Introduction

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G protein-coupled receptors (GPCRs) are seven transmembrane domain receptors that comprise a large and diverse family of membrane proteins. After continued or repeated agonist activation, many GPCRs undergo desensitization as a result of phosphorylation and uncoupling of the G protein (Ferguson, 2001). These receptors are typically endocytosed and recycled back to the membrane or degraded in lysosomes (Tsao and von Zastrow, 2001). Mammalian ₂-adrenergic receptors have been shown to undergo desensitization in the absence of internalization (Pippig et al., 1995); however, somatostatin receptor desensitization has been shown to require internalization (Beaumont et al., 1998).

The neurokinin-1 receptor (NK1R), also known as the substance P (SP) receptor, is a GPCR that rapidly desensitizes upon continuous or repeated exposure to substance P (Perrine et al., 2000). Agonist-induced internalization and recycling of the NK1R have been observed (Grady et al., 1995). The relationships of internalization to desensitization and of recycling to resensitization are not well understood. A previous study of ₂-adrenergic receptors suggested that desensitization is independent of internalization, whereas resensitization requires both internalization and recycling (Pippig et al., 1995). Similar conclusions were drawn about NK1Rs by Garland et al. (1996); however, the same recovery time periods were not used to study resensitization and recycling. Their study showed receptor resensitization within 30 min, but cells were allowed to recover for 240 min before recycling was tested. A direct correlation between receptor recycling and resensitization has not been made. To investigate the mechanisms of NK1R signaling, we have studied the effects of concanavalin A (Con A) and monensin on receptor desensitization, resensitization, phosphorylation, internalization, and recycling. Con A has been shown to inhibit internalization of ₂-adrenergic and somatostatin receptors (Pippig et al., 1995; Beaumont et al., 1998). Con A irreversibly cross-links glycosylated proteins, which is thought to inhibit receptor translocation (Beaumont et al., 1998). Monensin, a sodium ionophore, prevents the decrease in pH inside endosomes and has been shown to prevent recycling of ₂-adrenergic and somatostatin receptors (Pippig et al., 1995; Beaumont et al., 1998).

In our study, Chinese hamster ovary (CHO) cells stably expressing the rat NK1R (rNK1R) have been used to study the effects of Con A and monensin. Desensitization and resensitization were studied by measuring receptor-activated Ca²⁺ elevations. Radioactive and fluorescent ligand binding techniques were used to study internalization and recycling of the NK1R. Phosphorylation was assessed using radiolabeled orthophosphate and an immunoprecipitation protocol to isolate the rNK1R. By keeping agonist concentrations, duration of application, and duration of recovery periods consistent between the different assays, we have directly compared desensitization, resensitization, phosphorylation, internalization, and recycling of the rNK1R.

	Materials and Methods

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The protocols used to measure activation, desensitization, and resensitization are illustrated schematically in Fig. 1.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Summary diagram of the receptor activation protocol. Desensitization (A) and resensitization (B) protocols used for the SP activation experiments. W, 3× wash; Iono, ionomycin.

Receptor Activation. Activation of the NK1R was measured by observing SP-induced increases in intracellular Ca²⁺. CHO cells stably transfected with the cDNA of the rNK1R were obtained from Dr. James Krause and maintained as described previously (Raddatz et al., 1995). The receptor expression level is approximately 200,000 high-affinity NK1R binding sites/cell (Garland et al., 1996). Cells were cultured onto six-well plates (Corning Glassworks, Corning, NY) and loaded with 0.1% pluronic F-127 and Fura PE3 (Texas Fluorescence Labs, Austin, TX), a Ca²⁺ indicator dye, for 30 min at 37°C in an extracellular solution (ES) containing: 10 mM HEPES, 10 mM glucose, 115 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 2.3 mM CaCl₂. Ca²⁺ levels were measured using a FluoSTAR fluorescent plate reader (BMG Labtechnologies, Durham, NC) as the ratio of emission at 510 nm after excitation at 340 and 380 nm. A Ca²⁺ ionophore, ionomycin (10 µM), was added at the end of each experiment to normalize between wells. A typical Ca²⁺ tracing is shown in Perrine et al. (2000).

Desensitization. CHO cells stably expressing the rNK1R were pretreated with vehicle (ES) for 15 min after the incubation with Fura PE3. SP (100 nM) was applied for 5 min at 37°C to activate and desensitize the receptors (Vigna, 1999; Perrine et al., 2000). A second dose of SP (100 nM) was added to demonstrate the presence of a desensitized state.

Resensitization. Transfected cells were pretreated with vehicle (ES) for 15 min. SP (100 nM) was added for 5 min at 37°C to produce desensitization. The agonist was removed by rinsing three times, and the cells were allowed to recover for 30 or 60 min at 37°C in ES. After the 30- or 60-min recovery period, 100 nM SP was added to measure the amount of resensitization. This was followed by 10 µM ionomycin. Cells were pretreated with 0.25 mg/ml Con A for 15 min or 50 µM monensin for 10 min at 37°C. Con A or monensin was reapplied during the recovery periods.

Receptor Binding. Receptor localization under the various experimental conditions was analyzed by radioligand receptor binding. CHO cells stably expressing the rNK1R were cultured on T-25 culture flasks (Corning Glassworks). In each experiment, cells were pretreated with 0.25 mg/ml Con A (three flasks) for 15 min, 50 µM monensin (three flasks) for 10 min, or vehicle (three flasks) for 15 min at 37°C. SP (100 nM) was added to the flasks and incubated for 5 min at 37°C. The flasks were rinsed three times to remove the agonist. Cells from one flask for each of the three conditions were scraped and centrifuged at 4°C. ES, Con A, or monensin was reapplied to the appropriate remaining flasks. These flasks (two for each of the three pretreatments) were incubated at 37°C for 30 or 60 min to allow recovery. Cells from an additional flask were not treated with SP to provide a negative control.

1

Receptor Visualization. In addition to the radioligand binding experiment, we also visualized, by fluorescent labeling, the location of the NK1Rs during desensitization and resensitization. Oregon Green 488-SP (OG-SP) binds to and activates the rNK1R with a similar affinity as unlabeled SP (Bennett and Simmons, 2001). Fluorescence labeling of rNK1Rs with OG-SP was conducted to provide visualization of receptor localization. For these experiments, transfected CHO cells were cultured overnight on the three-well HTC supercured glass slides (Cel-Line Associates, Inc., Newfield, NJ). Cells were pretreated with 0.25 mg/ml Con A for 15 min, 50 µM monensin for 10 min, or vehicle for 15 min at 37°C. Each well was designated a specific treatment and triplicates were performed.

Receptor Phosphorylation. Some studies have suggested that GPCR phosphorylation causes desensitization and that receptor dephosphorylation leads to resensitization (Pippig et al., 1995; for review, see Ferguson, 2001). To investigate the mechanisms of desensitization and resensitization of the NK1R, we assessed the amount of ligand-induced receptor phosphorylation. rNK1R-expressing CHO cells were cultured on T-25 flasks until confluent. On the day of the experiment, cells were incubated with 200 µCi/ml [³²P]orthophosphate (PerkinElmer Life Sciences) in HEPES/Krebs' buffer (10 mM HEPES, 118 mM NaCl, 4.3 mM KCl, 1.17 mM MgSO₄, 1.3 mM CaCl₂, 0.34 mM NaHCO₃, and 11.7 mM glucose, pH 7.4) for 3 h at 37°C while shaking. Cells were pretreated with 0.25 mg/ml Con A for 15 min, 50 µM monensin for 10 min, or vehicle (two flasks/treatment) for 15 min at 37°C. After pretreatment, 100 nM SP was added for 5 min at 37°C. A flask of each condition was rinsed and Con A, monensin, or vehicle reapplied. These flasks were incubated at 37°C for 60 min without agonist to allow the cells to recover. Cells from the other flasks were scraped and immunoprecipitated to determine the amount of phosphorylation after agonist exposure.

Statistics. Statistical significance was determined using two-way analysis of variance followed by Student-Newman-Keuls analysis between treatment and time for the receptor activation, binding, and phosphorylation data.

Reagents. All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

	Results

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NK1 Receptor Desensitization to SP. The effects of Con A and monensin on receptor desensitization were analyzed by measuring intracellular Ca²⁺ levels. SP mobilizes intracellular Ca²⁺ via a G protein-dependent mechanism (Otsuka and Yoshioka, 1993). This SP-induced Ca²⁺ mobilization has been well characterized (Mochizuki-Oda et al., 1994; Perrine et al., 2000). Desensitization was measured as a decrease in the ability of SP to increase intracellular Ca²⁺ (Perrine et al., 2000) after a prior exposure to SP. In rNK1R-expressing CHO cells, an initial concentration of 100 nM SP (Fig. 2, solid columns) induced a 76 ± 3% loss of responsiveness (desensitization) to a second application of 100 nM SP (Fig. 2, cross-hatched columns). After pretreatment with either Con A or monensin the response to SP still desensitized. Cells treated with Con A showed 75 ± 3% desensitization and monensin-treated cells showed 71 ± 2% desensitization. Neither Con A nor monensin diminished the ability of the SP response of rNK1R-expressing CHO cells to desensitize.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Desensitization and resensitization of NK1R-expressing CHO cells. CHO cells expressing the rNK1R were pretreated with Con A, monensin, or vehicle. The initial SP concentration induced desensitization and provided an initial Ca2+ response (). The second dose of SP demonstrated the desensitized state of the NKIR (). Cells were desensitized with SP and allowed to recover for 30 () or 60 min (). A, illustrates data as a percentage of the ionomycin response. B, each response was taken as a percentage of the initial dose, so that for each condition, vehicle, Con A, or monensin, the initial response was set to 100%. Each column is the average of at least 10 samples. Under control conditions, a 5-min exposure to SP desensitized the rNK1R-expressing cells. There was a return of SP responsiveness after the 30- and 60-min recovery periods. Cells treated with monensin desensitized but did not resensitize. Treatment with Con A did not effect desensitization but decreased resensitization.

Resensitization of rNK1 Receptor Responsiveness to SP. Resensitization was observed as a return of the ability of SP to induce a Ca²⁺ response after desensitization. To measure resensitization, desensitization was first induced by a 5-min exposure to 100 nM SP. SP was removed, and the cells were allowed to recover for 30 or 60 min at 37°C. The SP responses after the 30- and 60-min recovery period are represented by the single-hatched and open columns, respectively, in Fig. 2. Under control conditions, SP responsiveness returned to 60 ± 2% of the initial SP response after a 30-min recovery period and to 75 ± 3% after a 60-min recovery. There was a significant increase (p < 0.05) in SP responsiveness after the recovery periods compared with the initial desensitized state. There was also a significant increase (p < 0.05) between the 30- and 60-min recovery periods. When the cells were treated with Con A, there was not a significant increase in SP responsiveness after 30 min compared with the desensitized state. There was a significant increase (p < 0.05) in responsiveness from the desensitized state after the 60-min recovery period in cells treated with Con A. The response of monensin-treated cells to SP did not resensitize after either recovery period.

SP-Induced Internalization of rNK1 Receptors. Desensitization and resensitization represent functional changes of the NK1R. It has been suggested that receptor translocation events may underlie these functional changes (Grady et al., 1995; Garland et al., 1996). Internalization of receptors may lead to desensitization of GPCRs, although desensitization of angiotensin II and dopamine D1 receptors have been shown to occur independently of internalization (Thekkumkara et al., 1995; Ng et al., 1995). Internalization and subsequent receptor recycling are thought to lead to agonist degradation and receptor dephosphorylation, which may permit resensitization.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Radioligand binding after Con A, monensin, and vehicle treatment. CHO cells stably expressing the rNK1R were pretreated with Con A, monensin, or vehicle. SP was added to induce internalization (). The single-hatched and open columns represent the 30- and 60-min recovery period, respectively. Data are expressed as the percentage of maximum binding (binding obtained from untreated cells). Each column represents a sample number of at least 15. SP caused a 46 ± 4% decrease in plasma membrane binding after 5 min, which is interpreted as internalization. An increase in receptor binding was not evident after either the 30- or 60-min recovery periods. Monensin-treated cells elicited similar responses as control cells, in that receptors were internalized but not recycled after the 60-min recovery period. Con A inhibited internalization, in that 101 ± 8% of the receptors remained at the plasma membrane.

NK1 Receptor Recycling. Resensitization was evident in CHO cells expressing rNK1Rs within 30 min after the removal of agonist (Fig. 2). We have examined whether this correlates with a return of receptors to the plasma membrane. Transfected CHO cells were exposed to SP (100 nM) for 5 min to induce internalization (Fig. 3, cross-hatched columns). The agonist was removed and the cells were allowed to recover for 30 or 60 min. The amount of radiolabeled SP binding was then measured, as represented by the single-hatched and open columns, respectively, in Fig. 3.

Agonist-Independent Effects of Con A and Monensin. In the studies presented above, the effects of Con A and monensin on the responses to SP were assessed. To test for the possibility that treatment with either of these compounds produced alterations in receptor number in the absence of agonist activation, the agonist-independent effects of Con A and monensin on membrane receptor binding have also been assessed.

Effectiveness of Agonist Washout. The Con A binding experiments demonstrated that SP was sufficiently removed during washout to prevent competition with radiolabeled SP in the binding assays. When the cells were treated with Con A for 20 min but not SP, binding was 105 ± 7%. Binding was 101 ± 8% when cells were pretreated with Con A and SP. Con A inhibits internalization, thus SP would not be internalized or degraded in Con A-treated cells. Because the percentage of binding of the radioligand was not different in these two experiments, the excess unlabeled SP was removed during the rinses and did not compete with radiolabeled SP in the binding assay.

Visualization of NK1 Receptors. In the receptor binding studies, the labeled receptors are those that are present on the plasma membrane after the experimental manipulations. We have recently shown that SP labeled with the fluorescent probe Oregon Green 488 mimics the actions of SP at the NK1R (Bennett and Simmons, 2001). OG-SP provides a means to visualize agonist-bound NK1Rs.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Effects of Con A and monensin on rNK1R internalization. OG-SP was added to vehicle-, Con A-, or monensin-treated rNK1R-expressing CHO cells for 5 min to induce internalization. Cells were rinsed and fixed. Untreated cells (A), cells treated with OG-SP at 4°C (B), cells treated with vehicle and OG-SP at 37°C (C), cells treated with monensin and OG-SP at 37°C (D), and cells treated with Con A and OG-SP at 37°C (E). F, cells treated with unlabeled SP at 37°C and poststained with OG-SP at 4°C to label membrane receptors. These receptors represent noninternalized receptors after agonist stimulation. Images were taken at a gain of 2 s and a 7-s exposure time. As seen in the radioligand binding assay, Con A, but not monensin, prevented internalization. Some receptors remained at the plasma membrane after the SP incubation, which was also observed in the binding assay.

View larger version (53K): [in this window] [in a new window]   Fig. 5.   Effects of Con A and monensin on rNK1R recycling. Pretreated CHO cells expressing the rNK1R were exposed to unlabeled SP to induce internalization and then allowed to recover without agonist for 30 min (A-C) or 60 min (D-F). Cells were poststained with OG-SP at 4°C to stain only plasma membrane receptors. A and D, cells exposed to SP and vehicle. B and E, cells treated with SP and monensin. C and F, cells treated with SP and Con A. Images were taken at a gain of 2- and a 7-s exposure time. As seen in the radioligand binding assay, receptors treated with Con A were still accessible to agonist after the recovery periods. Monensin treatments were not different than control conditions, in that receptors were not recycled within 60 min.

NK1 Receptor Phosphorylation. One of the primary molecular events that has been shown to underlie GPCR desensitization is receptor phosphorylation. Likewise, dephosphorylation has been suggested to lead to receptor resensitization. To better correlate the relationships between desensitization, internalization, resensitization, and recycling, we studied the effects of Con A and monensin on NK1R phosphorylation and dephosphorylation. CHO cells expressing rNK1Rs were incubated with radiolabeled orthophosphate, and the NK1Rs were successfully isolated from the CHO cells and ran at a similar size as observed previously (Roush et al., 1999). After a 5-min exposure, SP significantly increased (p < 0.05) the amount of NK1R phosphorylation to 3-fold above basal levels (Fig. 6, cross-hatched columns). The NK1R antagonist RP 67,580 decreased the amount of receptor phosphorylation after the 5-min exposure to SP. Neither monensin nor Con A significantly changed the amount of agonist-induced phosphorylation compared with vehicle-treated cells. After the recovery period, receptor phosphorylation returned to near basal levels in both vehicle- and monensin-treated cells (Fig. 6, open columns).

View larger version (52K): [in this window] [in a new window]   Fig. 6.   Phosphorylation of rNK1R-expressing CHO cells. A, basal phosphorylation, obtained from untreated receptors, is shown in lane 1. Lanes 2, 3, and 4 represent cells treated with SP and vehicle, monensin or Con A, respectively. Phosphorylation of vehicle-treated, but not SP-treated, receptors that were allowed to recover for 60 min is shown in lane 5. Lanes 6, 7, and 8 depict receptors treated with vehicle, Con A, or monensin, respectively, and SP for 5 min then allowed to recover for 60 min without agonist. B, agonist-independent effects of monensin and Con A on receptors that mimic the 5-min agonist treatment are shown in lanes 1 and 2, respectively. The effects of monensin and Con A after a 60-min recovery are shown in lanes 3 and 4, respectively. C, fraction of basal phosphorylation was calculated for each treatment and plotted above. The cross-hatched columns represent cells treated with vehicle or SP for 5 min without recovery. The open columns represent cells treated with SP and allowed to recover for 60 min. The NK1R antagonist RP 67,580 (RP) was added before SP exposure to test the specific actions of SP at the receptor. Columns represent the average from at least three experiments. SP caused a 3-fold increase in receptor phosphorylation from baseline, which was inhibited by the antagonist RP 67,580. The amount of agonist-induced phosphorylation decreased back to basal levels after the recovery period. Neither monensin nor Con A significantly altered the amount of phosphorylation. Monensin also did not alter NK1R dephosphorylation. The effects of Con A on NK1R dephosphorylation could not be determined (ND) due to the agonist-independent effects of Con A after the 60-min recovery period.

	Discussion

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The purpose of this study was to understand the relationships between desensitization, resensitization, receptor phosphorylation, internalization, and recycling of the NK1R after agonist activation. Desensitization and resensitization were measured as the ability of activated NK1Rs to elicit Ca²⁺ responses. Radioligand binding was used to measure internalization and recycling of NK1Rs. Con A and monensin were used to investigate the mechanisms of the above-mentioned processes.

Con A irreversibly cross-links glycosylated proteins, which is thought to inhibit receptor translocation (Beaumont et al., 1998). After application of SP in the presence of Con A, the response to SP desensitized, but the receptors remained localized at the cell membrane. After a 30-min recovery period, the Con A-treated receptors remained desensitized. After a 60-min recovery period a return of SP responsiveness was evident, although not as great as that observed under control conditions. Con A inhibited internalization but did not inhibit desensitization, which suggests that NK1R desensitization is independent of receptor internalization.

Monensin, a sodium ionophore, prevents the decrease in pH inside endosomes and has been shown to prevent recycling of ₂-adrenergic and somatostatin receptors (Pippig et al., 1995; Beaumont et al., 1998). In our experiments, plasma membrane binding was not different when cells were treated with monensin compared with control conditions. Monensin had no effect on desensitization or internalization but blocked receptor resensitization.

It is currently thought that receptor phosphorylation leads to desensitization, whereas dephosphorylation is an important step in recycling to the cell membrane. We have investigated the effects of Con A and monensin on rNK1R phosphorylation and dephosphorylation to correlate these processes with the effects on receptor translocation. SP induced a 3-fold increase in phosphorylation, which was inhibited by the rNK1R antagonist RP 67,580. After a 60-min recovery period, rNK1Rs were dephosphorylated. Neither monensin nor Con A altered rNK1R phosphorylation compared with vehicle-treated receptors. Monensin did not alter the ability of rNK1Rs to dephosphorylate. Con A elicited agonist-independent effects after the hour recovery period, which prevented us from determining the effect of Con A on NK1R dephosphorylation.

The results show that rNK1R desensitization, internalization, and phosphorylation occur within 5 min of agonist exposure. Receptor dephosphorylation and resensitization were evident within 1 h after agonist removal, but there was not an increase in cell membrane binding. In addition, monensin-treated receptors were dephosphorylated after the recovery period but were not resensitized, which suggests that receptor dephosphorylation is not the sole determinant of rNK1R resensitization. The monensin data also suggest that endosome acidification is not required for receptor dephosphorylation.

It has been suggested that resensitization of NK1Rs requires internalization and recycling (Garland et al., 1996). In that study, bafilomycin A, an acidotropic agent similar to monensin, was shown to inhibit resensitization. It had previously been shown that bafilomycin and other acidotropes, including monensin, prevented NK1R recycling (Grady et al., 1995). It was concluded that NK1R resensitization is dependent on recycling, however, the time periods for the study of resensitization and recycling were not the same. Receptor recycling was demonstrated after a 240-min recovery period without agonist, whereas resensitization was observed after 30 min without agonist.

We also observed NK1R resensitization after 30 min and saw an additional increase after 60 min without agonist; however, there was not an increase in plasma membrane binding after either the 30- or 60-min recovery period. We, therefore, conclude that the NK1R resensitization observed under our conditions is, at least to some extent, independent of receptor recycling.

Although our data suggest that NK1R resensitization does not require recycling, they do suggest that internalization is important. Con A inhibited internalization and decreased resensitization. Schmidlin et al. (2001) have reported similar results. They observed that NK1R resensitization was inhibited when dominant negative mutants of dynamin 1 and Rab5a were expressed to alter NK1 receptor trafficking. In unstimulated cells, dynamin 1 was colocalized with NK1Rs at the plasma membrane, whereas Rab5a was in the cytosol. After exposure to agonist dynamin 1 remained localized at the membrane, whereas Rab5a was found in endosomes along with NK1Rs. The dynamin 1 mutant caused NK1Rs to be retained at the plasma membrane. The Rab5 mutant prevented internalization of NK1Rs from superficial regions to perinuclear regions. Neither of the mutants altered desensitization, but both inhibited receptor resensitization. Both our data and that obtained by Schmidlin et al. (2001) suggest that internalization is not required in NK1R desensitization but is important in resensitization. In addition, we found that monensin, which prevents the acidification of endosomes, also prevented resensitization. Taken together, these data suggest that internalization and endosome acidification are important for resensitization.

Recently, Gray et al. (2001) reported that resensitization of 5-HT_2A receptors was independent of receptor internalization. They also investigated the dependence of resensitization on internalization in ₂-adrenergic receptors. Interestingly, the two receptor types showed different resensitization mechanisms. ₂-Adrenergic receptor resensitization required internalization, but 5HT_2A receptor resensitization did not. They also found that expression of a dominant-negative dynamin mutant and a truncated arrestin-2 mutant, both of which prevent internalization, did not hinder resensitization in human embryonic kidney-293 cells. On the other hand, in C6 glioma cells the mutants prevented 5-HT_2A receptor resensitization. Based on these results, they suggested a cell-surface mechanism for 5-HT_2A receptor resensitization in human embryonic kidney-293 cells that was not present in C6 glioma cells.

Our data show that there is an increase in function (resensitization) of rNK1Rs without an increase in plasma membrane binding (recycling); therefore, rNK1R resensitization does not require recycling. Our data and data from other laboratory groups suggest that internalization is important for NK1R resensitization. In addition, we found that resensitized receptors were dephosphorylated, which suggests a role for dephosphorylation in NK1R resensitization. Dephosphorylation alone does not bring about resensitization because monensin-treated cells were dephosphorylated but not resensitized. These results show that the return of NK1R responsiveness after desensitization is not accounted for by recycling of receptors back to the cell membrane.

The finding that NK1R binding and functional responses are not strictly correlated suggests that there are other components besides the receptor itself involved in the desensitization and resensitization of the response to SP. There are a number of potential sites at which this regulation could occur, the G proteins, phospholipase C, and the inositol trisphosphate receptor. Monensin and Con A could have effects at these alternative sites, independent of their effects on receptor translocation.

The goal of our study was to determine the effects of Con A and monensin on desensitization, resensitization, receptor phosphorylation, internalization, and recycling to better understand the relationships between these processes and their importance in regulating the NK1R. Interestingly, in addition to understanding the effects of Con and monensin, we discovered that NK1R resensitization occurs without an increase in receptor binding after internalization; therefore, NK1R resensitization precedes receptor recycling.

This reveals a novel mechanism of NK1R resensitization that does not require receptor recycling, but in which internalization is important. Further data are needed to investigate the role of internalization and endosome acidification and to determine the mediators involved in the resensitization pathway. Understanding the process of NK1R resensitization could yield novel drug developments for treatment of pain perception, inflammation, and depression, especially if the mechanism or portions of the mechanism are unique to the NK1R.