©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Truncation of the Thyrotropin-releasing Hormone Receptor Carboxyl Tail Causes Constitutive Activity and Leads to Impaired Responsiveness in Xenopus Oocytes and AtT20 Cells (*)

(Received for publication, September 9, 1994)

Noa Matus-Leibovitch (§) Daniel R. Nussenzveig (1) Marvin C. Gershengorn (1) Yoram Oron (¶)

From the Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel and the Division of Molecular Medicine, Department of Medicine, Cornell University Medical College, The New York Hospital, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

A large superfamily of membrane receptors mediate their physiological effects by coupling to G-proteins. (^1)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.


EXPERIMENTAL PROCEDURES

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(2), 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 (times10 or times40). 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(2), and [Ca] was assayed with times40 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-^3H]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-^3H]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(2) was purchased from Amersham. [methyl-^3H]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.


RESULTS

Functional Expression of WT and Mutant TRH-Rs

Oocytes injected with RNA transcribed in vitro from cDNA coding for WT TRH-R or C335Stop TRH-R mutant were tested for functional expression of the receptor. Twenty-four h after the injection of RNA, oocytes expressing the WT TRH-R exhibited typical TRH-induced chloride currents. The mean amplitude was 2688 ± 508 nA (N = 16), and the latency of the response was 2.6 ± 0.6 s (N = 6). Oocytes expressing the C335Stop mutant showed a lower TRH-evoked response (mean amplitude, 617 ± 79 nA, N = 23) and a modest increase in response latency (3.4 ± 0.7 s, N = 6). Paired comparison revealed a 40 ± 23% increase in latency (N = 6). To test whether the lower amplitudes reflected lower expression of the mutant receptor, we compared binding of the radiolabeled TRH analog, [methyl-^3H]TRH. Oocytes injected with RNA coding for the WT TRH-R bound 0.8 ± 0.5 fmol/oocyte (N = 4), whereas those injected with the C335Stop mutant receptor RNA showed significantly higher binding (4.9 ± 1.0 fmol/oocyte, N = 4). Hence, lower response amplitudes were observed despite an apparently higher number of receptors. These results are shown in Fig. 1.


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-^3H]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.



Kinetics of Calcium Mobilization

To further characterize the differences between the WT and the C335Stop mutant TRH-R, we assayed the kinetics of TRH-induced changes in Ca efflux and [Ca](i). The TRH-induced Ca efflux in oocytes expressing the C335Stop mutant exhibited a lower initial rate (Fig. 3A, note that the net increase over basal efflux is presented). This was more pronounced during the first 0.5-2.0 min of the response, whereas there was little difference between the two receptors when efflux after 5 min was compared (Fig. 4, left panel). The Ca efflux rate reflects changes in oocyte [Ca](i). We therefore assayed the kinetics of [Ca](i) transient in oocytes expressing either WT or C335Stop receptors. As shown in Fig. 3B, oocytes expressing mutant receptors showed a latency similar to that of oocytes expressing WT TRH-Rs (14.0 versus 14.9 s), a lower initial rate of [Ca](i) increase (49 versus 745 nM/min), and a lower fold stimulation over basal [Ca](i) levels (1.4 versus 6.6). The peak of the rapid chloride current response occurs within seconds of TRH exposure. Hence, the initial rates of the [Ca](i) rise mediate the physiological event, and later measurements may be irrelevant for the response and are misleading.


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).



Constitutive Activity of C335Stop TRH-R in Oocytes

The blunted responses in oocytes expressing C335Stop TRH-Rs may reflect poor receptor-G-protein coupling. However, as shown in Fig. 3B, the basal [Ca](i) was higher in oocytes that expressed the C335Stop mutant. We have, therefore, tested the basal Ca efflux rate and the basal [Ca](i). Basal Ca efflux was linear over an extended period of time(13) , and its value in oocytes injected with WT TRH-R RNA was 0.92 ± 0.22%/min of total label (N = 12). In oocytes expressing C335Stop TRH-Rs, this rate was 1.96 ± 0.48%/min of total label (N = 7). In a paired comparison of seven matched experiments, the basal efflux in oocytes expressing the C335Stop mutant was 209 ± 8% of the value found for the cells of the same batch injected with the WT TRH-R RNA (Fig. 4). Similarly, basal [Ca](i) was 65 ± 25 nM in oocytes expressing the WT TRH-R and 129 ± 30 nM in cells expressing C335Stop TRH-R. Hence, both parameters were 2-fold higher in oocytes expressing the C335Stop TRH-R (Fig. 4).

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](i) 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) . (^2)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.

Constitutive Activity of C335Stop TRH-R in AtT20 Cells

Previous reports suggested that the C335Stop mutation in the TRH-R affected the expression of the receptor on the cell surface but did not affect its signal transduction properties measured by phosphoinositide hydrolysis(10) . Therefore, the constitutive activity of C335Stop TRH-R observed in oocytes may have been due to a unique interaction with the endogenous amphibian G-proteins. To investigate this hypothesis, we assayed inositol phosphate formation and [Ca](i) in AtT20 cells transfected with either WT or C335Stop TRH-Rs. These AtT20 cells expressed similar numbers of receptors (250,000 ± 10,000 WT and 181,000 ± 59,000 C335Stop TRH-Rs/cell) of similar affinities (equilibrium dissociation constants of 0.8 and 1.9 nM for WT and C335Stop TRH-Rs, respectively). Cells transfected with the mutant receptor exhibited consistently higher levels of inositol phosphate formation and basal [Ca](i) values than those expressing the WT receptor. Inositol phosphate formation in the absence of TRH was 32% higher in AtT20 cells expressing C335Stop TRH-Rs than in cells expressing WT TRH-Rs (Table 2). Basal [Ca](i) was 144 ± 16 nM (n = 38, N = 5) in AtT20 cells expressing C335Stop TRH-Rs compared to 83 ± 9 nM (n = 40, N = 5) in cells expressing WT TRH-Rs. Paired comparison in five experiments demonstrated that the basal [Ca](i) was 1.8 ± 0.2-fold higher in cells expressing the mutant receptor (p < 0.005). When challenged with 5 µM TRH, cells expressing WT TRH-Rs responded with a 10-fold increase in [Ca](i) (822 ± 64 nM, n = 40), whereas cells transfected with the C335Stop mutant responded with a 1.6-fold [Ca](i) rise (277 ± 24 nM, n = 38). A large proportion of C335Stop TRH-R-transfected cells did not respond at all to the challenge with TRH. Representative responses of individual cells are shown in Fig. 6. These results indicated that mammalian cells transfected with the C335Stop receptor exhibit properties similar to those found in oocytes expressing this receptor mutant.




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).




DISCUSSION

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](i), 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](i)(22) but also to more proximal steps, as reflected by the dramatically lower rates of [Ca](i) 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](i) 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 beta(2)-adrenergic receptor results in its uncoupling from G(s). 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(3) 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(3) 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](i) 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](i) 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 alpha(1)-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.


FOOTNOTES

*
This work was supported by a grant of the United States-Israel Bi-National Science Foundation (to Y. O. and M. C. G.) and United States Public Health Service Grant DK43036 (to M. C. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
This work partially fulfills the Ph.D. thesis requirements of this author at The Sackler Faculty of Medicine, Tel Aviv University.

To whom correspondence and reprint requests should be addressed. Tel: 972-3-640-8753; Fax: 972-3-640-9113.

(^1)
The abbreviations used are: G-protein(s), guanine nucleotide-binding regulatory protein(s); GPCR(s), G-protein-coupled receptor(s); TRH, thyrotropin-releasing hormone; TRH-R(s), TRH receptor(s); C335Stop, cysteine codons at position 335 mutated to a stop codon; K338Stop, lysine at position 338 mutated to a stop codon; [Ca], cytosolic Ca concentration.

(^2)
M. Lupu-Meiri and Y. Oron, unpublished results.


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