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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 After TRH is withdrawn, cells are refractory to TRH.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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 2–3 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go and 2Go). 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 85–90% 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 45–50% (Fig. 1Go). 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 0–120 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 2–4 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, 20–30% of 3H-labeled MeTRH dissociated during the first minute at 37 C and 45–55% 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.

 
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. 2Go). 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. 3Go). 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 16–28 cells).

 
The IP3 response to TRH was not prevented when internalization was inhibited with K44E dynamin or with hypertonic sucrose (Fig. 4Go). 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. 2Go of Ref. 17). Values are the mean and range from duplicate dishes; where error bars are not visible, they fell within symbol size.

 
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. 4Go). 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. 5Go 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, 300–800% of basal, for at least 90 min after TRH was withdrawn (Fig. 5Go). 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. 5Go).



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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.

 
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. 6Go). 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 10–1000 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 0–90 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. 5Go. Shown are the mean and range from duplicate dishes; where error bars are not visible, they fell within symbol size.

 
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 20–40 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. 7Go). Under control conditions, 75% of wild-type receptors were apparently internalized and inaccessible at the start of the recovery period; recycling required 10–30 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 1.5–4 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. 8Go and 9Go. Values shown are the mean and range of results from duplicate dishes.

 
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. 8Go). 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 0–90 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.

 
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. 8Go). 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 1Go). 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 1Go). 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.


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Table 1. Recovery of the IP3 Response to TRH in Virus-Infected 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. 9aGo). 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. 9bGo). Refilling was relatively slow and incomplete over the time course of these experiments in cells expressing either the truncated or wild-type receptor (Fig. 9cGo).



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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.

 
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. 10Go). 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 = 16–20) 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 10–30 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 24–72 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Dulbecco’s PBS once and incubated with virus diluted in Dulbecco’s 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 18–24 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 92–98% of specifically bound 3H-labeled TRH from cells in hypertonic sucrose or cells expressing the truncated C335STOP receptor. This procedure removed only 20–30% 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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