Signal Transduction, Desensitization, and Recovery of Responses to Thyrotropin-Releasing Hormone after Inhibition of Receptor Internalization*
Run Yu and
Patricia M. Hinkle
Department of Pharmacology and Physiology and the Cancer Center
University of Rochester School of Medicine and Dentistry Rochester,
New York 14642
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ABSTRACT
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Three independent methods were used to block
internalization of the TRH receptor: cells were infected with vaccinia
virus encoding a dominant negative dynamin, incubated in hypertonic
sucrose, or stably transfected with a receptor lacking the C-terminal
tail. Internalization was blocked in all three paradigms as judged by
microscopy using a fluorescently labeled TRH agonist and biochemically.
The initial inositol trisphosphate (IP3) and
Ca2+ responses to TRH were normal when
internalization was inhibited. The IP3 increase
was sustained rather than transient, however, in cells expressing the
truncated TRH receptor, implying that the C-terminal tail of the
receptor may be important for uncoupling from phospholipase C. After
withdrawal of TRH, cells were refractory to TRH until both ligand
dissociation and resensitization of the receptor had occurred. When
surface-bound TRH was removed by a mild acid wash, which did not impair
receptor function, neither wild-type nor truncated receptors were able
to generate full IP3 responses for about 10
min. The rate of recovery was not altered by blocking internalization.
Recovery of intracellular Ca2+ responses also
depended on the rate of Ca2+ pool refilling. In
summary, in the continued presence of TRH, phospholipase C activity
declines quickly due to receptor uncoupling; this desensitization does
not take place for the truncated receptor. After TRH is withdrawn,
cells are refractory to TRH. Before cells can respond, TRH must
dissociate and a resensitization step, which takes place on the plasma
membrane and does not require the C-terminal tail of the receptor, must
occur.
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INTRODUCTION
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Many G protein-coupled receptors (GPCRs) undergo endocytosis after
the binding of an agonist. GPCRs internalize primarily by
clathrin-dependent pathways, although uptake by other routes has been
reported (1, 2). It is not known what conformational changes cause
GPCRs to become sequestered after agonist binding. Mutational analysis
of receptors has revealed that no single sequence or region of the
receptor is either necessary or sufficient for endocytosis of all
members of the receptor superfamily, and some GPCRs do not internalize
at all (1, 2). The impact of receptor sequestration on signal
transduction, desensitization, and resensitization is also uncertain.
Internalization appears to turn off the signaling capability of some
GPCRs (3). For ß-adrenergic receptors, the opposite seems to be true,
since sequestration is essential for the reactivation of previously
desensitized receptors (4, 5, 6). Blocking endocytosis has no obvious
effect on the function of several receptors that activate phospholipase
C (7, 8, 9, 10).
The pituitary receptor for TRH, a calcium-mobilizing receptor, is
unusual in the extent of its internalization. As much as 80% of the
TRH-receptor complex is sequestered in pituitary cells (11). The
transferrin receptor is known to undergo endocytosis via a classic
clathrin-dependent pathway (2). Transferrin colocalizes with a
fluorescently labeled TRH agonist, and the transferrin and TRH
receptors colocalize, indicating that the TRH receptor undergoes
endocytosis via clathrin-coated pits (12, 13). Internalization of the
TRH receptor occurs with a half-time of about 23 min after binding of
agonists, but the receptor does not undergo endocytosis after
activation of signal transduction pathways with drugs or after binding
of an inverse agonist, chlordiazepoxide (14). Several mutants of the
TRH receptor have been shown to be defective in internalization,
including receptors with mutations in the second putative transmembrane
region or the third intracellular loop, which do not couple to G
proteins, and a truncated receptor missing the entire intracellular
C-terminal region, which is capable of activating phospholipase C and
generating a calcium transient (12, 13, 15, 16).
There is currently no information about the function of ligand-driven
endocytosis of the TRH receptor. We have previously demonstrated that
the TRH receptor undergoes profound desensitization and characterized
the recovery of the inositol trisphosphate (IP3) and
intracellular free calcium ([Ca2+]i) responses and
intracellular Ca2+ pools (17). In the present study, we
have used three independent approaches to block TRH receptor
internalization and determined the impact on the initial signaling
step, desensitization and recovery of the response. We show that the
initial uncoupling of the receptor from phospholipase C does not
require internalization but does require the C-terminal tail of the
receptor. After TRH withdrawal, cells are refractory to a second
challenge with TRH until ligand dissociation and an additional
resensitization step have taken place. In contrast to the model
established for the ß-adrenergic receptor (4, 5, 6), we show that the
TRH receptor is resensitized without internalization.
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RESULTS
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Inhibition of Receptor Internalization
To study the function of internalization of the TRH receptor, we
used three independent methods to inhibit the process: 1) cells were
infected with vaccinia virus encoding a dominant negative dynamin
(K44E) that prevents pinching off of clathrin-coated pits (18); 2)
cells were incubated in hypertonic sucrose, which also causes
global inhibition of clathrin-dependent endocytic pathways (19);
and 3) the receptor was truncated before residue C335,
eliminating the 59 C-terminal amino acids; this truncated
receptor does not internalize in COS cells or AtT20 corticotropes (12, 15, 20). Internalization was assessed by resistance of bound
radiolabeled TRH to an acid/salt wash (21) and by microscopy after
binding of a fluorescently labeled TRH peptide (12). These studies were
performed using HEK293 cells stably expressing either the wild-type TRH
receptor (301 cells) or the truncated TRH receptor (421 cells). The
levels of receptor expression were approximately 250,000 receptors per
cell in the 301 cells and 500,000 in the 421 line (22). Infection with
vaccinia virus constructs and incubation with hypertonic sucrose did
not significantly alter the density of TRH-binding sites.
All three methods inhibited receptor endocytosis effectively (see Figs. 1
and 2
). To monitor internalization biochemically, we incubated cells
with radioligand at 0 C, washed to remove unbound hormone, and then
measured the rate of formation of acid/salt-resistant (internalized)
hormone over time. The rate and extent of internalization were the same
in control 301 cells or cells infected with either empty virus or virus
encoding wild-type dynamin; the half-time for internalization was 2.5
min, and 8590% of the ligand bound was internalized at steady state.
Expression of the dominant negative (K44E) dynamin and incubation in
hypertonic sucrose delayed the formation of an acid/salt-resistant
complex and reduced the maximal acid/salt-resistant fraction to
4550% (Fig. 1
). The truncated C335STOP
receptor, which was stably expressed in HEK293 cells, did not undergo
conversion to an acid/salt-resistant complex even after prolonged
incubation at 37 C (Fig. 1).

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Figure 1. Rate of Formation of an Acid/Salt-Resistant
TRH-Receptor Complex
Replicate dishes of cells were incubated with 2.5 nM
3H-labeled MeTRH for 1 h on ice and washed to remove
free hormone. To measure the rate of internalization of
3H-labeled MeTRH bound to surface receptors, dishes were
transferred to 37 C and at intervals from 0120 min, total specific
3H-labeled MeTRH binding and the fraction internalized were
measured in duplicate dishes as described in Methods.
Points show the mean of the percent of bound
3H-labeled MeTRH internalized; the errors fell within
symbol size. Control dishes for the different cell lines bound between
43,000 and 89,000 cpm/dish, corresponding to 24 pmol/mg protein, at
1 h. Left panel, 301 cells (301), 421 cells
expressing the C335STOP truncated TRH receptor (C335STOP), or 301 cells
in hypertonic sucrose (Sucrose). Right panel, 301 cells
infected with empty vaccinia virus (Empty virus) or virus encoding K44E
dynamin (K44E dynamin) or wild-type dynamin (Wildtype dynamin). In this
protocol, some 3H-labeled MeTRH dissociated before it
internalized, particularly during the first few minutes of incubation
at 37 C. In cells expressing the wild-type receptor, 2030% of
3H-labeled MeTRH dissociated during the first minute at 37
C and 4555% by 5 min. In cells expressing the truncated receptor,
less than 25% of 3H-labeled MeTRH dissociated in 120 min.
Similar results were obtained in a series of similar experiments where
cells were exposed continually to 3H-labeled MeTRH at 37 C
and internalization was followed over time.
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Figure 2. Internalization of Rhodamine-Labeled TRH
Cells were incubated with Rhod-TRH at 37 C for 10 min, washed, and
immediately visualized on a fluorescence microscope. An image (1a-6a)
was captured, and cells were incubated in a mild acid/salt solution
(0.5 M NaCl, 0.025 M HAc, 1 mM
CaCl2, pH 5.0) at 37 C for 10 min and another image was
obtained (1b-6b). All incubations were in HBSS or HBSS plus 0.4
M sucrose. Panels show Rhod-TRH staining of: 301 cells (1a
and 1b), 301 cells infected with empty vaccinia virus (2a and 2b),
vaccinia virus encoding wild-type (3a and 3b) or K44E dynamin (4a and 4b), 301 cells
incubated in HBSS supplemented with 0.4 M sucrose (5a and
5b) or 421 cells expressing the C335STOP TRH receptor (6a and 6b). In
experiments in which the mild acid wash was omitted, rhodamine-labeled
TRH could be seen to internalize after 60 min in cells expressing
dominant negative dynamin, but not in cells expressing the truncated
receptor or incubated in hypertonic sucrose. The images shown are
typical of those obtained in multiple experiments.
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The inhibition of receptor internalization by K44E dynamin, hypertonic
sucrose, and truncation of the receptor appeared more striking when
cells were stained with a fluorescently labeled TRH analog (Fig. 2
). Rhodamine-labeled TRH was visible in
intracellular vesicles within 10 min in cells expressing the wild-type
receptor, and infection with empty vaccinia virus or virus encoding
wild-type dynamin did not alter this pattern. Rhodamine-labeled TRH was
not observed in intracellular vesicles in cells expressing K44E
dynamin, in cells treated with hypertonic sucrose, or in cells
expressing the C335STOP receptor. Fluorescence was punctate and
apparently confined to the surface in cells treated with hypertonic
sucrose or cells expressing dominant negative dynamin.
Rhodamine-labeled TRH was uniformly distributed over the plasma
membrane in cells expressing the C335STOP receptor. When cells were
rinsed with a mild acid/salt solution, there was little loss of
fluorescent signal from control cells, cells expressing empty vector,
or cells expressing wild-type dynamin, supporting the conclusion that
most of the rhodamine-labeled TRH was in endocytic vesicles. In
contrast, nearly all of the rhodamine-labeled TRH was removed from
cells expressing K44E dynamin, cells expressing the truncated receptor,
or cells incubated in hypertonic sucrose, confirming that little
internalization had taken place.
Importance of Receptor Internalization for the
IP3 Response to TRH
The initial [Ca2+]i response to TRH was not changed
by inhibiting receptor internalization (Fig. 3
). We did find that surprisingly high
concentrations of TRH, at least 100 nM, were needed to
produce maximal [Ca2+]i and IP3 responses in
cells expressing the C335STOP receptor. The affinity of the truncated
receptor for MeTRH [dissociation constant (Kd)
1
nM] is twice that of the wild-type receptor in several
lines (20) including 421 cells (our unpublished observation).

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Figure 3. [Ca2+]i Responses to TRH after
Inhibition of Receptor Internalization
[Ca2+]i was monitored at 37 C in 301 and 421 cells; 1
µM TRH was added at 20 sec. Shown are typical traces for
301 cells infected with empty vaccinia virus or virus encoding K44E
dynamin, 301 cells incubated in hypertonic sucrose, and 421 cells
expressing the C335STOP TRH receptor. In all cases, cells were loaded
with Fura2 in normal HBSS. The peak increases in [Ca2+]i
(nM) were: 2345 ± 173 for uninfected 301 cells, 2145 ± 226
for 301 cells infected with empty virus, 1519 ± 240 for 301 cells
infected with K44E dynamin, 1907 ± 169 for 301 cells in sucrose,
or 2242 ± 226 for 421 cells (mean and SE of 1628
cells).
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The IP3 response to TRH was not prevented when
internalization was inhibited with K44E dynamin or with hypertonic
sucrose (Fig. 4
). The peak
IP3 level was reached 10 sec after TRH addition, and
IP3 then fell rapidly. The peak IP3
concentrations were comparable to those reached in either untreated
cells (17) or cells infected with a control vaccinia virus. In cells
expressing wild-type receptors, IP3 rapidly declined in the
continued presence of TRH. The rapid fall in IP3 is not the
result of exhaustion of phosphatidylinositol(4, 5)bisphosphate, because
other agonists stimulate a strong IP3 response when added
after TRH in 301 cells (17). 421 cells expressing the truncated TRH
receptor responded to 1 µM TRH with a 4- to 5-fold
increase in IP3, which is less than the 10- to 20-fold
response of cells expressing a lower number of wild-type receptors. We
have previously shown that a maximal [Ca2+]i response is
obtained at TRH concentrations far below those that give maximal
increases in IP3 in 301 cells (17), and similar findings
have been reported in pituitary cells (23). The finding that the
[Ca2+]i response to TRH is the same in 301 and 421 cells,
even though the IP3 response is less in the 421 cells, is
additional evidence that the amount of IP3 needed to cause
maximal release of IP3-sensitive Ca2+ stores is
relatively small.

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Figure 4. IP3 Concentrations after Addition of
TRH
301 and 421 cells were incubated with 1 µM TRH at 37 C,
and dishes were collected at intervals for measurement of
IP3 concentrations by RRA. Shown are IP3 levels
in: 301 cells infected with empty virus (Empty virus), 301 cells
infected with K44E dynamin (K44E dynamin), 301 cells in hypertonic
sucrose (Sucrose), or 421 cells expressing the C335STOP TRH receptor
(C335STOP TRHR). The responses of cells infected with empty virus were
not significantly different from the responses of uninfected control
cells (Fig. 2 of Ref. 17). Values are the mean and range from duplicate
dishes; where error bars are not visible, they fell
within symbol size.
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The IP3 response pattern was sustained rather than
transient in the 421 cells expressing the truncated TRH receptor. The
IP3 concentration remained elevated in the 421 cells for up
to 10 min in the continued presence of TRH (Fig. 4
). Although the
C335STOP receptor has been reported to have constitutive activity when
expressed in oocytes and AtT20 cells (20, 24), basal IP3
was not elevated in the 421 cell line, and addition of the inverse
agonist chlordiazepoxide for 5 min did not decrease IP3
(data not shown).
Desensitization and Recovery of the TRH-Mediated
IP3 Response
To follow decay of the TRH response after TRH withdrawal, we
treated cells with 1 µM TRH at 37 C for 10 min, washed
and then incubated the cells without hormone, and measured
IP3 concentrations at intervals. In cells expressing the
wild-type receptor, under all conditions tested (Fig. 5
and Ref.17), IP3
concentrations fell to near basal after TRH was removed and the cells
were washed. In cells expressing the truncated receptor, however,
IP3 remained very high, 300800% of basal, for at least
90 min after TRH was withdrawn (Fig. 5
). If the 421 cells were first
washed with mild acid to remove surface-bound hormone, as described
below, then the IP3 concentrations dropped quickly (Fig. 5
).

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Figure 5. IP3 Concentrations after Withdrawal of
TRH
Cells were incubated with 1 µM TRH at 37 C for 10 min and
washed with HBSS to remove free TRH. Dishes were then incubated at 37 C
in hormone-free medium or, where noted (+acid wash), first incubated
with the mild acid/salt solution (0.5 M NaCl, 0.025
M HAc, 1 mM CaCl2, pH 5.0) at 37 C
for 10 min to remove surface-bound TRH and then incubated in
hormone-free HBSS. The IP3 concentration was measured
immediately after TRH was removed and at intervals afterward and is
expressed as a percent of the basal, unstimulated value for control
dishes. Basal values, in picomoles/mg protein, were: Empty virus,
8.4 ± 0.5; K44E dynamin, 7.6 ± 0.6; C335STOP, 17.3 ±
1.5; Hypertonic sucrose, 14.3 ± 2.4. 301 cells infected with
empty virus responded essentially the same as uninfected cells. Points
give the mean and range from duplicate dishes; where error bars are not
visible, they fell within symbol size.
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We next asked how internalization affects the ability of cells to
respond to a second challenge with TRH. Cells infected with control
virus or virus encoding K44E dynamin were incubated with 1
µM TRH for 10 min, resulting in essentially complete
occupancy of receptors and extensive internalization of receptors in
control cells. The dishes were washed, allowed to recover for various
periods, and then challenged with different concentrations of TRH, and
the peak IP3 responses were measured. The
amplitude of the IP3 responses depended
on the recovery time and the concentration of TRH in the second
challenge, but the responses were unaffected by the inhibition of
internalization (Fig. 6
). Both control
and internalization-impaired cells were refractory to TRH immediately
after washout. The half-time for recovery was at least 10 min, and
complete recovery of the IP3 response required over 40 min.
Since the peak IP3 response increased with TRH dose over
the range 101000 nM, we would have been able to detect
changes that depended on the number of accessible, functional receptors
on the cell surface, i.e. the responses were not at a
maximum. The dose dependence of the response was unaffected by
expression of the dominant negative dynamin.

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Figure 6. Desensitization and Recovery of the IP3
Response to TRH in Virus-Infected Cells
301 cells infected with empty vaccinia virus (open
symbols) or virus encoding K44E dynamin (filled
symbols) were treated with 1 µM TRH for 10 min at
37 C, washed, and allowed to recover for 090 min. At intervals, 10,
100, or 1000 nM TRH was added, and the peak IP3
response was measured. Basal values, measured in parallel dishes at the
same time points, are shown in Fig. 5 . Shown are the mean and range
from duplicate dishes; where error bars are not visible,
they fell within symbol size.
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When 421 cells expressing the C335STOP receptor were exposed to TRH and
then washed in normal buffer, they did not show any IP3
response to a second TRH challenge for up to 90 min (data not shown).
We measured the rate of dissociation of specifically bound
3H-labeled TRH and found that TRH dissociated from the
truncated receptor much more slowly than from the wild-type receptor.
Less than 20% of receptor-bound 3H-labeled TRH had
dissociated from 421 cells expressing the C335STOP receptor after 90
min, whereas the half-time for dissociation was 2040 min for cells
expressing the wild-type receptor under all conditions (data not
shown). For this reason, we had to remove bound TRH before we could
assess the ability of the unoccupied receptor to respond to a second
TRH challenge. We developed a procedure in which cells were treated
with high salt at pH 5.0 for 10 min at 37 C to remove bound TRH. As
described in Methods, this mild acid wash removed 95% of
bound TRH without affecting the signaling capability of the receptor,
assessed by the normal IP3 responses of control cells
subjected to the same protocol. This procedure involved much milder
conditions than the acid/salt wash used to quantify
internalization.
To determine how many receptors were on the cell surface after
different recovery periods, we incubated cells with 1 µM
TRH for 10 min to occupy receptors; the cells were then washed and
recovery was allowed to proceed for different periods before washing
with mild acid to remove any hormone bound to surface receptors; the
cells were then incubated with 3H-labeled TRH on ice to
quantify available sites (Fig. 7
). Under
control conditions, 75% of wild-type receptors were apparently
internalized and inaccessible at the start of the recovery period;
recycling required 1030 min, in agreement with previous estimates for
pituitary cells (14). In contrast, in cells expressing the truncated
receptor, cells expressing K44E dynamin, and cells incubated in
hypertonic sucrose, nearly all receptors were accessible at the start
of the recovery period, again indicating that they had not undergone
extensive internalization.

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Figure 7. Accessible Receptors after TRH Exposure and
Recovery
Cells were incubated with 1 µM unlabeled TRH at 37 C for
10 min, washed, and then incubated for various times in HBSS or HBSS
plus 0.4 M sucrose before being washed with mild acid for
10 min to remove surface-bound TRH and placed on ice with 1
µM 3H-labeled TRH (950 nM
unlabeled TRH + 50 nM 3H-TRH) for 60 min to
quantify surface receptors. Maximal binding to control dishes was
between 2500 and 4300 cpm/dish for the different cell lines,
corresponding to 1.54 pmol/mg protein. For cells incubated in
hypertonic sucrose or 421 cells expressing the truncated receptor, the
mild acid wash was performed before the recovery period, to parallel
the protocol used in experiments shown in Figs. 8 and 9 . Values shown
are the mean and range of results from duplicate dishes.
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Immediately after TRH was removed from 421 cells by the mild acid wash,
1 µM TRH generated almost no increase in IP3,
i.e. the receptor was empty but not functional (Fig. 8
). The IP3 response
recovered with a half-time of
10 min, and a full IP3
response was observed by 25 min. These data imply that truncation of
the C-terminal 59 amino acids of the receptor, which include potential
protein kinase C and GPCR kinase phosphorylation sites, does not
preclude desensitization.

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Figure 8. Desensitization and Recovery of the IP3
Response to TRH after Inhibition of Internalization
301 cells in hypertonic sucrose or 421 cells expressing the
internalization-defective C335STOP TRH receptor were incubated with 1
µM TRH for 10 min at 37 C. Cells were then washed for 10
min at 37 C with a mild acid/salt solution (0.5 M NaCl,
0.025 M HAc, and 1 mM CaCl2, pH
5.0) to remove surface-bound TRH and allowed to recover for 090 min
at 37 C. At intervals, 1 µM TRH was added and the peak
IP3 response was measured. IP3 responses are
expressed relative to the control responses in naive cells, which were
170 ± 9 and 125 ± 20 pmol/mg protein for 301 cells in
sucrose and 421 cells, respectively. Shown are the mean and range from
duplicate dishes; where error bars are not visible, they
fell within symbol size.
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We also wanted to measure desensitization and recovery of the
IP3 response after blocking internalization with hypertonic
sucrose, but inhibition of endocytosis by sucrose was rapidly
reversible. When cells were switched from hypertonic sucrose to normal
medium, rhodamine-labeled TRH could be seen to internalize by
fluorescence microscopy, and bound 3H-labeled TRH increased
from 15.9 ± 0.3% to 88.7 ± 0.3% acid/salt resistant in 5
min. To measure resensitization in normal medium, therefore, we had to
remove bound TRH before removing the sucrose. To do this, we incubated
301 cells with TRH in hypertonic sucrose, incubated in mild acid to
remove bound hormone, and then allowed recovery to proceed for
different periods before readding TRH. Immediately after the removal of
bound hormone, a second TRH challenge elicited an IP3
response that was only 60% of control, although 95% of receptors were
empty (Fig. 8
). Complete recovery of responsiveness required
approximately 10 min. Resensitization occurred more quickly when ligand
was forcibly removed from the cell surface with the mild acid, showing
that ligand dissociation is important (Table 1
). Since the IP3 response to
a second challenge with TRH was reduced to 36% of the response of
naive cells, although 69% of receptors were empty, we concluded that
full resensitization requires more than just ligand removal (Table 1
).
Cells infected with control virus internalized the TRH-receptor
complex, and only 22% of receptors were on the surface and empty after
an acid wash. These cells responded to a second TRH challenge with an
IP3 response that was only 10% of the response shown by
naive cells.
Ca2+ Pool Refilling
Rates of refilling of intracellular Ca2+ pools were
measured in the three paradigms used to block internalization. The
Ca2+ pool was much more rapidly and completely refilled in
cells expressing the dominant negative dynamin than in cells infected
with empty virus (Fig. 9a
). Refilling was
also faster when internalization was inhibited by hypertonic sucrose,
whether sucrose was present during the refilling period (data not
shown) or not (Fig. 9b
). Refilling was relatively slow and incomplete
over the time course of these experiments in cells expressing either
the truncated or wild-type receptor (Fig. 9c
).

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Figure 9. Ca2+ Pool Refilling after Inhibition of
TRH Receptor Internalization
Left, 301 cells infected with empty vaccinia virus or
virus encoding K44E dynamin were treated with 1 µM TRH at
37 C for 10 min, washed, and then incubated in normal medium.
Center, 301 cells were treated with 1 µM
TRH for 10 min at 37 C in either HBSS or hypertonic sucrose. Cells were
then washed and incubated in the same medium. Right, 301
and 421 cells expressing the C335STOP TRH receptor were treated with 1
µM TRH at 37 C for 10 min in normal medium, washed for 10
min at 37 C with mild acid/salt solution (0.5 M NaCl, 0.025
M HAc and 1 mM CaCl2, pH 5.0) to
remove surface-bound TRH, and then incubated in normal medium. At
intervals, the intracellular Ca2+ pool size was measured as
described in Methods. The values are expressed relative
to the Ca2+ pool size for parallel dishes not initially
exposed to TRH. Points represent the mean and SE (n =
20); where error bars are not visible, they fell within
symbol size. Control Ca2+ pool sizes, measured as the
increase in 340/380 ratios after ionomycin, were: empty virus,
6.46 ± 0.68; K44E dynamin, 4.23 ± 0.77; 301 cells in normal
medium, 4.01 ± 0.33; in hypertonic sucrose, 1.80 ± 0.08;
301 cells after acid/salt, 2.58 ± 0.18; 421 cells after
acid/salt, 3.28 ± 0.70.
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Effect of Dominant Negative Mutant Dynamin on the
[Ca2+]i Response to TRH
As shown above, the IP3 response recovered normally
when internalization was inhibited by K44E dynamin or hypertonic
sucrose, but the Ca2+ pool refilled faster. This suggested
that the [Ca2+]i response to TRH might also recover
faster when internalization of the TRH receptor was inhibited. This was
in fact observed when the ability of TRH to stimulate
[Ca2+]i was measured in 301 cells infected with empty
virus or virus encoding K44E dynamin (Fig. 10
). The IP3 response to
either 1 nM or 1 µM TRH recovered faster in
cells expressing K44E dynamin than in control cells. Under both
conditions, Ca2+ pools had refilled and the IP3
response to TRH had recovered fully before [Ca2+]i
responses to TRH were completely restored. The activity of the
IP3 receptor may had been reduced after agonist treatment.
It has been reported that the responsiveness of IP3
receptors can be reduced rapidly by agonist treatment due to either
down-regulation (25) or phosphorylation (26).

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Figure 10. Recovery of the [Ca2+]i Response to
TRH in Virus-Infected Cells
301 cells were infected with (open symbols) empty
vaccinia virus or (filled symbols) virus encoding K44E
dynamin. Cells were incubated with 1 µM TRH at 37 C for
10 min; the peak [Ca2+]i response to this initial TRH
exposure is shown as "Naive Control". Cells were then washed and
incubated in hormone-free medium. At intervals, either 1 nM
(circles) or 1 µM (squares)
TRH was added, and the peak [Ca2+]i response was
measured. Values shown are the mean and SE (n =
1620) of [Ca2+]i measured at the peak after TRH
addition; [Ca2+]i was between 80 and 200 nM
before TRH was added for all points.
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DISCUSSION
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Ligand-driven internalization of receptors has been well
documented for many GPCRs (1, 2, 4), but its function is unknown in
most cases. In this study, we used three independent approaches to
block endocytosis and specifically tested the hypothesis that
internalization affects desensitization or resensitization of the TRH
response (3, 5, 6). We studied the IP3 response because
IP3 is the proximal messenger in the Ca2+
signaling pathway and most directly reflects receptor-G protein
coupling. In addition, the amount of IP3 produced in
response to TRH increases with the number of receptors on the cell and
with the concentration of agonist (27, 28) over a broad range, so we
did not need to be concerned that a few active receptors would give a
maximal response after partial inhibition of receptor endocytosis.
Multiple steps are involved in receptor-mediated endocytosis (2), and
the micrographs of Rhod-TRH staining suggest that the different methods
used to block internalization in this study inhibit the process at
different steps. It is generally accepted that receptors congregate
into clathrin-coated pits on the plasma membrane. Clathrin provides the
force to form coated pits, and dynamin pinches off the pits to generate
endocytic vesicles (29). Internalization of the ß-adrenergic receptor
depends on agonist until the receptors have clustered into coated
microdomains, at which point the process becomes independent of agonist
(30). The smooth distribution of Rhod-TRH on cells transfected with the
C335STOP TRH receptor suggests that the agonist-induced conformational
changes needed for the receptor clustering were absent. Since the
receptor did stimulate a normal calcium transient, coupling to
Gq/11 is not sufficient to drive the truncated TRH receptor
to coated pits. The granular nature of Rhod-TRH staining on cells
treated with sucrose indicates that the receptors did cluster, in
agreement with the finding that hypertonic sucrose disrupts the
clathrin lattice but does not prevent adaptin from aggregating on the
plasma membrane (19). TRH receptor internalization was significantly
delayed by the dominant negative dynamin, confirming the function of
dynamin in pinching off coated vesicles during ligand-dependent
receptor endocytosis (29). Dominant negative dynamin has recently been
shown to inhibit the internalization of ß-adrenergic (31) and
epidermal growth factor receptors (32).
There are limitations to each of the three independent methods used to
block the internalization of the TRH receptor. Removal of the
C-terminal tail of the receptor could interfere with many aspects of
signal transduction, as well as internalization, and the truncated
receptor has been reported to display unusual cycling (13, 20) and
constitutive activity (20, 24) in other cells, although no evidence for
constitutive activity was observed in our studies. Hypertonic sucrose
(19) and K44E dynamin (18, 29) block all endocytosis via
clathrin-dependent vesicles, and this inhibition may perturb signaling
itself. The results obtained with all of the different approaches were
consistent, however.
The results of our studies show that internalization of the
TRH-receptor complex is not essential for the desensitization of the
TRH response. Like the TRH receptor, the angiotensisn II (7, 8, 9) and
muscarinic m3 (10) receptors, which are also coupled to
Gq/11, can desensitize without undergoing endocytosis.
Our findings are in sharp contrast to results showing that
internalization is important for resensitization of the ß-adrenergic
receptor (5, 6), which couples to adenylyl cyclase via Gs.
The ß-adrenergic receptor is phosphorylated by receptor-specific
kinases (ßARKs) and cAMP-dependent protein kinase; ß-arrestin binds
the phosphorylated receptor and uncouples it (4). Resensitization
requires dephosphorylation, and there is evidence that this takes place
in endocytic vesicles where a phosphatase active at acidic pH
dephosphorylates the receptor before it recycles to the membrane in
active form (33). The TRH receptor can clearly be resensitized on the
plasma membrane, because once ligand had been removed, resensitization
took place at a normal or accelerated rate when internalization was
blocked by hypertonic sucrose, dominant negative dynamin, or receptor
truncation. Bogatkewitsch et al. (3) reported that
internalization delays resensitization of the m4 muscarinic receptor,
which is coupled to Gi. It is unclear whether
internalization will prove to be nonessential for the resensitization
of all GPCRs coupled to Gi and Gq/11 and
essential for all GPCRs coupled to Gs, or whether
individual receptors in each class will behave differently. The data
described here and in prior work are consistent with the following
sequence of events during sustained or intermittent administration of
TRH.
1. In the continued presence of TRH, phospholipase C activity declines
over time due to uncoupling of the receptor. IP3
concentrations fall almost to basal levels within 1 min in cells
expressing the wild-type receptor whether internalization takes place
or not. In contrast, this early form of desensitization does not take
place for the C335STOP receptor, which lacks both the potential
palmitoylation sites at Cys335 and Cys337 and potential phosphorylation
sites in the C-terminal tail. IP3 remains high in cells
expressing the truncated receptor as long as TRH is present, although
it falls quickly if TRH is forcibly removed. One possible explanation
is that uncoupling the receptor from phospholipase C requires
phosphorylation of residues in the C-terminal tail of the receptor.
This interpretation is supported by the finding that the GnRH receptor,
which has no C-terminal tail, does not undergo early desensitization
(34, 35), nor does the PAF receptor if it is mutated to remove the
C-terminal region (36). The C-terminal region of the NK1 receptor
likewise confers early desensitization (37). Most native
Gq/11-coupled receptors display the same kind of rapid
desensitization in the continued presence of agonist as the native TRH
receptor (1, 38, 39), although exceptions have been reported (40).
After agonist is removed from the extracellular medium, the cell cannot
immediately respond to the readdition of TRH with an increase in
IP3. It is likely that several steps must occur before
responsiveness is restored.
2. Ligand must fall off the receptor. Receptors must be on the surface
and empty to respond to a second challenge with hormone, since
receptors that are still occupied are turned off. In our experiments,
the rate of hormone dissociation limited the rate of resensitization.
It seems likely that ligand dissociation will generally be slow and
contribute to the time needed for resensitization of peptide hormone
receptors, because peptides tend to bind with high, nanomolar
affinities and dissociate slowly. This may differ from the situation
with neurotransmitter receptors, because neurotransmitters tend to bind
with lower affinities and dissociate quickly.
3. Some additional resensitization process must occur. This step, which
remains poorly defined, does not require receptor internalization and
can take place on the plasma membrane. To isolate this step in the
resensitization process, it was necessary to remove TRH from surface
receptors forcibly with mild acid, and this process required a 10-min
incubation at 37 C. For this reason it was not possible to compare the
extent of desensitization at zero time, or measure the rate of
resensitization from zero time, in all paradigms. The ability to
respond to TRH was restored over about 1030 min in all cases. Since
the delayed form of desensitization is observed with the C335STOP
receptor, it does not involve phosphorylation of the C-terminal
tail of the receptor. The GnRH receptor, which has no C-terminal tail,
likewise undergoes a refractory period after withdrawal and readdition
of agonist (41), and there are several examples of mutated receptors
that do not respond to a second challenge with agonist even though they
are not phosphorylated (42, 43). The refractory period to TRH does not
result from an overall inhibition of phospholipase C due to the action
of downstream kinases, because the response to other agonists was not
impaired, i.e. heterologous desensitization was not observed
(17).
4. IP3 acts on the IP3 receptor and releases
Ca2+, which requires that the intracellular
Ca2+ pool be at least partially full. As shown previously
(17), pool refilling normally limits the response of 301 cell to
repeated applications of TRH. Pool refilling takes place faster in
cells expressing the dominant negative dynamin and in hypertonic
sucrose, and under these conditions, recovery of the Ca2+
response to TRH is also accelerated. In the continued presence of TRH,
intracellular Ca2+ pools do not refill in GH3
cells, lactotropes (44), or in the transfected cell models used
here.
One obvious question left unanswered by these studies is why receptor
cycling occurs, if it is not required to turn on or turn off the
initial signal cascade. When a dominant negative dynamin was used to
block epidermal growth factor receptor internalization, a wide variety
of effects were noted (32), and it is quite possible that
internalization has effects on TRH actions other than the immediate
signaling events studied here. We speculate that cycling is important
for regulation of receptor concentration, often termed down-regulation.
TRH causes a decrease in the density of TRH receptors on pituitary
cells over a period of 2472 h (45), and similar down-regulation is
observed for many other GPCRs (1). Although most internalized TRH
receptors are recycled, a small fraction may be sorted to the lysosomal
pathway and degraded with each round of endocytosis, leading to a new,
lower steady state concentration of receptors.
Pituitary cells probably encounter hypothalamic releasing factors in
pulses, and desensitization of the TRH receptor is likely to be
important in controlling TRH responsiveness in vivo. We have
described both a rapid desensitization while agonist is present and a
refractory period after agonist is removed. Overall, TRH responsiveness
is reduced by as much as 95%. Our results suggest that uncoupling of
the receptor from phospholipase C requires the C-terminal tail of the
receptor, but the refractory period after agonist withdrawal does not.
Experiments are in progress to clarify the molecular mechanism of the
desensitization and resensitization of the TRH receptor.
 |
MATERIALS AND METHODS
|
---|
Materials
G418 and LipofectAmine were purchased from GIBCO (Grand Island,
NY). Sources of all other reagents are the same as in previous studies
(17). HEK293 cell lines stably expressing the wild-type mouse TRH
receptor, 301 cells, have been described previously (22, 46). HEK293
cells stably expressing a mouse TRH receptor truncated after residue
334 (C335STOP) were obtained as follows. Plasmid pAd/CMVmTRHRC335STOP
encoding the C335STOP TRH receptor (15) was a gift from Dr. Marvin
Gershengorn, Cornell University Medical School (New York, NY). Plasmid
pCBRneo encoding the bombesin receptor and neomycin resistance (47) was
a gift from Dr. Thomas Segerson, Oregon Health Sciences University
(Portland, OR). HEK293 cells were cotransfected with the two plasmids
using LipofectAmine, selected in G418, and screened for
3H-MeTRH binding.
Methods
Vaccinia virus encoding a dominant negative (K44E) dynamin (18)
and empty vaccinia virus were a gift from Dr. Gary Thomas at the Vollum
Institute (Portland, OR). For infection, 293 cells were washed with
Dulbeccos PBS once and incubated with virus diluted in Dulbeccos
PBS (1:20,000) at room temperature for 30 min. Cells were washed and
incubated in normal medium overnight. All cells appeared to have been
successfully infected based on the presence of a rounded morphology and
failure of cells to internalize rhodamine-labeled TRH the next day.
Experiments were done 1824 h after infection. Ca2+
imaging and calibration of [Ca2+]i were carried out
essentially as described by Nelson and Hinkle (48). Methods for cell
culture and measurement of intracellular Ca2+ pools and
IP3 were performed as described (17). In all experiments,
cells were incubated in HBSS or, where hypertonic sucrose is indicated,
in HBSS supplemented with 0.4 M sucrose.
To measure radioligand binding, cells plated in 35-mm dishes were
washed twice with HBSS and incubated at 37 C in buffer containing
3H-labeled TRH or 3H-labeled MeTRH with or
without a 1000-fold molar excess of unlabeled hormone. Dishes were then
washed three times with HBSS. In some cases, cells were then incubated
in ice-cold 0.5 M NaCl/0.2 M HAc (pH 2.8) for 1
min, and radioactivity in the acid wash and the cells was quantitated.
The acid/salt-resistant hormone is often equated with internalized
hormone (49). After this strong acid treatment, cells were unable to
bind radiolabeled TRH.
Cells were stained with a rhodamine-labeled TRH agonist (12) as
follows. Cells plated on coverslips were rinsed twice with HBSS and
incubated at 37 C with Rhod-TRH, which was prepared as described
previously (12) and diluted to a concentration of
50 nM.
Cells were rinsed, placed in a chamber in HBSS, and viewed on a Nikon
inverted fluorescence microscope with filters selective for
rhodamine.
In some experiments, it was necessary to remove surface-bound TRH under
conditions that preserved subsequent IP3 responses to TRH.
To accomplish this, we washed cells that had been incubated in TRH
three times with HBSS and then incubated them in a mild acid solution
containing 0.5 M NaCl, 0.025 M HAc, and 1
mM CaCl2 (pH 5.0) at 37 C for 10 min. At the
end of incubation, cells were washed once with HBSS. Incubation in high
salt at pH 5.0 removed 9298% of specifically bound
3H-labeled TRH from cells in hypertonic sucrose or cells
expressing the truncated C335STOP receptor. This procedure removed only
2030% of 3H-labeled TRH from 301 cells that had been
incubated at 37 C in normal medium to internalize the hormone-receptor
complex. After the high salt pH 5.0 treatment, the number and affinity
of 3H-labeled TRH binding sites was normal, and the
IP3 response to TRH was normal. Although it would have been
preferable to remove 3H-labeled TRH at low temperature or
to remove it more quickly, we were unable to find conditions that would
allow us to do so and still retain subsequent responsiveness.
 |
ACKNOWLEDGMENTS
|
---|
The authors are grateful to Dr. Gary Thomas for the gift of
vaccinia virus vectors.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Patricia M. Hinkle, Department of Pharmacology and Physiology, University of Rochester Medical Center, Box 711, Rochester, New York 14642.
This work was supported in part by NIH Grant DK-19974 and Cancer Center
Core Research Grant CA11198, and by a Pharmaceutical Manufacturers
Association Advanced Predoctoral Fellowship and Wilmot Fellowship to
Run Yu.
Received for publication January 4, 1998.
Accepted for publication February 2, 1998.
 |
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