(Received for publication, September 9, 1994)
From the
We studied the activity of a truncated thyrotropin-releasing
hormone receptor (TRH-R), which lacks the last 59 amino acids of the
carboxyl tail, where Cys-335 was mutated to a stop codon (C335Stop)
(Nussenzveig, D. R., Heinflink, M., and Gershengorn, M. C.(1993) J.
Biol. Chem. 268, 2389-2392). In Xenopus laevis oocytes expressing C335Stop TRH-Rs, TRH binding was higher,
whereas chloride current, Ca
efflux, and
[Ca
]
responses evoked
by TRH were 23, 39, and 21%, respectively, of those in oocytes
expressing wild type mouse pituitary TRH-Rs (WT TRH-Rs). In oocytes
expressing C335Stop TRH-Rs, basal
Ca
efflux and [Ca
]
were twice those in oocytes expressing WT TRH-Rs; chelation
of Ca
caused a rapid increase in holding current,
which is consistent with basal activation; and coexpression with other
receptors caused inhibition of the responses to the other cognate
agonists. In AtT20 pituitary cells stably expressing C335Stop TRH-Rs,
thyrotropin-releasing hormone (TRH)-independent inositol phosphate
formation was 1.32 ± 0.11-fold higher, basal
[Ca
]
was 1.8 ±
0.2-fold higher, and the [Ca
]
response to TRH was much lower than in cells expressing WT
TRH-Rs. We conclude that a TRH-R mutant truncated at Cys-335 exhibits
constitutive activity that results in desensitization of the response
to TRH.
A large superfamily of membrane receptors mediate their
physiological effects by coupling to G-proteins. ()The tools
of molecular biology allow the investigation of the molecular basis of
receptor function. Among these receptors, the domains that determine
coupling to G-proteins have been intensively studied.
It has been proposed that Cys residue(s) in the carboxyl cytoplasmic tail of the receptor may affect the efficacy of coupling to the appropriate G-proteins. It has been demonstrated that the Cys residue in some receptors may be palmitoylated and that palmitoylation improves receptor-G-protein coupling(1, 2, 3) .
Different effects of mutations in the carboxyl-terminal domain of GPCRs on receptor internalization have been reported(4, 5, 6, 7, 8, 9, 10) . Hence, receptors mutated in the carboxyl tail may exhibit different profiles of plasma membrane/internal membrane distribution as well as diminished coupling efficacy.
In this report, we describe a study of
two carboxyl-terminal mutants (10) of the receptor for
TRH(11) . Using the Xenopus laevis oocyte expression
system, which allows excellent sensitivity and temporal resolution of
response, we demonstrate that truncation of the cytoplasmic tail,
including deletion of the
Cys-Asn
-Cys
sequence, causes
altered expression of TRH-R in oocyte plasma membrane. Moreover, the
truncated receptor exhibits constitutive, TRH-independent activity.
Similar results were obtained in AtT20 cells transfected with either WT
or C335Stop TRH-Rs. This effect has not been previously reported for
mutations in the carboxyl terminus of GPCRs.
The expression of TRH-R in Xenopus oocytes and the
electrophysiological methods have been described in detail previously (12, 13) . Oocytes injected with RNA transcribed from
cloned mouse pituitary TRH-R DNA or mutants truncated at Cys or Lys
residues (10) (C335Stop and
K338Stop, 5-10 ng/oocyte) were used. All electrophysiological
experiments were performed on denuded oocytes clamped at -100 mV
to minimize potassium currents.
To measure the duration of the latency, oocytes maintained under two-electrode voltage clamp were rapidly exposed to the desired concentration of TRH. This was attained by injecting a large volume (approximately 1 ml) of the TRH solution in ND96 medium (for composition, see (13) ) into a 0.1-ml chamber. The oocyte was exposed to the desired concentration of the hormone within <1 s (described in detail in (14) ).
Ca
efflux was performed on oocytes
injected with 20,000-100,000 cpm of
CaCl
, as described previously(13) .
Basal
Ca
efflux was linear for at least
1 h(13) , and all measurements were performed 2 h after the
injection of the label, i.e. the time required for the
homogeneous distribution of
Ca
in
oocytes (data not shown).
Ratio imaging of oocyte cytosolic calcium
concentration ([Ca]
)
was performed using the Magical system (Applied Imaging) as follows.
Oocytes were injected with 60 pmol of Fura 2 pentapotassium salt (in a
30-nl volume) 30-90 min before the assay. The cell was then
placed in a special perfusion chamber (volume, <10 µl) with the
equator facing the epifluorescence objective (
10 or
40).
The microscope was focused on the membrane at the equator, and
successive 340/380 nm frames were acquired at 0.3-2.0 s/pair
without averaging. Oocyte [Ca
]
was calculated from a standard curve generated from 340/380
nm ratio values obtained with 0 and 10 mM calcium.
Intermediate values were calculated by applying the equation of
Grynkiewicz et al. (15) .
Inositol phosphate and
[Ca]
determinations in
AtT20 cells were performed as follows. AtT20 cells that were stably
transfected with either the WT or the C335Stop TRH-Rs were grown in
Dulbecco's modified Eagle's medium containing 5% Nu-Serum
(Collaborative Research). Cells at intermediate density were analyzed
24-96 h after plating. Inositol phosphate formation was measured
as described(11) . For
[Ca
]
determination,
cells were grown on coverslips and loaded with Fura 2-AM (3
µM) in medium for 30 min at 37 °C. The cells were
washed with phosphate-buffered saline containing 1.8 mM CaCl
, and [Ca
]
was assayed with
40 oil-immersion objective at 20
°C. TRH (5 µM) was added rapidly in a large volume,
and the 340/380 nm frame pairs were acquired at 0.3-s intervals.
[methyl-H]TRH (15-25
nM) binding in oocytes was performed essentially as described
previously for muscarinic receptors(16) . Briefly, denuded
oocytes were incubated for 3-4 h in groups of 10 cells in 0.2 ml
of ND96 containing the labeled TRH analog at 0 °C. Nonspecific
binding was determined in the presence of 20 µM TRH. At
the end of the incubation period, oocytes were washed 4 times with 4 ml
of ice-cold ND96, and each cell was separately counted in 4.5 ml of
Hydroluma (Lumac). Mean nonspecific binding was subtracted from the
total binding for each oocyte in each experiment. The nonspecific
binding was 45% of the total binding (N = 10), a
relatively low fraction when intact oocytes are
concerned(16, 17) . The results were presented as the
mean ± S.E. (fmol/oocyte). In AtT20 cells,
[methyl-
H]TRH (0.1-10 nM)
binding was performed as described(10) .
Fura 2
pentapotassium salt and Fura 2-AM was purchased from Molecular Probes.
TRH, bombesin, acetylcholine, and collagenase were the products of
Sigma. CaCl
was purchased from Amersham.
[methyl-
H]TRH was purchased from DuPont
NEN. All other chemicals were of analytical grade.
All experiments were repeated several times. For experiments using oocytes, a number of different oocytes (denoted by n) obtained from different donors (denoted by N) were used. Results are presented as the mean ± S.E. Statistical significance was determined by paired or unpaired t test.
Figure 1:
Amplitude, latency, and receptor number
in oocytes expressing the WT or the C335Stop TRH-R. Oocytes were
injected with 5-10 ng each of RNA transcribed in vitro from DNA coding for either the WT TRH-R (open bars) or
the C335Stop mutant (solid bars).
[methyl-H]TRH binding, amplitudes, and
latencies of responses to 1 µM TRH were assayed 24 h after
injection of RNA. Each value represents the mean ± S.E. of
32-220 determinations on individual oocytes from 4-23
different donors.
Comparison of the dose-response
relationships in oocytes expressing WT and C335Stop TRH-Rs revealed an
apparent shift of the curve to higher doses for the truncated receptor (Fig. 2A). However, the magnitude of the responses
obtained in oocytes expressing the mutant receptor did not allow
kinetic analysis of maximal amplitude and half-maximal effects. A
similar comparison of TRH-induced increases in Ca
efflux during the first 2 min of the
response also showed a lower maximal effect (3.9 ± 0.7 versus 12.9 ± 0.8% of total label at 10 µM TRH) for C335Stop TRH-R than for WT receptor (Fig. 2B).
Figure 2:
Dose-response relationship in oocytes
expressing the WT or the C335Stop mutant TRH-R. Oocytes of the same
donors expressing either receptor were assayed for TRH-induced chloride
current (A) or for a TRH-induced net increase in Ca
efflux (B). The amplitude of
the TRH-induced chloride current was measured at the peak of the
response 10-30 s after the challenge with the hormone (see also Fig. 5). The TRH-induced increase in
Ca
efflux was measured over the first 2 min of exposure to TRH (1.0
µM) after the subtraction of basal efflux values.
Representative experiments are shown. Each point represents
the mean ± S.E. of 6-10 determinations on individual
oocytes.
Figure 5:
Kinetics of holding current in oocytes
expressing the WT (left panel) or the C335Stop mutant TRH-R (right panel). Oocytes expressing either receptor were
voltage-clamped at -100 mV, and the holding current was measured
after the removal of Ca from the medium (+ 0.1
mM EGTA). At the time indicated by the second arrow,
the solution was adjusted so that
[Ca
]
= 1.8
mM. The third, bold arrow indicates the
addition of TRH (1 µM). Please note the difference in
vertical calibration between the two trace
records.
Figure 3:
Kinetics of Ca
efflux and [Ca
]
in oocytes expressing the wild type or the C335Stop mutant
TRH-R. Oocytes expressing either receptor were assayed for either
TRH-induced
Ca
efflux (A) or
TRH-induced [Ca
]
transient (B). The arrow (B) denotes
the addition of TRH (1 µM), which was present for the
remainder of the experiment. Net
Ca
efflux rate values for each time point represent the mean
± S.E. of 24-30 determinations on three different donors
(basal efflux values were subtracted).
[Ca
]
transients were
measured by Fura 2 340/380 nm fluorescence ratio imaging as described
under ``Experimental Procedures.'' Latency includes the dead
time of TRH addition (approximately 12 s). Representative experiments
are shown.
Figure 4:
Ca
efflux
and cytosolic [Ca
]
in
oocytes expressing the WT or the C335Stop mutant TRH-R. Oocytes
expressing either receptor were assayed for
Ca
efflux (left panel) or
[Ca
]
(right
panel). Results were expressed as the percent of control values (i.e. values in oocytes expressing the WT TRH-R). Open
bars represent basal efflux or
[Ca
]
values. Solid
bars represent a TRH-induced (1 µM) increase over
basal. The numbers above the bars denote the duration
of measured stimulated efflux in min. The TRH-induced increase in
[Ca
]
was measured at
the peak of the response (see Fig. 3B).
To further localize the domain responsible for the characteristics
of the C335Stop mutant, we have examined an additional TRH-R mutant
that is truncated at Lys. Oocytes that expressed K338Stop
TRH-R exhibited responses (3430 ± 1291 nA, N =
3) that were comparable with those mediated by WT TRH-R (2688 ±
508, N = 16). Similarly, the basal
Ca
efflux rate (0.64 ± 0.2%/min
of total label) and the total TRH-induced
Ca
efflux (8.6 ± 2.9% of total label, 1.5-min stimulation
with the hormone) were similar to the values obtained in oocytes
expressing the WT TRH-R (0.9 ± 0.2%/min and 8.7 ± 1.1% of
total label, respectively; n = 26, N =
7). These data suggest that TRH-R truncated at Lys
behaved identically to the WT receptor and that the behavior of
the C335Stop mutant was due to the deletion of the
Cys
-Asn
-Cys
sequence.
The
consistently higher basal [Ca]
and
Ca
efflux suggested that the
C335Stop mutant receptor had a constitutive, hormone-independent
activity. To test this hypothesis, we monitored holding current in
oocytes expressing the WT or the C335Stop receptor in calcium-free
medium. Previous data on muscarinic and inositol
1,4,5-trisphosphate-induced responses showed that the agonist or the
second messenger promote calcium entry or loss, depending on the
presence or absence of calcium in the
medium(18, 19, 20) . Oocytes maintained in
calcium-free medium tend to deteriorate and display a continuously
increasing depolarizing current(21) . (
)This
deterioration was much more rapid and extensive in oocytes that
expressed the C335Stop mutant. In Fig. 5, the left tracing describes the slow deterioration due to the removal of calcium
from the medium in an oocyte that expressed the WT TRH-R. Upon the
addition of 1.8 mM Ca
, the holding current
stabilized, and a typical TRH response was observed. In an oocyte that
expressed the C335Stop TRH-R (Fig. 5, right tracing),
the removal of calcium resulted in a dramatic depolarizing current that
stabilized at a much higher value when 1.8 mM Ca
was added. A challenge with TRH resulted in a blunted response.
When these phenomena were quantitated, the mean rate of increase of
holding current was 60 ± 30 nA for the WT receptor and 838
± 412 nA (n = 6, N = 2) for the
C335Stop mutant during the first 2 min after Ca
removal. Hence, the holding current resulting from Ca
removal was 14 times higher in oocytes expressing the C335Stop
TRH-R.
It has been reported that prolonged exposure of oocytes to an agonist results in long-term desensitization targeted, among other sites, on chloride channels(22) . Hence, it was likely that the low response to TRH in oocytes expressing the C335Stop TRH-R may have been a result of desensitization of the signal transduction pathway at the chloride channel and possibly other steps, or the low response to TRH could have been due to decreased coupling efficacy of C335Stop TRH-R. To attempt to distinguish between these possibilities, we coexpressed C335Stop mutant and WT receptors in the same oocytes. Were the mutant receptor only lesioned in its coupling to the G-protein, one would have predicted an additive effect. On the other hand, if the blunted response mediated by the C335Stop mutant was due to desensitization, a less than additive response would be expected. Indeed, in oocytes injected with a mixture of the WT and the C335Stop TRH-R RNAs, the response to TRH was only 34% of the response amplitude measured in oocytes expressing the WT receptor alone (Table 1). Thus, the presence of the C335Stop receptor resulted in desensitization of the WT TRH-R-mediated response.
To further validate this putative desensitization of the response to the stimulation of WT TRH-Rs by the constitutively active coexpressed C335Stop mutant, we coexpressed C335Stop TRH-R with either m1 muscarinic or gastrin-releasing peptide receptors. In both cases, coexpression of C335Stop TRH-R significantly inhibited (by 41 and 73% of control, respectively) the responses to the stimulation of the second expressed receptor (Table 1). In order to test whether this phenomenon was due to a negative dominant effect of coexpression of two types of receptors in the same cell, we coexpressed WT TRH-R and gastrin-releasing peptide receptor in the same oocytes and compared the amplitudes of the responses to each agonist to those obtained in oocytes injected with either message alone. In oocytes injected with WT TRH-R RNA alone, the response to 1 µM TRH was 1485 ± 464 nA, whereas in oocytes injected with both TRH-R and gastrin-releasing peptide receptor RNAs, the response to TRH was 1656 ± 590 nA. Similarly, in oocytes of the same batch injected with gastrin-releasing peptide receptor RNA alone, the response to 0.1 µM bombesin was 879 ± 241 nA, though in cells injected with both RNAs, the response to bombesin was 1450 ± 605 nA. These results clearly demonstrate that an increase in the amount of two different receptor-coding RNAs does not cause a reduced response to either receptor agonist.
Figure 6:
Basal and TRH-stimulated
[Ca]
in AtT20 cells.
AtT20 cells stably transfected with either WT or C335Stop TRH-R were
loaded with Fura 2, and [Ca
]
was assayed as described under ``Experimental
Procedures.'' Representative tracings of two cells expressing
either receptor are shown. The arrow denotes the time of
addition of TRH (5 µM).
Truncation of a GPCR that deletes the distal part of the carboxyl-terminal cytoplasmic tail, including the Cys residue(s) potentially subject to palmitoylation, has been previously shown to have two effects on receptor function: impaired coupling to G-proteins and changes in the internalization or cycling of the receptor. Evidence for these two effects has been reported for several receptors of this family(1, 2, 3, 4, 5, 6, 7, 8, 9) . We have recently shown that TRH-stimulated internalization of C335Stop TRH-R is inhibited also(10) .
Xenopus oocytes injected with RNA coding for the C335Stop mutant exhibited ligand binding that was 6 times higher than in cells expressing the WT TRH-R. It should be noted that the significant right shift of the dose-response curve in the C335Stop mutant may reflect a lower affinity of this mutant, and the values reported here should be viewed as an indication of the minimal range of receptor expression. This was consistent with lower internalization, i.e. higher proportion of the receptors found in the plasma membrane(10) .
In
addition to these confirmatory findings, we presented evidence strongly
suggesting that the C335Stop mutant of TRH-R possesses constitutive,
agonist-independent activity in oocytes. This evidence includes
elevated basal [Ca]
, higher
Ca
basal efflux rates, and greater
sensitivity to calcium withdrawal from the medium of oocytes expressing
C335Stop TRH-Rs compared with oocytes expressing WT TRH-Rs. Although
mutations that result in constitutive activity of GPCRs have been
reported(23) , they were effected in different domains of the
receptor. Constitutively active GPCRs have been reported with mutations
in the carboxyl-terminal domain of the third cytoplasmic loop and in
the transmembrane
segments(24, 25, 26, 27) . Our data
suggest that there may be additional sites involved in the constraint
of the receptor in its inactive conformation. In this respect, our
findings are novel and could be examined in other GPCRs.
We could not compare WT and C335Stop TRH-Rs at the same expression level due to our inability to obtain measurable responses in oocytes expressing low levels of C335Stop TRH-Rs on the one hand and due to the difficulty in obtaining high levels of WT TRH-Rs on the other. Hence, it could be argued that the constitutive activity exhibited by the C335Stop TRH-R could have been a result of a higher level of membrane expression of this mutant. This explanation, however, is not likely. First, very high levels of WT TRH-R expression in transfected cells do not produce either constitutive activity or desensitization (28) . Second, in oocytes of different donors, there is no correlation between the number of expressed TRH-Rs and the amplitude of the response. In the same donor, injecting higher amounts of WT TRH-R RNA results in increased responses(14, 29) . Moreover, in the same donor, there is a correlation between the receptor number and the response amplitude(30) .
The data obtained with the K338Stop
mutant suggest that the deletion of the
Cys-Asn
-Cys
sequence is
responsible for the changes in the TRH-R characteristics seen in the
C335Stop mutant. Further mutations in this domain are necessary to
fully characterize the residue(s) involved in impaired coupling and/or
constitutive activity observed in the C335Stop TRH-R.
Previous
evidence for GPCRs expressed in oocytes, including TRH-R, points to
pronounced desensitization following receptor activation (12) .
Hence, the decreased responses in oocytes expressing the mutant
receptor could reflect desensitization due to the constitutive activity
of C335Stop TRH-R. This desensitization could be attributed to
inactivation of chloride channels by a higher
[Ca]
(22) but also to
more proximal steps, as reflected by the dramatically lower rates of
[Ca
]
increase. Indeed,
coexpression of WT and C335Stop TRH-Rs resulted in 66% inhibition of
the response observed in oocytes expressing WT TRH-R alone. This result
is consistent with desensitization of the response by the prolonged
elevation of [Ca
]
caused by the
constitutively active C335Stop TRH-R. We cannot exclude the possibility
that the more highly expressed C335Stop receptor mutant inhibits
responses mediated by coexpressed WT TRH-R by competing for the
G-protein(s). This possibility, however, appears unlikely. First,
coexpression of TRH-Rs and gastrin-releasing peptide receptors did not
lead to a decrease in the response to either agonist. Moreover, the
injection of increasing amounts of RNA coding for the WT TRH-R (up to
100 ng/oocyte) results in larger responses(14) , indicating
that the G-protein(s) is not limiting in oocytes. Second, the other
results presented here are compatible with constitutive activity and
desensitization rather than with competition for G-protein(s).
The
C335Stop mutant lacks 59 residues of the carboxyl tail, including the
two cysteine residues that are potential sites for palmitoylation. In
some GPCR systems(1, 2, 3) , deletion or
mutation of an analogous Cys(s) caused uncoupling from G-proteins.
Valiquette et al.(31) have shown that point mutation
of Tyr in the carboxyl tail of the
-adrenergic receptor results in its uncoupling from
G
. Their report suggests that other changes in the
cytoplasmic tail of GPCRs may result in receptor uncoupling. Hence, it
is feasible that the low responses observed in oocytes expressing high
levels of the C335Stop TRH-R may reflect impaired coupling to
G-protein(s) as well as desensitization due to constitutive activity.
What proportion of the decrease in the response can be assigned to the
impaired coupling of the truncated receptor, and what proportion can be
assigned to the desensitization resulting from constitutive receptor
activation? This question is difficult to answer without additional
mutants that could differentiate between the two phenomena. However,
were desensitization the sole cause of the decreased responses evoked
with C335Stop TRH-Rs, we would expect the responses in oocytes
coexpressing both receptors to be very similar to those found in
oocytes expressing C335Stop TRH-Rs alone. The intermediate value
suggests that C335Stop TRH-R possesses both constitutive activity and
impaired coupling to the G-protein.
We have previously examined the
C335Stop mutant in transfected mammalian cells(10) . Our
results indicated that a higher proportion of the mutant receptors is
in the plasma membrane after binding agonist, indicating that, in this
respect, oocytes treat the mutated receptor in the same way as
mammalian cells. In mammalian cells, the increase in inositol phosphate
accumulation induced by TRH stimulation of the C335Stop mutant TRH-R
was not different from that found for the WT TRH-R(10) . This
discrepancy between the two expression systems could be due to
differences in the G-proteins or other downstream components of the
signal transduction cascade or due to differences in desensitization
between mammalian and amphibian cells. On the other hand, it is also
possible that this could be due to differences in the methodology used
for assaying receptor-mediated responses. In mammalian cells,
receptor-mediated increases in inositol phosphates were examined after
60 min of continuous stimulation with TRH. It is feasible that this
type of assay did not reveal receptor properties that are apparent only
during the initial phase of receptor stimulation. For example, Aizawa
and Hinkle (32) have reported that TRH-induced prolactin
secretion in GH cells exhibited a dramatic decrease after
the first 2-3 min of stimulation. Similarly, we have previously
reported that TRH-induced phosphatidylinositol 4,5-bisphosphate
hydrolysis in GH
cells is a transient phenomenon observable
primarily during the first 2 min of stimulation; the accumulation of
inositol 1-phosphate, however, proceeded for an extended period of
time(33) .
To examine this hypothesis, we assayed the change
in [Ca]
upon a challenge with
TRH in AtT20 cells stably transfected with WT or C335Stop TRH-Rs. The
response to TRH was blunted, similar to what was found in oocytes. The
desensitization exhibited by cells expressing the mutant receptor,
therefore, appeared to be proximal to calcium mobilization. Hence, the
decrease in responses due to constitutive activity of the C335Stop
mutant and the subsequent desensitization may be a general phenomenon
that is observable only during the initial period of stimulation.
Last, although our initial observation was made in oocytes,
constitutive activity of C335Stop TRH-R was measurable in mammalian
cells also. The basal levels of inositol phosphate formation and
[Ca]
in AtT20 cells that stably
expressed the mutant receptor were increased 32 and 67%, respectively,
above those values observed in cells expressing the WT TRH-R. This
modest increase should be compared with increases in inositol phosphate
formation found with other GPCRs that have been reported to be
constitutively active. With the series of constitutively active
-adrenergic receptors(23) , one-third caused
less than a 50% increase in basal inositol phosphate production, and
the maximum increase was to only 200% above control. Also, two mutants
of the thyrotropin (thyroid-stimulating hormone) receptor found in
patients with hyperthyroidism stimulated cyclic AMP production 2.4- and
2.8-fold but did not measurably increase inositol phosphate formation,
even though these receptors increased cyclic AMP and inositol phosphate
formation to the same extent as WT receptors when they were activated
by thyrotropin(26) . Thus, it appears that agonist-independent
stimulation of second messenger formation by constitutively active
GPCRs that couple to inositol phosphate formation is not as marked as
with receptors that signal via cyclic AMP. In fact, we did not discern
the basal activity of C335Stop TRH-R in our initial study(10) .
It was the sensitivity of the oocyte system that first identified the
constitutive activity of C335Stop TRH-R. Indeed, we think these
findings support the idea that the Xenopus oocyte system is an
excellent investigative tool for studies of signal transduction with
high sensitivity and sub-second temporal resolution. Thus, Xenopus oocytes exhibit properties that make them better model systems
than mammalian cells for study of certain aspects of GPCR function.