point (zero hour). The wt rGnRHR number recovered
thereafter (27 h) but did not overshoot the control value, with 10%
reduction of specific binding at 7 h compared with the control. In
contrast, the rGnRHR-Ctail receptor number was regulated in a
monophasic fashion during the incubation period. The receptor was
progressively down-regulated during 7 h of incubation. After
2 h, both wt and rGnRHR-Ctail receptors were similarly
down-regulated, with a 25% decrease in specific binding compared with
that of control at the initial time. Instead of recovery of wt GnRHR
after 2 h incubation, the rGnRHR-Ctail remained down-regulated,
with a 55% decrease in specific binding at 7 h compared with that
observed at the initial time. These results indicate that addition of
an intracellular C terminus to rGnRHR changes the pattern of homologous
regulation of rGnRHR from a biphasically down- and up-regulated pattern
to a monophasic down-regulated pattern without recovery.

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Figure 3. Homologous Regulation of the GnRHR in
GH3 Cells Expressed wt rGnRHR or Chimeric rGnRHR-Ctail
Seventy two hours after transfection of GH3 cells, cells
were incubated with 10 nM GnRH for the indicated times. The
GnRH was removed, and the binding of [125I]Buserelin was
assessed as described in Materials and Methods. Data
shown are the mean of triplicate treatments, represented by specific
binding in counts per min (upper panel) and in the
percentage of control at initial incubation time (lower
panel). Each experiment was repeated at least three times, with
similar results.
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Addition of an Intracellular C-Terminus Augments rGnRHR-Mediated
Inositol Phosphate Production Which Is Uncoupled from the Increase in
Receptor Binding Sites
A dose-response study of Buserelin-stimulated IP production is
shown in Fig. 4
. Two hours of stimulation
with Buserelin resulted in a significant, dose-dependent response in IP
production from GH3 cells expressing wt rGnRHR and chimeric
rGnRHR-Ctail. The response of IP production from GH3 cells
expressing chimeric receptor was higher than that observed for
GH3 cells expressing wt receptor, with EC50 of
8.25 x 10-11 M for rGnRHR-Ctail and
EC50 of 1.56 x 10-10 M for
wt rGnRHR. However, this difference (
2-fold in EC50) in
IP production between wt rGnRHR and rGnRHR-Ctail was not proportional
to the 5-fold increase in receptor binding sites of rGnRHR-Ctail
compared with wt rGnRHR. Two hours of treatment with
10-13-10-9 M Buserelin did not
stimulate IP production from GH3 cells transfected with wt
cfGnRHR. However, a significant increase in IP production was observed
at higher doses (10-8-10-7 M) of
Buserelin. There was no measurable elevation in IP production from
GH3 cells transfected with C terminus-truncated cfGnRHR
(cfGnRH-t329) at 10-13-10-7 M
Buserelin treatment.

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Figure 4. Dose-Response of Buserelin-Stimulated IP Production
in Transfected GH3 Cells
Forty eight hours after transfection of GH3 cells with wt
rGnRHR, wt cfGnRHR, chimeric rGnRHR-Ctail, or truncated cfGnRHR
(cfGnRHR-t329), the cells were preloaded with 4 µCi/ml
[3H]inositol for 18 h. The cells were treated with
the indicated concentrations of Buserelin for 2 h. Total IP
production was determined by ion exchange chromatography. The data
shown are the means of triplicate determinations, represented by the
percentage of control (treated with medium alone). Error bars show the
SEM. Each experiment was repeated at least three times with
similar results.
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Addition of an Intracellular C Terminus Does Not Affect
rGnRHR-Mediated cAMP Production and PRL Release
Incubation with 10-13-10-7 M
Buserelin for 24 h stimulated cAMP release in a dose-dependent
manner in GH3 cells expressing either wt rGnRHR or chimeric
rGnRHR-Ctail (Fig. 5
). However, there was
no significant difference in Buserelin-stimulated cAMP release between
GH3 cells expressing wt rGnRHR and GH3 cells
expressing chimeric rGnRHR-Ctail. Similarly, a 24-h Buserelin treatment
stimulated PRL release in a dose-dependent manner in GH3
cells expressing wt and chimeric receptors (Fig. 6
), and there was no significant
difference between the response of wt receptor and chimeric receptor.
No significant elevation of responses of cAMP release or PRL release
above basal levels was observed from GH3 cells transfected
with cfGnRHR at 10-13-10-8 M of
Buserelin treatment.

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Figure 5. Dose-Response of Buserelin-Stimulated cAMP Release
in Transfected GH3 Cells
Forty eight hours after transfection of GH3 cells with wt
rGnRHR, wt cfGnRHR, or chimeric rGnRHR-Ctail, the cells were incubated
with the indicated concentrations of Buserelin and 0.2 mM
MIX for 24 h. The samples were heated at 95 C for 5 min with 1
mM theophylline, and their cAMP contents were determined by
RIA. The data shown are the means of triplicate determinations,
represented by the percentage of control (treated with medium alone).
Error bars show the SEM. Each experiment was repeated at
least three times with similar results.
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Figure 6. Dose Response of Buserelin-Stimulated PRL Release
in Transfected GH3 Cells
Forty eight hours after transfection of GH3 cells with wt
rGnRHR, wt cfGnRHR, or chimeric rGnRHR-Ctail, the cells were incubated
with the indicated concentrations of Buserelin for 24 h. The
medium was collected, and PRL release was measured by RIA. The data
shown are the means of triplicate determinations, represented by the
percentage of control (treated with medium alone). Error bars show the
SEM. Each experiment was repeated at least three times with
similar results.
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Truncation of a Portion of the Added C Terminus in rGnRHR Reduces
the Number of Receptor Binding Sites and Attenuates Homologous
Down-Regulation
To construct rGnRHR containing different lengths of the C
terminus, the chimeric rGnRHR-Ctail was truncated at either residue
Arg337, Asn343, or Ser350,
respectively, in the added intracellular C terminus. This resulted in a
rGnRHR construct containing an 11-amino acid C terminus
(rGnRHR-Ctail-t337), a 17-amino acid C terminus (rGnRHR-Ctail-t343), or
a 24-amino acid C terminus (rGnRHR-Ctail-t350) (Fig. 1
).
The rGnRHR-Ctail and three truncated rGnRHR-Ctail were transiently
expressed in GH3 cells. The GH3 cells were then
continuously incubated with 10 nM GnRH for the indicated
times (Fig. 7
), and receptor binding to
[125I]Buserelin was assessed. Compared with the
rGnRHR-Ctail, the three truncated receptors show 3- to 8-fold reduced
specific binding for [125I]Buserelin at the initial time
point of incubation (Fig. 7
, upper panel), with 3-fold
reduction for rGnRHR-Ctail-t350 (longest tail), 4-fold reduction for
rGnRHR-Ctail-t337 (shortest tail), and 8-fold reduction for
rGnRHR-Ctail-t343 (medium length of tail). These results indicate that
truncation of the C terminus of rGnRHR-Ctail reduced the number of
receptor binding sites; however, this reduction was not directly
related to the length of C terminus. In addition, truncation of the C
terminus of rGnRHR-Ctail also changed the pattern of homologous
regulation of rGnRHR (Fig. 7
, lower panel). Similar to the
biphasic pattern of homologous regulation of wt rGnRHR, the specific
binding of rGnRHR-Ctail-t337 was reduced by 47% after a 1-h incubation
with GnRH. The rGnRHR-Ctail-t337 receptor number recovered thereafter
(27 h), but did not overshoot the control value, with 5% and 10%
reduction of specific binding at 5 h and 7 h, respectively,
compared with the control at the initial time. The specific binding of
rGnRHR-Ctail-t343 was gradually reduced over 15 h in the presence of
GnRH, with a 28% reduction at the 5-h time point. The specific binding
of rGnRHR-Ctail-t343 slightly recovered at the 7-h time point. The
specific binding of rGnRHR-Ctail-t350 was modestly down-regulated
during 17 h incubation of GnRH, with a 21% reduction at the 7-h time
point.

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Figure 7. Homologous Regulation of the GnRHR in
GH3 Cells Expressed rGnRHR-Ctail and Three C Terminus
Truncated Receptors
Seventy two hours after transfection of GH3 cells, cells
were incubated with 10 nM GnRH for the indicated times. The
GnRH was removed and the binding of [125I]Buserelin was
assessed as described in Materials and Methods. Data
shown are the mean of triplicate treatments, represented by specific
binding in counts per min (upper panel) and in the
percentage of control at initial incubation time (lower
panel). Each experiment was repeated at least three times, with
similar results.
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DISCUSSION
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Mammalian GnRHR is unique among GPCRs and distinct from
nonmammalian GnRHRs, since the former lack an intracellular C-terminal
tail. In the present study, addition of the C terminus did not affect
receptor-binding affinity, but significantly elevated the receptor
expression at the cell surface. Truncation of the added C terminus
impaired the elevated receptor binding. Addition of the C terminus
altered the pattern of receptor regulation from biphasic down- and
up-regulation to monophasic down-regulation alone and significantly
enhanced the extent of down-regulation. This alteration in receptor
regulation was reversible by truncation of the added C terminus.
Addition of the C terminus significantly augmented the IP response to
Buserelin, but this augmentation was not coupled to the elevation of
receptor- binding sites. Addition of the C terminus did not affect the
Buserelin-stimulated cAMP and PRL release. GH3 cells
transfected with wild-type cfGnRHR did not show measurable binding to
Buserelin and significant IP, cAMP, and PRL responses to Buserelin
(10-13-10-9 M). GH3
cells transfected with C terminus-truncated cfGnRHR showed no IP
response to Buserelin (10-13-10-7
M). These results suggest that introduction of cfGnRHR
intracellular C terminus to rGnRHR has a significant impact on rGnRHR
expression and regulation and efficiency of receptor coupling to G
protein in GH3 cells. The results imply that due to the
absence of the C terminus, mammalian GnRHR might have evolved distinct
receptor expression levels and patterns of receptor regulation needed
to adapt to physiological requirements.
The role of the intracellular C-terminal tail of GPCRs on the receptor
cell surface expression is unclear, as truncation of C terminus of
different GPCRs results in varied levels of receptor expression. In
some GPCRs, the truncation of C terminus did not affect receptor number
at the cell surface (19, 21, 37), whereas in some other GPCRs, the C
terminus-truncated receptor showed either reduced (17, 24, 27, 35) or
increased binding sites (30) but no difference in binding affinity
compared with wt receptor. However, the effect of the truncation of the
C-terminal tail on the number of receptor-binding sites was dependent
on the site where the truncation occurred. Studies in a number of GPCRs
showed that truncation of the distal portion of the C-terminal tail,
which usually includes the Ser/Thr-enriched region, did not
significantly alter the receptor-binding capacity, while truncation of
a large portion or the entire C-terminal tail typically impaired or
abolished the receptor expression at the cell surface due to the
intracellular localization of truncated receptor (17, 24, 35). These
results suggest that part of the C-terminal tail is involved in the
trafficking and routing of the receptor to the plasma membrane.
Since mammalian GnRHR normally lacks the intracellular C-terminal tail,
it is a useful model with which to examine the impact of extension of a
C terminus on receptor expression and function. The present results
show that addition of a C-terminal tail significantly enhances the
rGnRHR expression at the cell surface. This enhancement can be reversed
by truncation of a portion of the added C-terminal tail; however, the
mechanism involved in this action of the added C terminus remains
unknown. The absence of an intracellular tail in mammalian GnRHR is
likely to be accompanied by structural accommodations in other parts of
the receptor, forming the intact receptor conformation required for
correct expression and function. The RT-PCR showed that addition of the
nucleotide sequence encoding the C-terminal tail did not affect mRNA
levels transcribed from the mutant plasmid construct. Therefore, the
structural determinants in the added C terminus may contribute to the
changes in receptor conformation that favor more efficient
receptor-membrane interaction and receptor insertion into the membrane.
The reduction in receptor binding sites after truncation of added
C-tail may be explained by the increase in intracellular localization
of truncated receptor as that demonstrated in other C tail-truncated
GPCRs (17, 24, 35). A recent study shows that truncation of the
cytoplasmic tail of the LH receptor results in an increase in the
relative number of mobile LH receptors on the cell surface (42),
supporting the role of an intracellular tail on the receptor movement
and localization in the plasma membrane. In addition, it was reported
that the capacity of high-affinity cfGnRHR sites (1, 678 fmol/mg
protein) is much higher compared with those reported in rats (43).
Whether the presence of a C terminus in cfGnRHR contributes to this
difference in receptor binding capacity remains an open question.
Mammalian GnRHR was shown to undergo biphasic homologous regulation by
physiological concentrations of GnRH (44). Initially, down-regulation
of receptors is observed (0.54 h posttreatment) followed by an
increase in the number of GnRHRs (9 h posttreatment). In the present
study, GH3 cells transiently expressing wt rGnRHR also
showed a biphasic pattern of regulation of GnRHR. This regulation by
GnRHR is similar to that reported for primary pituitary cells (44) and
is also similar to previous results from GH3 cells stably
expressing rGnRHR [GGH3 cells (45)]. The ability of the
GnRHR to be homologously regulated in GH3 cells suggests
that GnRHR regulation does not require cell-specific components and may
not involve regulation at the transcriptional level, as the expression
of the GnRHR in GH3 cells is driven by a cytomegalovirus
promoter.
Introduction of the intracellular tail of cfGnRHR altered the pattern
of homologous regulation of rGnRHR and markedly enhanced the extent of
homologous down-regulation of GnRHR. These results suggest that
structural changes in the receptor due to addition of C terminus had a
significant impact on receptor regulation. Conversely, truncation of
the added C terminus to rGnRHR impaired receptor regulation, indicating
that the role of the C terminus is reversible. Notably, truncation at
position 350 or 343 (which deletes 28 and 35 residues, respectively, of
the added C terminus) markedly impaired the extent of down-regulation
but did not significantly alter the pattern of receptor regulation.
Truncation at position 337 of rGnRHR-Ctail, which deletes six
additional residues including the Cys-Phe-Cys motif (two potential
palmitoylation sites) from rGnRHR-Ctail-t343, not only impaired the
extent of regulation but also altered the pattern of regulation, from a
monophasic down-regulation pattern back to a biphasic down- and
up-regulation as shown for wt rGnRHR. These results indicate that the
Cys-X-Cys motif may contribute to the change of receptor regulation
pattern. Similarly, two putative palmitoylation sites, Cys-X-Cys, in
the C terminus of TRH receptor, appear to be involved in the
agonist-induced internalization (23).
The mechanism of homologous regulation of GnRHR is unclear. It is
evident that down-regulation of GnRHR occurs, in part, by physical
internalization of agonist-occupied receptors (46), and up-regulation
of GnRHR requires calcium mobilization and protein synthesis (44, 47, 48). The initial down-regulation of GnRHR is temporally associated with
desensitization of gonadotropes to GnRH (46). Regulation of the
ß-adrenergic receptor (ßAR) involves G protein, phosphorylation of
receptor by protein kinase A (PKA), and a decline in mRNA stability
resulting from elevated cAMP levels as well as a second signal
transduction pathway activated by the agonist (49). In
ß2AR, mutation of the consensus sequence for
phosphorylation by PKA in the third intracellular loop abolished
cAMP-induced receptor phosphorylation and significantly delayed the
rate and reduced the extent of down-regulation of receptor numbers by
cAMP (50). It was suggested that phosphorylation of ß2AR
enhances the rate of down-regulation by shortening the receptor
half-life in the membrane. However, whether agonist-stimulated
phosphorylation of the sites in the C terminus by PKA is involved in
receptor down-regulation is unknown. Mutation of four Ser and Thr
residues in the C terminus in ß2AR (51) or mutation of
Tyr residue in NPLIY motif in the junction between the C terminus and
the transmembrane segment of ß2AR (52) abolished
agonist-stimulated receptor phosphorylation and internalization, but
did not affect long-term down-regulation. However, mutation of two Tyr
residues in the middle of the C terminus of ß2AR
dramatically decreased the agonist-stimulated down-regulation of the
receptor, but did not affect sequestration of the receptor (53). These
results suggest that the C terminus is involved in receptor regulation,
and differential structural determinants in the C terminus are
implicated in receptor regulation and internalization. In the present
study, addition of a C terminus, which contains 10 Ser and Thr
residues, may introduce extra phosphorylation sites into the receptor,
leading to increased receptor phosphorylation and enhanced receptor
down-regulation. On the other hand, the potential conformational change
in the receptor due to the addition of the C terminus may result in
decline in receptor stability in the membrane and contribute to the
enhanced down-regulation. In nonmammalian vertebrate, GnRH-stimulated
homologous receptor down-regulation has been demonstrated (54).
However, the time course of GnRHR regulation has not been examined in
nonmammalian species, and whether biphasic receptor regulation is also
present in nonmammalian GnRHR is unknown. The mechanism for alteration
in the pattern of receptor regulation due to the addition of a C
terminus remains to be investigated.
The intracellular C-terminal tail has been implicated in
agonist-stimulated internalization and/or rapid desensitization in most
GPCRs examined (2). Truncation of the intracellular C-terminal tail or
mutations of potential phosphorylation sites in the intracellular tail
attenuates or abolishes agonist-induced receptor internalization and/or
delays the onset of rapid desensitization (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). While mammalian
GnRHR lacks an intracellular C terminus, rapid desensitization (<15
min) is evident in the primary pituitary cells continuously exposed to
GnRH (55); agonist-stimulated internalization of GnRHR has been
observed to occur within 1015 min (48). Results from these studies
indicate that mammalian GnRHR can internalize and undergo rapid
desensitization without the presence of an intracellular C terminus,
suggesting that different mechanism from that used by other GPCRs may
be used by the GnRHR system. In the present study, we did not examine
whether the introduction of an intracellular C terminus can affect
rGnRHR internalization. In ß2AR, since mutations of the C
terminus abolished agonist-stimulated receptor phosphorylation and
internalization but did not affect long-term down-regulation, it was
suggested that receptor internalization is dissociated from a slowly
evolving down-regulation process (51). However, because of the
difference in a C terminus and consequent difference in the mechanism
of internalization between GnRHR and other GPCRs, we cannot exclude the
possibility that addition of the C terminus alters receptor
internalization, which contributes, in part, to the alteration in
receptor regulation.
In GGH3 cells, GnRHR is coupled to Gq/11
,
resulting in activation of phospholipase C and IP turnover (7, 8); the
GnRHR also appears to be coupled to adenylate cyclase-mediated PRL
release through Gs
(9, 10). In the present study,
GH3 cells transiently transfected with rGnRHR or with
chimeric rGnRHR-Ctail showed a significant and dose-dependent increase
in IP production and cAMP and PRL release after Buserelin stimulation.
These results suggest a similar G protein-coupling pattern for rGnRHR
transiently expressed in GH3 cells as established for
continuous GGH3 cell lines; addition of C terminus to
rGnRHR did not appear to affect the pattern of coupling of this
receptor to G protein (Gq/11 and Gs). The
intracellular C terminus has been shown to be involved in G protein
coupling in several GPCRs (15, 16, 17). However, truncation of the C
terminus of a number of GPCRs caused an attenuation of receptor
internalization without affecting G protein coupling (20, 21, 23, 25),
suggesting that the C terminus may not contribute to the receptor
conformation required for the sites for G protein coupling. In
addition, GH3 cells expressing rGnRHR-Ctail receptor showed
a significantly higher increase in Buserelin-stimulated IP production
(2-fold in EC50) compared with that from GH3
cells expressing wt rGnRHR. This elevation in IP production may result
from the increase in receptor-binding sites due to the addition of a C
terminus. However, the elevation in IP production was not proportional
to the increase (5-fold) of receptor-binding sites caused by addition
of the C terminus. Furthermore, GH3 cells expressing
rGnRHR-Ctail and expressing wt rGnRHR show an indistinguishable
response of cAMP and PRL release to Buserelin stimulation. These
results suggest that the conformational change of the receptor due to
the addition of a C terminus preferentially impairs the efficiency of
receptor coupling to G protein. In addition, the enhanced receptor
down-regulation due to the addition of a C terminus could also be
responsible for the decreased signal transduction. The differential
effects of the addition of a C terminus on receptor-mediated IP
production and cAMP release suggests differential requirements for
receptor conformation for coupling to Gs and
Gq/11.
 |
MATERIALS AND METHODS
|
---|
Materials
rGnRHR cDNA in pcDNA1 was generously provided by Dr. W. W.
Chin (56). The African catfish GnRHR was prepared as described (39).
The expression vector pcDNA3.1 was purchased from Invitrogen (San
Diego, CA). Natural sequence GnRH was provided by the National
Pituitary Agency. Buserelin
(D-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH)
was a kind gift from Hoechst-Roussel Phamaceuticals (Somerville, NJ).
Myo-[3H] inositol was purchased from Dupont (New England
Nuclear, Boston, MA). DMEM, OPTI-MEM, lipofectamine, and PCR reagents
were purchased from Life Technologies (Grand Island, NY). Restriction
enzymes, modified enzymes, and competent cells for subcloning were
purchased from Promega (Madison, WI). Other reagents were of the
highest degree of purity available from commercial sources.
Methods
Generation of Mutant Receptor Constructs
wt rGnRHR cDNA in pcDNA1 was subcloned into pcDNA3.1 at
BamHI and XhoI restriction enzyme sites. Chimeric
receptor (rGnRHR-Ctail) containing wt rGnRHR and intracellular C
terminus of cfGnRHR was constructed by overlap extension PCR, a
procedure used to join DNA fragments that contain an overlap region
(57). To construct the chimera, the fragments originating from each
receptor were amplified in separate reactions, each containing one
receptor as template. rGnRHR sequence, including 5'-untranslated region
and complete coding region but not stop codon, was amplified from the
wt rGnRHR cDNA in pcDNA3.1, using a 20-mer vector primer (T7)
corresponding to sequence within the T7 polymerase promoter of pcDNA3.1
vector and a 42-mer primer that is the reverse complement of
5'-CCA CTT ATA TAT GGG TAT TTC TCT TTG/ACG CCA TCG TTC CGT.
This primer is comprised of 27 bases from the rGnRHR template
(underlined) and a 15-base adaptor from the 5'-sequence for
cfGnRHR intracellular C terminus. The sequence for the intracellular C
terminus of cfGnRHR was amplified from wt cfGnRHR cDNA in pcDNA3, using
a 18-mer pcDNA3.1/BGH reverse primer (BGH-rev) complementary to
sequence within the BGH polyadenylation signal of pcDNA3.1 vector and a
34-mer primer, 5'-GGG TAT TTC TCT TTG/ACG CCA TCG TTC CGT
GCC G. This primer is comprised of 19 bases from the 5'-sequence for
cfGnRHR intracellular C terminus and a 15-base adaptor
(underlined) from rGnRHR template. The two chimeric primers
used in each reaction were complementary (overlap region) for 30 bases,
with the junction (indicated as a slash) between rGnRHR and
cfGnRHR sequence. The result of the two PCR reactions was the
amplification of one fragment of the rGnRHR sequence with a 15-base
cfGnRHR sequence end, and one fragment of cfGnRH sequence for
intracellular C terminus with a 15-base rGnRHR sequence end, yielding
30 bases of overlap region between two fragments. The two fragments
were gel purified and used as templates in a third PCR reaction with
only the two outer primers, T7 and BGH-rev. The third PCR reaction
produced a full-length chimeric receptor cDNA, presumably by the
formation of heteroduplexes between complementary ends of the two
templates. The junction of chimeric receptor is between the last amino
acid (Leu327) of rGnRHR and the first residue
(Thr329) of cfGnRHR intracellular C terminus, forming the
sequence
-Phe325-Ser326-Leu327/Thr328-Pro329-Ser330-.
A truncated cfGnRHR mutant (cfGnRHR-t329) was created by substitution
of the codon for the first residue (Thr329) of wt cfGnRHR
intracellular C terminus with a stop codon (TAA) using the overlap
extension PCR as described above. Briefly, two fragments were amplified
separately from the same template (wt cfGnRHR) using primer set, T7 and
a 35-mer primer 5'-CG GAA CGA TGG TTA AAA GAA GCC GTA TAT
TAC TGG, and BGH-rev and a 24-mer primer 5'-C GGC TTC TTT
TAA CCA TCG TTC CG, respectively. The sequence
underlined in the primers corresponds to or is complementary
to the introduced stop codon (TAA). The two fragments were then used as
templates in a third PCR reaction with primer set, T7 and BGH-rev. The
third PCR reaction produced a full-length cfGnRHR with stop codon after
amino acid Phe328, yielding a truncated cfGnRHR that lacks
intracellular C terminus. The chimeric rGnRHR-Ctail was further
truncated to create rGnRHR with different lengths of intracellular C
terminus; three truncated rGnRHR-Ctail, designated as
rGnRHR-Ctail-t337, rGnRHR-Ctail-t343, and rGnRHR-Ctail-t350, were made
by substitution of stop codon (TAA) for the residue Arg337,
Asn343, and Ser350 in the C terminus,
respectively, using the overlap extension PCR as described above. The
internal primers are 5'-GAC TTG TCC TAA TGT TTC TGT TGG AG and 5'-ACA
GAA ACA TTA GGA CAA GTC GGC ACG for rGnRHR-Ctail-t337, 5'-TGT TGG AGG
TAA CAA AAT GCT TCA GCC and 5'-AGC ATT TTG TTA CCT CCA ACA GAA ACA TC
for rGnRHR-Ctail-t343, and 5'-TCA GCC AAA TAA CTG CCA CAC TTC TCT G and
5'-GTG TGG CAG TTA TTT GGC TGA AGC ATT TTG for rGnRHR-Ctail-t350.
All mutant receptor cDNAs (chimeric and truncated receptor cDNAs) were
flanked by the restriction sites present in the polylinker of pcDNA3.1
vector. The cDNAs were thus digested with BamHI and
XhoI and subcloned into the same sites of pcDNA3.1 vector.
The identity of all mutant constructs and the correctness of all
PCR-derived coding sequences were verified by Dye Terminator Cycle
Sequencing according to the manufacturers instructions (Perkin Elmer,
Foster City, CA). For transfection, large-scale plasmid DNAs containing
wt or mutant receptor cDNAs were prepared by double-banded CsCl
gradient centrifugation. The purity and identity of plasmid DNAs were
further verified by restriction enzyme analysis.
Transient Transfection of GH3 Cells
Wt and mutant receptors were transiently expressed in GH3
cells (45). GH3 cells were maintained in growth medium
[DMEM containing 10% FCS (Hyclone Laboratories, Logan, UT) and 20
µg/ml gentamicin (Gemini Bioproducts, Calabasas, CA)] in a
humidified atmosphere (37 C) containing 5% CO2. Cells
(105 per well) were seeded in 24-well plates (Costar,
Cambridge, MA). Twenty four hours after plating, the cells were
transfected with 0.8 µg plasmid DNA/well using 2 µl lipofectamine
in 0.25 ml OPTI-MEM. Five hours later, 0.25 ml DMEM containing 20% FCS
was added to each well. Twenty four hours after the start of
transfection, the medium was replaced with fresh growth medium, and the
cells were allowed to grow for 48 h before functional assays (IP
production; cAMP and PRL release) were done. For receptor binding, the
same transfection procedure was followed except that 20 µg plasmid
DNA and 50 µl lipofectamine were used to transfect the cells in
75-cm2 flasks (Costar) when they are 6080% confluent.
For studies of down-regulation of GnRHR, the same transfection
procedure was followed except that 2 µg plasmid DNA/well and 5 µl
lipofectamine in 1 ml OPTI-MEM were used to transfect cells (5 x
105/well) seeded in six-well plates (Costar), when they
were 6080% confluent.
Quantification of IPs
Forty eight hours after the start of transfection, the cells
transfected with wt or mutant receptor DNAs were washed with DMEM-0.1%
BSA and incubated in 0.5 ml DMEM (without inositol) containing 4
µCi/ml [3H]inositol for 18 h at 37 C. After the
preloading period, cells were washed twice in DMEM (inositol free)
containing 5 mM LiCl and stimulated with Buserelin at
indicated doses in 0.5 ml DMEM-LiCl for 2 h at 37 C. The treatment
solution was removed, and 1 ml 0.1 M formic acid was added
to each well. The cells were frozen and then thawed to disrupt cell
membranes. IP accumulation was determined by Dowex anion exchange
chromatography and liquid scintillation spectroscopy, as previously
described (58).
Quantification of cAMP
Forty eight hours after the start of transfection, the cells
transfected with wt or mutant receptor DNAs were washed with DMEM
containing 0.1% BSA (Irvine Scientific, Santa Ana, CA) and 20 µg/ml
gentamicin. The cells were then stimulated for 24 h with Buserelin
(10-13-10-7 M) in DMEM-0.1%
BSA-20 µg/ml gentamicin containing 0.2 mM
methylisobutylxanthine (MIX) to prevent degradation of cAMP. After
stimulation, the medium from each well was collected in tubes
containing sufficient theophylline for a final concentration of 1
mM. The samples were heated (95 C) for 5 min to destroy
phosphodiesterases. RIA of cAMP was performed by a modification of the
method of Steiner et al. (59), with the addition of the
acetylation step described by Harper and Brooker (60). cAMP antiserum
C-1B [prepared in our laboratory (61)] was used at a titer of 1:5100.
This antiserum showed less than 0.1% cross-reaction with cGMP,
2',3'-cAMP, 5'-cAMP, 3'-cAMP, ADP, GDP, ATP, CTP, MIX, or
theophylline.
Quantification of PRL Release
Forty eight hours after the start of transfection, the cells
transfected with wt or mutant receptor DNAs were washed twice with DMEM
containing 0.1% BSA and 20 µg/ml gentamicin (DMEM-BSA-Gentamicin).
The cells were then incubated with different doses of Buserelin in a 1
ml volume of DMEM-BSA-Gentamicin at 37 C for 24 h. The medium was
collected, and the PRL release in medium was measured by RIA, using
materials obtained from the Hormone Distribution Program of the
National Pituitary Agency, NIDDK. PRL was radioiodinated by standard
procedures (62). Intra- and interassay variances were 5% and 7%,
respectively.
Receptor Binding and Down-Regulation
Intact cell binding was assessed in a range of concentrations of
[125I]Buserelin, prepared as previously reported (63), in
DMEM-0.1% BSA. Seventy two hours after the start of transfection, the
cells transfected with wt or mutant receptor DNAs were scraped and
resuspended in warm DMEM-BSA. Cells then were pelleted and washed twice
with ice-cold DMEM-BSA. One hundred microliters of the cell suspension
(1 x 106 cells) were added to each tube, and the
assay was allowed to come to equilibrium (3 h) at 4 C at a final volume
of 150 µl. Binding was terminated by overlayering each sample on 2 ml
DMEM-0.3 M sucrose at 4 C and centrifuging at 2,000 x
g for 10 min at 4 C in Sorvall SM-24 rotor. The supernate
was aspirated. The cell pellet was resuspended in 1 ml PBS, and
radioactivity was determined using a 10-channel
-counter (Packard
Instruments, Meriden, CT). For studies of down-regulation of the GnRHR,
72 h after start of the transfection, cells were washed twice with
DMEM-BSA, treated with 10 nM GnRH (a desensitizing dose) or
medium alone for the indicated times, and washed three times (4
ml/well) at 23 C with DMEM-BSA to remove excess GnRH. The medium was
decanted and replaced with 2 ml [125I]Buserelin/well at a
concentration of 0.4 µCi/ml. Binding was assessed after 30 min (23
C). Nonspecific binding was determined in the presence of 10
µM unlabeled GnRH. Binding was terminated by decanting
the radioligand-containing medium and placing the cells on ice. Cells
were washed twice with ice-cold DMEM-BSA. Cells were then collected by
scraping in 1 ml DMEM-BSA containing 2.5 mM EGTA (4 C)
twice. The cell lysate was layered over 2 ml 0.3 M sucrose
in DMEM, and the cell pellet was collected and its radioactivity was
counted as described above.
Data Analysis
Data shown are the mean of triplicate assay wells and are
presented as the mean ± SEM of replicates in each
experiment. The SEM was typically less than 10% of the
mean. The data were analyzed by Students t test,
P < 0.05 being considered significant. Each experiment
was repeated three or more times to ensure the reproducibility of the
findings.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. W. W. Chin for providing the rGnRHR
cDNA. We thank Dr. Alfredo Ulloa-Aguirre and Dinesh Stanislaus for
their advice.
 |
FOOTNOTES
|
---|
Address requests for reprints to: P. Michael Conn, Oregon Regional Primate Research Center, 505 Northwest 185th Avenue, Beaverton, Oregon 97006.
This study was supported by NIH Grants HD-19899, HD-00163, and
HD-18185.
Received for publication September 24, 1997.
Accepted for publication October 30, 1997.
 |
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