Protein kinase C-mediated desensitization of the neurokinin 1 receptor

Olivier Déry, Kathryn A. Defea, and Nigel W. Bunnett

Departments of Surgery and Physiology, University of California San Francisco, San Francisco, California 94143-0660


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An understanding of the mechanisms that regulate signaling by the substance P (SP) or neurokinin 1 receptor (NK1-R) is of interest because of their role in inflammation and pain. By using activators and inhibitors of protein kinase C (PKC) and NK1-R mutations of potential PKC phosphorylation sites, we determined the role of PKC in desensitization of responses to SP. Activation of PKC abolished SP-induced Ca2+ mobilization in cells that express wild-type NK1-R. This did not occur in cells expressing a COOH-terminally truncated NK1-R (NK1-Rdelta 324), which may correspond to a naturally occurring variant, or a point mutant lacking eight potential PKC phosphorylation sites within the COOH tail (NK1-R Ser-338, Thr-339, Ser-352, Ser-387, Ser-388, Ser-390, Ser-392, Ser-394/Ala, NK1-RKC4). Compared with wild-type NK1-R, the t1/2 of SP-induced Ca2+ mobilization was seven- and twofold greater in cells expressing NK1-Rdelta 324 and NK1-RKC4, respectively. In cells expressing wild-type NK1-R, inhibition of PKC caused a 35% increase in the t1/2 of SP-induced Ca2+ mobilization. Neither inhibition of PKC nor receptor mutation affected desensitization of Ca2+ mobilization to repeated challenge with SP or SP-induced endocytosis of the NK1-R. Thus PKC regulates SP-induced Ca2+ mobilization by full-length NK1-R and does not regulate a naturally occurring truncated variant. PKC does not mediate desensitization to repeated stimulation or endocytosis of the NK1-R.

substance P; tachykinins; downregulation; G protein-coupled receptors


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BIOLOGICAL RESPONSES TO AGONISTS of G protein-coupled receptors (GPCRs) for neurotransmitters and hormones are closely regulated by mechanisms that operate at the level of the ligands and their receptors (4, 24). These mechanisms prevent uncontrolled stimulation, which may otherwise lead to disregulation and disease. At the level of the ligand, the balance between release and uptake or degradation determines the availability of agonists to interact with cell surface receptors. At the level of the receptor, several mechanisms terminate signal transduction. These mechanisms include homologous desensitization to repeated application of the same agonist, heterologous desensitization to repeated challenge with different agonists, and receptor endocytosis. Desensitization is principally mediated by uncoupling receptors from heterotrimeric G proteins. G protein receptor kinases (GRKs) and second messenger kinases such as protein kinase C (PKC) and protein kinase A play important roles in desensitization (see Ref. 4 and references therein). Agonists of many GPCRs induce translocation of GRKs and second messenger kinases to the plasma membrane where they phosphorylate activated receptors. beta -Arrestins also translocate to the plasma membrane where they interact with GRK-phosphorylated receptors and thereby uncouple them from G proteins to mediate desensitization. beta -Arrestins also couple receptors to clathrin and thereby mediate endocytosis, which contributes to desensitization by depleting GPCRs from the plasma membrane (14, 19). These regulatory mechanisms have been characterized in detail for only a few GPCRs, and the relative importance of these processes differs from one receptor to another (4). Although truncation of the intracellular COOH tails and mutation of potential phosphorylation sites within these regions can impair uncoupling and endocytosis of certain GPCRs (2, 3, 5, 7, 18, 25, 34, 35, 38), the receptor domains that are critical for these processes have not been fully characterized.

We investigated regulation of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P (SP). An understanding of regulation of the NK1-R is of interest because this receptor mediates neurogenic inflammation, nociception, and the regulation of gastrointestinal secretion and motility (30). Biological responses to SP are rapidly attenuated in the continued presence of agonists and diminish to repeated challenge, indicating that the NK1-R rapidly desensitizes (8, 16, 28). However, the mechanisms of this desensitization are incompletely understood. GRKs and beta -arrestins may mediate desensitization, because GRK-2/3 extensively phosphorylates the NK1-R (23), and SP induces translocation of GRK-2/3 and beta -arrestins 1/2 from the cytosol to the plasma membrane where they interact with the NK1-R (1, 28, 29). PKC also participates, because activators of PKC induce phosphorylation of the NK1-R and inhibit SP signaling (32, 40), and SP stimulates membrane translocation of PKCbeta 2 (1, 27). However, the domains of the NK1-R that are important for uncoupling and endocytosis are not fully defined. The COOH-terminal tail of the NK1-R contains 26 serine and threonine residues that could be phosphorylated by GRKs or PKC and which may interact with beta -arrestins. Truncation of the COOH tail to remove some of these sites diminishes SP-induced desensitization and endocytosis of the NK1-R (5, 25, 33). Since a naturally occurring variant of the NK1-R lacking most of the COOH tail has been identified (9, 15, 22), an understanding of the importance of the COOH tail in receptor regulation is of considerable interest.

We examined the role of PKC in SP-induced uncoupling and endocytosis of the NK1-R by using activators and inhibitors of PKC and by receptor mutation to remove putative PKC phosphorylation sites with the COOH tail. Our aims were to 1) generate cell lines expressing truncation mutants of the NK1-R and point mutants of potential PKC sites; 2) determine the consequences of mutation for uncoupling and endocytosis of the NK1-R; and 3) investigate the effects of PKC activators or inhibitors on NK1-R signaling and endocytosis. Our results show that PKC plays an important role in attenuating SP-induced Ca2+ mobilization.


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Reagents. SP was from Phoenix Pharmaceutical (Belmont, CA). [125I]SP (2,000 Ci/mmol) was from Amersham Pharmacia Biotech (Uppsala, Sweden). Phorbol 12,13 dibutyrate (PDBu), 4alpha -phorbol 12,13-didecanoate (PDD), GF-109203X, and bisindolylmaleimide V were from Calbiochem (San Diego, CA). Fura 2-AM, pluronic acid, and propidium iodide were from Molecular Probes (Eugene, OR). The synthesis of cyanine 3.8-labeled SP (Cy3-SP) has been described (10). The monoclonal M2 antibody directed against the FLAG epitope (DYKDDDDK) was from International Biotechnologies (New Haven, CT). Goat-anti-mouse IgG labeled with FITC was from Caltag Laboratories (Burlingame, CA). GeneEditor in vitro site-directed mutagenesis system was from Promega (Madison, WI). The expression vector pcDNA3 was from Invitrogen (Carlsbad, CA). Oligonucleotides were from Genemed Biotechnologies (San Francisco, CA). Lipofectin, DMEM, and PBS were from Life Technologies (Gaithersburg, MD). G418 was from Gemini Bio-Products (Calabasas, CA). Kirsten sarcoma virus-transformed rat kidney epithelial cells (KNRK) were from American Type Culture Collection (Rockville, MD). Other reagents were from Sigma Chemical (St. Louis, MO).

Description of NK1-R mutants. Of the 99 residues in the COOH tail of rat NK1-R, 26 are serine and threonine. We truncated the NK1-R to remove portions of the COOH tail (Fig. 1). NK1-Rdelta 324, the most truncated mutant that may correspond to a naturally occurring variant, lacks 83 of the COOH-terminal residues, including all serine and threonine. NK1-Rdelta 342 and NK1-Rdelta 354, respectively, lack 65 and 53 residues and possess only 3 and 7 serine and threonine residues. The NK1-R COOH tail includes two serine/threonine clusters, one in region 338-360 close to the transmembrane domain, and the other near the COOH terminus in region 376-403. NK1-Rdelta 342 lacks both clusters, and NK1-Rdelta 354 lacks the cluster 338-360. Point mutations of full-length NK1-R were made to disrupt potential PKC phosphorylation sites (Fig. 1). Because phosphorylation by PKC requires the presence of a basic residue near phosphorylated serine and threonine, we considered potential phosphorylation sites to be serine or threonine residues adjacent to, or one residue from, arginine or lysine. We mutated 8 of the 26 serine and threonine residues in the COOH tail. In NK1-RKC1, Ser-338 and Thr-339 were mutated to alanine. In NK1-RKC2, we mutated Ser-352, which lies within a stretch of 12 residues between NK1-Rdelta 342 and NK1-Rdelta 354. In NK1-RKC3, Ser-387, -388, -390, -392, and -394 were mutated to alanine. In NK1-RKC4, all of the 8 mutations of NK1-RKC1, NK1-RKC2, and NK1-RKC3 were introduced.


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Fig. 1.   Truncation and point mutants in the intracellular COOH tail of the rat neurokinin 1 receptor (NK1-R). Filled circles denote mutated residues.

Generation of NK1-R mutants. All mutants were derived from rat NK1-R with the NH2-terminal FLAG epitope. This construct was subcloned into pcDNA3 to generate the wild-type NK1-R (NK1-Rwt). The FLAG epitope does not affect signaling, desensitization, or trafficking of the NK1-R (39). Truncation mutants were generated by introduction of a stop codon after residues position 324, 342, and 354 to form NK1-Rdelta 324, NK1-Rdelta 342, and NK1-Rdelta 354 as described (5). Potential PKC consensus sites in the COOH tail were mutated using a GeneEditor site-directed mutagenesis kit. The primers used to obtain the mutants were KC1, 5'-GGGCTGGAAATGAAAGCCGCCCGGTACCTCCAGACA-3', which carries the mutations Ser-338, Thr-339/Ala (bold), and a KpnI restriction site (underlined); KC2, 5'-CAGAGCAGCGTATACAAGGTCGCCCGCCTGGAGACCACC-3', Ser-352/Ala mutation (bold), AccI site (underlined); and KC3, 5'-CTCACCTCCAACGGCGCCGCTCGAGCCAACGCCAAGGCCATGACAGAAAGCTCC-3', Ser-387, Ser-388, Ser-390, Ser-392, Thr-394/Ala (bold), XhoI site (underlined). The mutant KC4 was obtained using all three primers and contained all the corresponding mutations. After transformation of JM109 bacteria, clones were screened by restriction analysis and DNA sequencing.

Transfection and cell culture. KNRK cells expressing wild-type and truncated NK1-Rs were generated and characterized as described (13, 17, 29, 39). KNRK cells were transfected with cDNAs encoding the point mutants using Lipofectin. After 7-10 days, cells expressing high levels of receptor were sorted using flow cytometry (13, 29). A minimum of 24 clones was transferred in 24-well plates on coverslips and analyzed for expression by immunofluorescence using the FLAG M2 antibody and by binding of Cy3-SP. Selected clones were maintained in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.4 mg/ml G418. Clones were selected to express similar levels of NK1-R, as determined by binding experiments using radiolabeled SP, binding of Cy3-SP, and flow cytometry using the FLAG antibody.

Measurement of [Ca2+]i. NK1-R signaling was assessed by measuring SP-induced Ca2+ mobilization (16). Cells were incubated with 2.5 µM fura 2-AM and 0.2% pluronic acid for 20 min at 37°C, washed, and fluorescence was measured at 340- and 380-nm excitation and 510 nm emission in a spectrophotometer (F-2000; Hitachi Instruments, Irvine, CA). The ratio of the fluorescence at the two excitation wavelengths, which was proportional to the intracellular Ca2+ concentration [Ca2+]i, was calculated. For concentration-response analyses, cells were exposed to a single application of graded concentrations of SP. To determine the EC50, the sigmoidal shape of the dose-response curve was transformed by a logit/log representation where logit (response) = log[response/(1-response)] is plotted against log(dose). A linear regression was used to obtain the best fit, and the EC50 was calculated as the concentration for which the logit = 0, corresponding to the inflection point of the sigmoidal curve. To examine desensitization, cells were exposed to a first challenge of SP or vehicle (control) for 2 min, washed, and were then reexposed to a second challenge of SP 5 min after the first. Observations were from more than three experiments.

Binding and endocytosis of [125I]SP. The rate of internalization of the NK1-R was determined by binding assays with [125I]SP (17). Cells were incubated in Hanks' balanced salt solution with 0.1% BSA and 50 pM [125I]SP for 1 h at 4°C, washed, and incubated at 37°C for 0 to 30 min. Cells were washed with ice-cold PBS and incubated in 250 µl of ice-cold 0.2 M acetic acid containing 0.5 M NaCl (pH 2.5) on ice for 5 min to separate acid-labile (cell surface) from acid-resistant (internalized) label. Radioactivity was counted in the acid fraction, and the fraction internalized in the cells was first detached by lysing the cells with 0.5 N NaOH overnight at 4°C. Nonspecific binding was measured by preincubation of the cells with 1 µM SP and subtracted to obtain specific binding. Observations were in triplicate in n > 3 experiments.

Fluorescence microscopy. To localize the NK1-R and examine endocytosis, we used Cy3-SP (20, 29). Cells were incubated in DMEM with 0.1% BSA containing 10-100 nM Cy3-SP for 1 h at 4°C for equilibrium binding. They were washed in DMEM-BSA at 4°C and either fixed immediately or incubated in SP-free medium at 37°C for various times to permit receptor endocytosis and trafficking to proceed. Cells were fixed with 4% paraformaldehyde in PBS, pH 7.4, for 20 min at 4°C, washed, and mounted. Cells were observed with an MRC 1000 laser scanning confocal microscope (Bio-Rad Laboratories, Hercules, CA).

Statistical analysis. Results are expressed as means ± SE and are compared with Student's t-test or ANOVA and Student-Newman-Keuls test, with P < 0.05 considered significant.


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Truncated and mutant receptors were functional. We verified that all cell lines expressed functional receptors by measuring SP-induced Ca2+ mobilization. SP stimulated a similar rapid increase in [Ca2+]i in cell lines expressing wild-type, truncated, and point mutant NK1-Rs with comparable efficacies (not shown). In cells expressing NK1-Rwt, SP induced a Ca2+ response with an EC50 of 0.6 nM (Fig. 2). In cells expressing the truncated receptors, EC50 was 0.2, 0.1, and 0.2 nM for the delta 324, delta 342, and delta 354, respectively (Fig. 2A). We obtained similar results with the point mutants for which the EC50 of KC1, KC2, KC3, and KC4 were 0.2, 0.1, 0.1, and 0.1 nM, respectively (Fig. 2B). Thus SP stimulates Ca2+ mobilization in cells expressing truncated and point mutant NK1-Rs, which lack serine and threonine residues in their COOH tails, with three- to sixfold higher potency than in cells expressing NK1-Rwt.


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Fig. 2.   Substance P (SP)-induced Ca2+ mobilization in KNRK cells expressing wild-type (wt) and mutant NK1-R. Cells were exposed to single concentrations of SP. Results are expressed as a percentage of the maximal response to SP and are means of n = 3 observations. A: concentration-response curves for wild-type NK1-R (NK1-Rwt) and the truncated mutants delta 324, delta 342, and delta 354. B: concentration-response curves for NK1-Rwt and point mutants KC1, KC2, KC3, and KC4. A logit/log representation was used to fit curves. [Ca2+]i, intracellular Ca2+ concentration.

PKC inhibits SP-induced Ca2+ mobilization. Activation of PKC with phorbol esters strongly inhibits signaling by several GPCRs (18, 34, 35, 37, 38, 40). To determine the role of PKC in regulating the NK1-R, we preincubated cells with 1 µM PDBu for 10 min, which activated the classical and novel subtypes of PKC, and then challenged cells with SP at concentrations close to the EC50 (0.1-0.3 nM), which similarly increased [Ca2+]i. In cells expressing NK1-Rwt, PDBu abolished SP-induced Ca2+ mobilization (Fig. 3A and Fig. 4). This effect of PDBu was prevented by preincubation with 0.1 µM GF-109203X for 20 min, which inhibits PKC. Preincubation with GF-109203X alone did not affect the magnitude of SP-induced Ca2+ mobilization. The inactive enantiomers PDD and bisindolylmaleimide V had no effect (not shown). Thus in cells expressing NK1-Rwt, activation of PKC prevents SP-induced Ca2+ mobilization.


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Fig. 3.   Effects of activation and inhibition of protein kinase C (PKC) on SP-induced Ca2+ mobilization. Cells were preincubated with vehicle (control), 1 µM phorbol 12,13-dibutyrate (PDBu) for 10 min, 0.1 µM GF-109203X for 20 min, or both PDBu and GF-109203X and challenged with SP at a concentration close to the EC50 (0.1 nM SP for NK1-Rwt, delta 324, delta 342, delta 354, KC2, and KC3; 0.3 nM SP for KC1 and KC4). A: responses for NK1-Rwt and truncated mutants delta 324, delta 342, and delta 354. B: responses for NK1-Rwt and point mutants KC1, KC2, KC3, and KC4. Lanes 1, no pretreatment; lanes 2, pretreatment with PDBu; lanes 3, pretreatment with PDBu and GF-109203X; lanes 4, pretreatment with GF-109203X. Results are the maximal responses to SP and are expressed as percentage of Ca2+ response without pretreatment; n = 3 observations. *P < 0.05 compared with control (lanes 1); dagger P < 0.05 compared with pretreatment with PDBu (lanes 2).



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Fig. 4.   Effects of activation and inhibition of PKC on SP-induced Ca2+ mobilization in cells expressing NK1-Rwt, delta 324, or KC4. Cells were preincubated with vehicle (control), 10 µM PDBu for 10 min, or 0.1 µM GF-109203X for 20 min and challenged with SP (arrows) at a concentration close to the EC50 (0.1 nM SP for NK1-Rwt, delta 324; 0.3 nM SP for KC4). Representative traces from triplicate observations are shown.

To identify receptor domains that are required for regulation of SP-induced Ca2+ mobilization by PKC, we studied the effects of activators and inhibitors of PKC in cells expressing mutant receptors. One group of mutants behaved almost identically to NK1-Rwt. In cells expressing NK1-RKC2 and NK1-RKC3, as in cells expressing NK1-Rwt, PDBu strongly inhibited SP-induced Ca2+ mobilization (Fig. 3B). Pretreatment with GF-109203X fully reversed this inhibition in cells expressing NK1-RKC1, confirming involvement of PKC. GF-109203X only partially reversed PDBu-induced inhibition in cells expressing NK1-RKC2, but the reason for this partial reversal is unknown. A second group of mutant receptors was not affected by the activation of PKC. In cells expressing NK1-Rdelta 324, delta 342, and KC4, in marked contrast to cells expressing NK1-Rwt, PDBu had no effect on SP-induced Ca2+ mobilization (Fig. 3, A and B, and Fig. 4). GF-109203X alone had no effect on SP-induced Ca2+ mobilization in these cells. A third group of mutant receptors showed an intermediate response to activation of PKC. In cells expressing NK1-Rdelta 354 and KC1, PDBu reduced, but did not abolish, SP-induced Ca2+ mobilization (Fig. 3, A and B). Thus domains in the COOH tail of the NK1-R that are absent in the truncation mutants NK1-Rdelta 324 and delta 342 and the point mutant KC4, but partially present in NK1-Rdelta 354 and KC1, are necessary for PKC-induced inhibition of SP-induced Ca2+ mobilization.

PKC partially mediates desensitization of SP-induced Ca2+ mobilization. Homologous desensitization of GPCRs is manifested by the attenuation of responses in the continued presence of agonists and by diminished responses to repeated application of the same agonist (4). To determine the role of PKC in these processes, we examined the duration of SP-induced Ca2+ mobilization and desensitization of Ca2+ transients to repeated challenge with SP in cells expressing wild-type and mutated NK1-R in the presence or absence of PKC inhibitors. We compared cells expressing NK1-Rwt with those expressing NK1-Rdelta 324 or KC4, since these mutants exhibited the most marked differences in responsiveness to PDBu compared with NK1-Rwt.

To evaluate the effects of PKC inhibitors on the duration of the Ca2+ response, cells expressing NK1-Rwt, delta 324, or KC4 were challenged with 1 nM SP with or without preincubation with GF-109203X (0.1 µM, 20 min). The half-life for [Ca2+]i to return to prestimulated levels was measured. In cells expressing NK1-Rwt, the t1/2 was 8.8 ± 0.3 s in the absence and 12.9 ± 0.4 s in the presence of GF-109203X (P = 0.003; Fig. 4). In marked contrast, in cells expressing NK1-Rdelta 324, the t1/2 was 66 ± 12 s in the absence and 66 ± 5 s in the presence of GF-109203X (P = 1; Fig. 4). Similarly, in cells expressing NK1-RKC4, the t1/2 was 16.5 ± 1.0 s in the absence and 15.0 ± 1.0 s in the presence of GF-109203X (P = 0.34; Fig. 4). Thus inhibition of PKC prolongs SP-induced Ca2+ mobilization by NK1-Rwt by 35%, but not by NK1-Rdelta 324 or KC4, which were unresponsive to activation of PKC. Notably, SP-induced Ca2+ mobilization was prolonged sevenfold in NK1-Rdelta 324 cells and twofold in NK1-RKC4 cells, compared with cells expressing NK1-Rwt.

We also evaluated desensitization of the NK1-Rwt, delta 324, and KC4 to repeated challenge with graded concentrations of SP. Cells were exposed to SP (0.01-100 nM) or vehicle (control) for 2 min, washed, and were challenged again with SP (1 nM) 5 min after the first challenge. SP stimulated Ca2+ mobilization with varying potencies for NK1-Rwt, delta 324, and KC4 (see Fig. 2). To allow comparisons between these receptors, we determined the extent of desensitization of the second challenge of 1 nM SP (expressed as percentage of the response in vehicle-treated control cells) to graded first challenges of SP at concentrations that produced similar Ca2+ responses of all receptors (expressed as percentage of the maximal response; Fig. 5A). SP desensitized NK1-Rwt, delta 324, and KC4 in a concentration-dependent manner. Approximately 50% desensitization was obtained to concentrations of SP that induced 50-65% of the maximal response (Fig. 5A). We have previously reported, in cells expressing NK1-Rwt, that exposure to 10 nM SP has little effect on thapsigargin-induced Ca2+ mobilization (8), indicating that the reduced response to a second SP challenge is due to homologous desensitization of the NK1-R rather than depletion of intracellular Ca2+ stores. Thus NK1-Rdelta 324 and KC4 undergo homologous desensitization to repeated challenge with SP to a similar extent as NK1-Rwt.


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Fig. 5.   Homologous desensitization of Ca2+ mobilization to repeated challenge with SP in cells expressing NK1-Rwt, delta 324, or KC4. A: cells were exposed to vehicle (control) or 0.01-100 nM SP for 2 min, washed, and challenged with 1 nM SP at 5 min after the first challenge. The abscissa shows the magnitude of the Ca2+ response to the first challenge with graded concentrations of SP, expressed as percentage of the maximal response. The ordinate shows the magnitude of the Ca2+ response to the second challenge with 1 nM SP, expressed as percentage of the response in control cells treated with vehicle; n = 3 observations. B: NK1-Rwt cells were preincubated with vehicle (control) or GF-109203X (0.1 µM, 20 min) before challenge with 5 nM SP (arrows). Representative traces from triplicate observations are shown.

To further evaluate the role of PKC in homologous desensitization to repeated challenge with SP, we treated NK1-Rwt cells with GF-109203X (0.1 µM, 20 min) or vehicle (control). In control cells, responses to a second challenge with 5 nM SP were 21 ± 5% of the first response to 5 nM SP (Fig. 5B). In cells treated with GF-109203X, responses to a second challenge with 5 nM SP were 26 ± 11% of the first response to 5 nM SP. Similarly, GF-109203X did not prevent homologous desensitization on NK1-Rdelta 324 or KC4 (not shown). Thus inhibition of PKC does not attenuate homologous desensitization of SP-induced Ca2+ mobilization in cells expressing wild-type NK1-R, NK1-Rdelta 324, or NK1-RKC4.

PKC does not regulate endocytosis of the NK1-R. Agonist-induced endocytosis of GPCRs may contribute to desensitization by depleting receptors from the plasma membrane. For several GPCRs, serine and threonine residues in the COOH tail are required for endocytosis (2, 21). We have previously reported that truncation of the NK1-R at 324 and 342, but not at 354, inhibits SP-induced endocytosis of the NK1-R and that tyrosine residues in the COOH tail may contribute to endocytic motifs (5). Moreover, in cells expressing NK1-Rdelta 324, SP does not cause translocation of beta -arrestin to the plasma membrane, suggesting that NK1-Rdelta 324 does not interact with beta -arrestins, which may explain the impaired endocytosis and desensitization (11). To evaluate the importance of serine and threonine residues in the COOH tail and of the role of PKC, we studied SP-induced endocytosis of NK1-Rwt and point mutants lacking serine and threonine residues in the COOH tail in the presence of the PKC inhibitor GF-109203X.

To quantify the rate of endocytosis, cells were incubated with [125I]SP for 1 h at 4°C, washed, and incubated for 2-30 min at 37°C. Cells were washed with acid to separate cell surface (acid labile) from internalized (acid resistant) label. In NK1-Rwt cells, after 1 h at 4°C, 89 ± 4% of specifically bound SP was at the cell surface and 11 ± 2% was internalized. Warming to 37°C resulted in rapid endocytosis, with 36 ± 4% internalized at 2 min, 68 ± 4% at 5 min, 76 ± 2% at 10 min, and 52 ± 4% at 30 min (Fig. 6). The rate of endocytosis of [125I]SP was the same for NK1-Rwt and for NK1-RKC1, KC2, KC3, and KC4 (Fig. 6). Thus after 5 min at 37°C, 68 ± 4% of specific binding was internalized in cells expressing NK1-Rwt, 52 ± 5% for KC1, 58 ± 2% for KC2, 56 ± 4% for KC3, and 59 ± 2% for KC4. To localize the NK1-R, we incubated cells with Cy3-SP for 1 h at 4°C, washed, incubated cells at 37°C for 15 or 30 min, and observed them by confocal microscopy. In NK1-Rwt cells, after 1 h at 4°C, Cy3-SP was confined to the plasma membrane (Fig. 7). After 15 or 30 min at 37°C, Cy3-SP was detected in endosomes located immediately beneath the plasma membrane and in a perinuclear region, and labeling of the plasma membrane was markedly diminished. At 4°C, Cy3-SP was confined to the plasma membrane of cells expressing point mutant NK1-Rs (Fig. 7). After 15 and 30 min at 37°C, Cy3-SP was detected in superficial and perinuclear endosomes in cells expressing point mutants. Preincubation with 10 µM GF-109203X for 20 min did not affect endocytosis of SP (not shown). Thus mutation of serine and threonine residues at potential PKC sites in the COOH tail of the NK1-R and inhibition of PKC had no effect on SP-induced endocytosis of the NK1-R. These results suggest that PKC does not regulate NK1-R endocytosis.


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Fig. 6.   Endocytosis of [125I]SP in cells expressing NK1-Rwt or point mutants KC1, KC2, KC3, and KC4. Cells were incubated with [125I]SP for 60 min at 4°C, washed, and incubated at 37°C for 0-30 min. Cells were washed with acid to separate cell surface from internalized SP. Results are expressed as the percentage of the specifically bound radioactivity; n = 3 experiments.



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Fig. 7.   Endocytosis of Cy3-SP in cells expressing NK1-Rwt or point mutants KC1, KC2, KC3, and KC4. Cells were incubated with Cy3-SP for 60 min at 4°C, washed, incubated for 0, 15, or 30 min at 37°C, and then fixed. At 4°C, SP was found at the plasma membrane and all cell lines (arrowheads). After 30 min at 37°C, SP was found in superficial or perinuclear endosomes in all cells (arrows). Results are representative of 3 experiments. Scale bar = 10 µm.


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REFERENCES

Acute activation of PKC abolished SP-induced Ca2+ mobilization by NK1-Rwt. In marked contrast, PKC activators did not affect signaling by truncated NK1-Rdelta 324 or by NK1-RKC4 lacking eight potential PKC sites in the COOH tail. Inhibition of PKC prolonged SP-induced Ca2+ mobilization by 35% in cells expressing NK1-Rwt but had no effect in cells expressing NK1-Rdelta 324 or NK1-RKC4. SP-induced Ca2+ mobilization was prolonged by sevenfold in NK1-Rdelta 324 cells and by twofold in NK1-RKC4 cells, compared with cells expressing NK1-Rwt. However, desensitization of Ca2+ responses to repeated application of SP and SP-induced endocytosis were unaffected by NK1-R truncation, deletion of potential PKC sites, or by administration of PKC inhibitors. Thus our results show that PKC plays a major role in determining the magnitude and duration of SP-induced Ca2+ mobilization by NK1-Rwt, and serine and threonine residues within the COOH tail are necessary for this regulation. PKC does not contribute to homologous desensitization or to SP-induced endocytosis of the NK1-R. Importantly, PKC does not regulate signaling by a naturally occurring truncated variant, which is likely to be of functional importance in tissues that predominantly express this receptor.

PKC regulates the magnitude and duration of SP-induced Ca2+ mobilization. Receptor phosphorylation and beta -arrestin-mediated uncoupling from heterotrimeric G proteins principally mediate desensitization of GPCRs. Homologous desensitization is manifested by attenuation of the magnitude and duration of responses after a single challenge with agonist and diminished responsiveness to repeated challenge. Several observations from the present study suggest that PKC contributes to attenuation of the magnitude and duration of SP-induced Ca2+ mobilization. First, in cells expressing NK1-Rwt, PDBu abolished SP-induced Ca2+ mobilization. This effect was due to activation of PKC, since it was reversed by GF-109203X, which selectively inhibits the classical and novel subtypes of PKC, and an inactive enantiomer of PDBu had no effect. Second, GF-109203X increased the duration of the signal by 35% in cells expressing NK1-Rwt. Third, deletion of potential PKC sites in the COOH tail of the NK1-R (notably in NK1-Rdelta 324 and KC4) reversed the inhibitory effect of PDBu and prolonged the Ca2+ response to SP by two- to sevenfold, compared with NK1-Rwt. Finally, deletion of these sites increased the potency of SP signaling by three- to sixfold. This increase in potency may indicate diminished desensitization of Ca2+ signaling or increased affinity for SP. Alternatively, NK1-Rwt may be constitutively phosphorylated on one or more potential PKC sites, the removal of which would lead to more efficient coupling. However, constitutive phosphorylation of the NK1-R has not been observed (32, 40). In support of the conclusion that PKC regulates NK1-R, others have shown that activation of PKC inhibits SP signaling in transfected cells and in acinar cells from the parotid gland that naturally expresses the NK1-R (37, 40). Moreover, activation of PKC inhibits agonist-induced signaling of other GPCRs, including protease-activated receptor 2, gastrin-releasing peptide receptor, thromboxane A2 receptor, purinergic P2Y2 receptor, and angiotensin II type 1A receptor (6, 18, 34, 35, 38, 41). Proteinase-activated receptor 2 resembles NK1-R since mutation of serine and threonine residues in the COOH tail increases the magnitude and duration of signaling and reverses the inhibitory effects of PDBu (6, 12).

To identify domains of the NK1-R that are necessary for regulation by PKC, we studied truncation and point mutants. Of the truncation mutants, PDBu only partially inhibited SP-induced Ca2+ mobilization in cells expressing NK1-Rdelta 354, but had no effect in NK1-Rdelta 342 or NK1-Rdelta 324 cells. Thus the COOH tail between residues 325 and 354 is important for regulation by PKC. Mutation of Ser-338 and Thr-339 (NK1-RKC1) also impaired the ability of PDBu to inhibit SP-induced Ca2+ mobilization, suggesting the importance of Ser-338 and Thr-339 for regulation by PKC. In contrast, mutation of Ser-352 (NK1-RKC3) and of Ser-387, Ser-388, Ser-390, Ser-392, and Thr-394 (NK1-RKC3) had no effect. However, mutation of all of the aforementioned residues (NK1-RKC4) abolished PDBu inhibition of SP-induced Ca2+ mobilization. These results show that several domains within the COOH tail of the NK1-R are required for inhibition of SP-induced Ca2+ mobilization by PDBu. The difference in the extent of PDBu inhibition between NK1-RKC1 (partial inhibition) and NK1-RKC4 (full inhibition) suggests that Ser-338 and Thr-339 are necessary. In addition, Ser-352 is also required because its presence in NK1-Rdelta 354 conveys partial sensitivity to PDBu, and it is absent in NK1-Rdelta 342, which is insensitive to PDBu. Thus our results suggest that Ser-338, Thr-339, and Ser-352 are required for PDBu-induced inhibition of SP signaling.

The most straightforward interpretation of our results is that PKC phosphorylates the COOH tail of the NK1-R (Ser-338, Thr-339, and Ser-352), which disrupts coupling of the NK1-R to signaling proteins. In support of this interpretation, phorbol esters strongly stimulate phosphorylation of the NK1-R (32, 40), and SP induces translocation of PKCbeta 2 to the plasma membrane where it may phosphorylate the NK1-R (1, 27). However, the sites of PKC-induced phosphorylation of the NK1-R remain to be determined directly by phosphopeptide mapping, and the PKC subtype that phosphorylates the NK1-R in functionally important cells is unknown.

PKC does not mediate desensitization of the NK1-R to repeated stimulation. Although PKC can regulate the magnitude and duration of SP-induced Ca2+ mobilization, we found no evidence that PKC mediates desensitization to repeated challenge with SP. Exposure of NK1-Rwt cells to graded concentrations of SP inhibited responses to a second challenge 5 min later, and this desensitization was unaffected by GF-109203X and is thus not mediated by PKC. Furthermore, mutation of potential PKC sites in NK1-Rdelta 324 and KC4 did not prevent desensitization. In support of our results, PDBu phosphorylates the NK1-R at sites that are different from those that are phosphorylated by SP, and PKC inhibitors do not affect SP-stimulated phosphorylation (32, 40). SP-induced phosphorylation of the NK1-R has been observed in KNRK cells used in the present study (Ref. 40 and Bunnett, unpublished observations), although the sites of phosphorylation remain to be determined. The most likely mechanism of homologous desensitization is GRK-stimulated phosphorylation of the NK1-R and interaction with beta -arrestins. In favor of this hypothesis, GRK-2/3 strongly phosphorylate the NK1-R at unknown sites (23), and SP triggers the rapid translocation of GRK-2/3 and beta -arrestins 1/2 from the cytosol to the plasma membrane of transfected cells and neurons that naturally express the NK1-R (1, 28, 29). Second messenger kinases play variable roles in desensitization of other GPCRs. Thus PKC does not mediate desensitization of the gastrin-releasing peptide receptor (41), but protein kinase A can desensitize the beta 2-adrenergic receptor (31).

Our observation that NK1-Rdelta 324, like NK1-Rwt, undergoes normal homologous desensitization to repeated stimulation was unexpected, because others have reported that diminished desensitization of truncated NK1-Rs expressed Xenopus oocytes and Chinese hamster ovary cells (25, 33). The reason for this discrepancy remains to be determined but could be related to levels of expression of GRKs, beta -arrestins, and NK1-Rs in the different cell types or the concentrations of SP used to induce desensitization. However, desensitization was similar for NK1-Rwt, delta 324, and KC4, regardless of the concentration of SP. Thus domains other than the COOH terminus may be necessary for homologous desensitization of the NK1-R.

Comparisons between NK1-Rdelta 324 and KC4 provide insight into the relative importance of PKC and other mechanisms of desensitization. NK1-Rdelta 324 is not sensitive to PKC agonists, and compared with NK1-Rwt, exhibits a sevenfold prolonged Ca2+ transient, does not interact with beta -arrestins (11), and shows diminished endocytosis (5) and homologous desensitization (25, 33). NK1-RKC4 is also insensitive to PKC agonists and exhibits a twofold prolonged Ca2+ transient, yet undergoes normal endocytosis and homologous desensitization compared with NK1-Rwt. As in cells expressing NK1-Rwt, but in contrast to cells expressing NK1-Rdelta 324 (11), SP induces rapid translocation of beta -arrestin 1 from the cytosol to the plasma membrane in cells expressing NK1-RKC4 (Bunnett, unpublished observations). This result suggests that NK1-RKC4 can interact with beta -arrestins and presumably undergo GRK-dependent phosphorylation, which is required for interaction with beta -arrestins. Together, these findings suggest that PKC-independent mechanisms (GRK phosphorylation and beta -arrestin binding) are of principal importance for homologous desensitization of NK1-Rwt.

PKC does not mediate SP-induced endocytosis of the NK1-R. Truncation of the NK1-R inhibits SP-induced endocytosis of the NK1-R, indicating that COOH-terminal domains are necessary for trafficking (5). However, acute administration of GF-109203X did not affect endocytosis of NK1-Rwt, and endocytosis of SP proceeded at the same rate in cells expressing NK1-R mutants lacking potential PKC sites within the COOH tail compared with NK1-Rwt. Therefore, PKC does not participate in SP-induced endocytosis of the NK1-R. In support of our results, PKC does not regulate endocytosis of the purinergic P2Y2 receptor (18). In addition to their role in uncoupling GPCRs from heterotrimeric G proteins, beta -arrestins also serve to couple receptors to clathrin for endocytosis (14, 19). Expression of dominant negative mutants of beta -arrestin strongly inhibits SP-induced endocytosis of the NK1-R (13), and NK1-Rdelta 324 does not interact with beta -arrestins, which may explain the diminished endocytosis (11). Thus SP-induced endocytosis of the NK1-R proceeds by a beta -arrestin-dependent mechanism that requires GRK-mediated phosphorylation of the NK1-R rather than by PKC-induced phosphorylation.

Physiological implications. Desensitization of signal transduction is important in preventing the continued stimulation of cells in an uncontrolled manner. Defects in the mechanisms that terminate signaling by SP, such as deletion of neutral endopeptidase that degrades extracellular SP, result in exaggerated neurogenic inflammation (26, 36). Our results show that PKC regulates the duration and magnitude of SP-induced Ca2+ mobilization and may thereby contribute to desensitization of signaling. Thus defects in PKC expression may also contribute to uncontrolled SP signaling. Although GRKs and beta -arrestins are the most likely mediators of desensitization and endocytosis, their specific roles in desensitization remain to be determined.

Regulation by PKC requires the intact COOH terminus of the NK1-R, since PKC does not affect SP-induced Ca2+ mobilization in cells expressing NK1-Rdelta 324. NK1-Rdelta 324 is internalization defective (5), does not efficiently couple to mitogenic signaling pathways (11), and is resistant to homologous desensitization compared with full-length NK1-R (25, 33). Thus it will be important to compare the roles of full-length and truncated NK1-R in cells that naturally express these receptors.


    ACKNOWLEDGEMENTS

We thank Michelle Lovett for technical assistance, Paul Dazin for assistance with flow cytometry, and Dr. Eileen F. Grady for helpful comments.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-39957.

Address for reprint requests and other correspondence: N. W. Bunnett, Univ. of California San Francisco, 521 Parnassus Ave., C-317, San Francisco, CA 94143-0660 (E-mail: nigelb{at}itsa.ucsf.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 September 2000; accepted in final form 6 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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