Endosomal Endothelin-converting Enzyme-1

Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and β-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. β-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that endothelin-converting enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, β-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls β-arrestin-dependent signaling of endocytosed GPCRs.

lular agonist concentration and receptor coupling to heterotrimeric G proteins control signal transduction. Cell-surface metalloendopeptidases, exemplified by neprilysin, degrade neuropeptides in the extracellular fluid to regulate receptor activation (1,2). G protein receptor kinases phosphorylate activated GPCRs to promote their interaction with ␤-arrestins, which uncouple receptors from G proteins and mediate desensitization (3). However, activated GPCRs internalize, and endocytosed receptors continue to signal by G protein-independent mechanisms. ␤-Arrestins mediate endocytosis and intracellular signaling of GPCRs. ␤-Arrestins couple GPCRs to clathrin and AP2 to mediate endocytosis (4,5) and are scaffolds that recruit Src, Raf-1, and mitogen-activated protein kinases (MAPKs) to GPCRs in endosomes forming a MAPK signalosome, which determines the location and function of activated extracellular signal-regulated kinases (ERKs) (6 -9). Compared with our understanding of receptor regulation at the plasma membrane, little is known about the mechanisms that regulate GPCR signaling and trafficking at the endosomal membrane. Specifically, it is not known whether agonist interaction with GPCRs or GPCR interaction with ␤-arrestins is necessary for receptor signaling in endosomes. Regulation of these interactions could determine the duration of ␤-arrestin-mediated ERK activation, with important functional implications (10,11).
We hypothesized that mechanisms that regulate GPCRs at the plasma membrane also control receptor signaling and trafficking at the endosomal membrane. We recently reported that the metalloendopeptidase endothelin-converting enzyme-1 (ECE-1) is present at the endosomal membrane and that ECE-1 degrades internalized neuropeptides within acidified early endosomes (12)(13)(14). This degradation disrupts the peptide-GPCR-␤-arrestin complex to promote ␤-arrestin translocation back to the cytosol and receptor recycling to the cell-surface, which mediates resensitization. Whether this process regulates signaling of receptors at the endosomal membrane and subsequent translocation of signals to the nucleus and throughout the cytosol is completely unknown.
We report that ECE-1, by degrading neuropeptides in endosomes, regulates the stability and activity of peptide-GPCR-␤-arrestin-MAPK signalosomes to control the duration of ERK activation and subsequent activation of transcription factors, one of which mediates programmed cell death. We examined the role of ECE-1 in regulating signaling by substance P (SP). SP is expressed in the nervous and immune systems and interacts with the neurokinin 1 receptor (NK 1 R) to control neurogenic inflammation, pain, neurodegeneration, smooth muscle contraction, and exocrine secretions (15). SP induces NK 1 R interaction with ␤-arrestins at the plasma membrane, which mediates desensitization and causes ␤-arrestin-dependent endocytosis of the SP-NK 1 R-␤-arrestin complex to early endosomes containing ECE-1 (13,16). ␤-Arrestins recruit Src, MEK, and ERKs to the NK 1 R at the endosomal membrane, which mediates the effects of SP on gene expression and cell survival (6). However, nothing is known about the regulation of the stability and activity of the SP-NK 1 R-␤-arrestin-MAPK signalosome in endosomes.
Measurement of [Ca 2ϩ ] i -[Ca 2ϩ ] i was measured in populations of cells using Fura2-AM (Invitrogen) as described (12,20). To assess resensitization, cells were incubated with vehicle or SP (10 nM, 0 or 10 min, 37°C), washed, and recovered in SP-free medium for 2 h. Cells were challenged with SP (10 nM), and [Ca 2ϩ ] i was measured.
ERK Activation in Mouse Spinal Cord-Mice were anesthetized by isoflurane (2.5%) and SM-19712 (17.5 g/5 l) or saline (control, 5 l) were injected into the subarachnoid space on the midline between the L4 and L5 vertebrae using a Hamilton syringe with a 30-gauge needle. After 30 min animals received intraplantar injections (5 l) of vehicle (ethanol:Tween 80: saline (1:8:1)) or capsaicin (10 g). After 10 and 60 min, spinal cord was frozen in liquid nitrogen, homogenized in Camiolo buffer containing 10 mM NaF and 0.1 mM Na 3 VO 4 , and processed for Western blotting.
Cytotoxicity Assay-Cell death was assessed using a cytotoxicity detection kit (Roche Applied Science) to measure the lactate dehydrogenase (LDH) activity. Released and total cellular LDH activity were measured. Results are expressed as LDH release as a percentage of the total cellular LDH and as the -fold increase over control of LDH activity released in response to stimulation with SP.
Reverse Transcription-PCR-RNA from mouse spinal cord (L4-L6) and myenteric ganglia (ϳ50) of rat ileum was isolated using Trizol (Invitrogen) and treated with DNase I (Ambion, Austin, TX). RNA was reverse-transcribed using standard protocols with random hexamers and TaqMan reverse transcription reagents (Applied Biosystems). Subsequent PCR reactions used primers specific for rat or mouse ECE-1 isoforms (sequences available on request). Control reactions omitted reverse transcriptase. PCR products were separated by electrophoresis, stained with ethidium bromide, and sequenced to confirm identity.
Statistics-Data are presented as the SE. of n Ն 3 experiments or animals. Results were compared by Student's t test, with p Ͻ 0.05 considered significant (*).

SP Activates ERKs by Epidermal Growth Factor Receptor (EGFR)-and ␤-Arrestin-dependent Pathways-GPCRs activate
ERKs by mechanisms operating at the plasma membrane and in endosomes (10,11). One mechanism involves activation of cellsurface metalloendopeptidases, which release membrane-tethered ligands that activate the EGFR (27). To determine the contribution of the EGFR to SP-stimulated ERK activation, we examined the effects of the tyrosine kinase inhibitor AG1478 on SP-induced phosphorylation of ERK2 (pERK2). HEK-NK 1 R cells treated with vehicle or AG1478 were stimulated with SP (10 nM, 0 -30 min), and pERK2 and ERK2 were quantified by Western blotting. In unstimulated cells, pERK2 was low (Fig.  1A). In vehicle-and AG1478-treated cells, SP stimulated a prompt increase in pERK2 within 2 min (Fig. 1A). However, the magnitude of the increase in pERK2 over basal was reduced in AG1478-treated cells (5.5 Ϯ 1.2-fold increase, 2 min) compared with vehicle-treated cells (-fold increase, 2 min, 12 Ϯ 0.9, Fig.  1A). Thus, SP rapidly activates ERK2 in part by transactivating the EGFR. To exclude this pathway, subsequent experiments used cells treated with AG1478 (unless indicated), which abolished EGF-stimulated ERK2 activation (supplemental Fig. 1A).

ECE-1 Regulates ERK Signaling
To confirm that ECE-1 regulates SP-induced activation of ERK1 in a similar manner to ERK2, we also examined the effects of ECE-1 inhibition and ECE-1 overexpression on SP-stimulated ERK1 activation. Whereas ECE-1 inhibition was markedly sustained in cells treated with SM-19712, ECE-1 overexpression attenuated SP-induced activation of ERK1 (supplemental Fig. 4, A  and B). Thus, ECE-1 regulates the duration of ERK1 and ERK2 signals.
The Stability of SP in Endosomes Determines the Duration of SP-induced ␤-Arrestin-dependent ERK Activation-At endosomal pH 5.5, ECE-1 cleaves SP (RPKPQQFFGLM-NH 2 ) at Gln 6 -Phe 7 , Phe 7 -Phe 8 , and Gly 9 -Leu 10 (13,30). The SP analogue SMSP, a full and selective NK 1 R agonist (33), has a substitution (Gly 9 3 Sar 9 ) at an ECE-1 cleavage site. We hypothesized that SMSP is resistant to ECE-1 degradation and that ECE-1 does not regulate SMSP-stimulated ERK activation. To compare degradation rates, we incubated SP and SMSP with ECE-1 at pH 5.5 and 7., and used mass spectrometry to determine the rate of peptide degradation, product formation, and metabolism. ECE-1 degraded SP at pH 5.5 but not at pH 7.4 (Fig. 5, A  and B). The first identified product was 1 RPKPQQFFG 9 , which FIGURE 5. ECE-1 degrades SMSP with reduced efficiency and only partially regulates SMSP-induced ERK2 activation. A, kinetics of ECE-1 degradation of SP and SMSP. Peptides were incubated with ECE-1 at pH 5.5 or pH 7.4. Abundance of substrate and products was measured as the relative contribution of their respective ions to the total ion current in the MALDI-TOF MS spectrum as a function of time. Tripling the ECE-1 concentration gave similar rates of degradation of SMSP compared with SP. B, kinetics of ECE-1 degradation of SP and generation and metabolism of products. Degradation at pH 5.5 was monitored as in B. Displayed are the mass-to-charge ratio (m/z) of the detected peptide ions and their ion intensity during the reaction. Sequences of substrates and products and their (m/z) values are indicated in red. Sequence assignments were confirmed by MS/MS. C, HEK-NK 1 R cells were incubated with AG1478 and vehicle or SM-19712, challenged with SMSP (10 nM, 0 -10 min), washed, and incubated in SMSP-free medium (0 -120 min). SMSP caused similar initial ERK2 activation in vehicle-and SM-19712-treated cells, and the magnitude of the sustained response to SMSP was only slightly increased by SM-19712 (n Ն 3). did not accumulate and was rapidly metabolized to 1 RPKPQQ 6 and 7 FFG 9 . The mechanism of the generation of another major product, 1 RPKPQQF 7 , is unclear. Thus, ECE-1 first degrades SP at Gly 9 -Leu 10 , and 1 RPKPQQFFG 9 is also rapidly degraded at Gln 6 -Phe 7 . Although ECE-1 degraded SMSP at pH 5.5 but not pH 7.4, the Sar 9 Met(O 2 ) 11 -SP modification dramatically decreased the rate at which ECE-1 degraded SMSP (Fig. 5B). A 3-fold higher ECE-1 concentration was required to give similar degradation rates for SMSP (195 nM) compared with SP (65 nM). 1 RPKPQQFFSar 9 was the first product, which was rapidly metabolized to 1 RPKPQQ 6 . Surprisingly, 1 RPKPQQF 7 was not formed from SMSP. SM-19712 prevented degradation of both SP and SMSP (not shown). Consistent with the diminished susceptibility of SMSP to degradation by ECE-1, SM-19712 enhanced SMSP-induced ERK2 activation by only 1.9 Ϯ 0.1fold at 120 min (Fig. 5C), compared with a 4 -9-fold increase in SP-induced ERK2 activation at the same time (Figs. 3A and 4, A  and B). Thus, agonist susceptibility to degradation by ECE-1 in acidified endosomes determines the duration of ERK activation.

ECE-1 Regulates ERK Activation in Spinal
Neurons-We sought to determine whether ECE-1 regulates SP-induced ERK activation in vivo. The NK 1 R is prominently expressed in nociceptive neurons in the spinal cord. SP, released from central projections of primary spinal afferent neurons in the dorsal horn of the spinal cord, causes NK 1 R endocytosis (34) and activates ERK in nociceptive neurons (35), which results in inflammatory hyperalgesia (36). We determined if ECE-1 is also expressed in mouse spinal cord using reverse transcription-PCR, Western blotting, and immunofluorescence. Reverse transcription-PCR amplified mRNA transcripts for ECE-1a-d, and ECE-1 expression was confirmed by Western blotting (ϳ120 kDa) (supplemental Fig. 6, A and B). ECE-1-like immunoreactivity (LI) was prominently colocalized with NK 1 R-LI in the soma and fibers of neurons of the dorsal horn of the spinal cord (Fig. 6A). Preabsorption of the ECE-1 antibody with membranes from HEK-ECE-1c-GFP cells inhibited staining compared with membranes from cells expressing empty vector (supplemental Fig. 6C), confirming specificity. Thus, ECE-1 is appropriately localized to regulate SP-induced ERK activation in spinal neurons.
Intraplantar injection of capsaicin stimulates SP release in the dorsal horn, which causes NK 1 R endocytosis (34) and ERK activation (35). To determine whether ECE-1 regulates SP-induced ERK activation in nociceptive neurons, vehicle or SM-19712 were administered intrathecally to mice followed by intraplantar injection of vehicle or capsaicin. We examined ERK2 activation in the spinal cord (L4-L6) by Western blotting. Capsaicin similarly stimulated ERK2 activation in vehicle-and SM-19712-treated mice (1.3-fold increase over basal at 10 min, Fig. 6B). In vehicle-treated mice, pERK2 returned to sub-basal levels after 60 min (0.7-fold of basal at 60 min, Fig. 6C). In contrast, pERK2 was sustained in SM-19712-treated mice (1.3fold increase over basal at 60 min, Fig. 6C). Thus, ECE-1 attenuates the effects of endogenous SP on ERK2 activation in spinal nociceptive neurons.

ECE-1 Regulates SP-induced Activation of Transcription Factors and Nur77-mediated Cell
Death-SP and the NK 1 R cause neurodegeneration in the central nervous system and induce non-apoptotic programmed cell death of HEK cells and striatal neurons (37)(38)(39). The transcription factor and nuclear receptor Nur77 mediates cell death, and ERK2 (not ERK1) induces threonine phosphorylation (Thr(P)) of Nur77 (40). SP up-regulates and phosphorylates Nur77, and SP-induced cell death requires ␤-arrestin-dependent activation of MEK2 and ERK2 and ERK2-mediated phosphorylation Nur77 (37). We hypothesized that ECE-1, by regulating the duration of ␤-arrestin-mediated ERK2 signaling, controls SP-induced Nur77 phosphorylation and cell death. To determine whether ECE-1 regulates expression and phosphorylation of Nur77, we treated HEK-NK 1 R cells with vehicle, AG1478, or AG1478 and SM-19712, challenged with SP (10 nM, 0 -10 min), and washed and incubated cells in SP-free medium (2-4 h). Nur77 was immunoprecipitated, and Western blots were analyzed for Nur77 and Thr(P). Nur77 and Thr(P) were not detectable in unstimulated cells (Fig. 7, A and B). In vehicle-treated cells, SP stimulated expression and phosphorylation of Nur77 after 120 and 240 min. SM-19712 induced a minor up-regulation of Nur77 but markedly enhanced Nur77 phosphorylation (-fold increase, 240 min, 10.5 Ϯ 6.6, Fig. 7, A and B).
By terminating ERK2 activation, ECE-1 may prevent the cytotoxic actions of SP. To examine this possibility, HEK-NK 1 R cells were treated with vehicle, AG1478, or AG1478 and SM-19712, challenged with SP (10 nM, 0 -10 min), and washed and incubated in SP-free medium (24 h). LDH release was measured to assess cell death. In cells not treated with SP, AG1478 or SM-19712 or both AG1478 and SM-19712 had no effect on LDH release measured at 24 h (Fig. 7C). Brief exposure to SP alone (with or without AG1478) also had no effect on LDH release measured 24 h later (Fig. 7C). However, in cells
SP stimulates NK 1 R-dependent expression of the transcription factor c-Fos in many cells, including nociceptive spinal neurons (41). To examine the contribution of ERK to SP-induced c-Fos expression, we treated HEK-NK 1 R cells with the MEK inhibitor UO126 or vehicle. Cells were challenged with SP (10 nM, 0 -10 min), washed, and incubated in SP-free medium (120 min). c-Fos and ␤-actin were analyzed by Western blotting. c-Fos was not detected in unstimulated cells or after brief (10 min) stimulation with SP (Fig. 7D). c-Fos was readily detected in vehicle-treated cells stimulated with SP and then incubated in SP-free medium (120 min). UO126 strongly inhibited c-Fos (-fold decrease, 120 min, 5.6 Ϯ 0.5, p Ͻ 0.05), which is, thus, dependent on the MEK pathway (not shown). To determine the role of ECE-1 in SP-induced c-Fos expression, we treated cells with SM-19712 or vehicle. SM-19712 markedly enhanced SP-induced expression of c-Fos (-fold increase, 120 min, 2.2 Ϯ 0.3, Fig. 7D). Thus, ECE-1 regulates SP-induced c-Fos expression.
SP can induce NK 1 R-mediated neurodegeneration (37)(38)(39), and myenteric neurons from the distal intestine are absent from mice lacking ECE-1 (42). To determine whether ECE-1 regulates death of myenteric neurons, cultured neurons were treated with vehicle or SM-19712, challenged with SP (1 M, 0 -10 min), and washed and incubated in SP-free medium (24 h). In neurons not treated with SP, there was considerable basal LDH release after 24 h, which is probably related to serum deprivation (Fig. 8C, left panel). This basal LDH release was unaffected by SM-19712. Brief challenge with SP alone did not further increase basal LDH release measured 24 h later (Fig. 8C,  right panel). However, in neurons treated with SM-19712, SP caused a significant increase in LDH release (1.2 Ϯ 0.04-fold). The small size of this effect may be related to the fact that only a small proportion of myenteric neurons expressed the NK 1 R. Moreover, the cultures contained glial cells, myocytes, and fibroblasts, which also do not express NK 1 R. However, transient exposure to SP induces neuronal death only when ECE-1 is inhibited. These results suggest ECE-1, by attenuating ERK2 activation, can protect against SP-induced neuronal death.

DISCUSSION
Although internalized GPCRs continue to signal in endosomes by ␤-arrestin-dependent ERK activation, it is not known whether agonist occupation of receptors or receptor interaction with ␤-arrestins is required for this sustained signaling and whether agonist degradation attenuates these signals. Our results show that ECE-1 degrades SP in acidified early endosomes to disrupt the endosomal SP-NK 1 R-␤-arrestin-Src MAPK signalosome. SP degradation attenuates ERK2 activation in the cytosol and nucleus and terminates the nuclear actions of ERK2, which include activation of Nur77 death receptor (Fig. 9). Thus, SP-NK 1 R interaction is necessary for sustained ␤-arrestin-dependent ERK2 signaling from endosomes.
Mechanisms of SP-induced ERK Activation-Because the consequences of ERK1/2 activation depend on subcellular location and duration of activation (10,11), it is important to understand the mechanisms controlling the location and duration of the ERK1/2 signal. SP causes a rapid and sustained activation of ERK1/2 by temporally and spatially distinct mechanisms. Rapid activation depends on transactivation of EGFR, as the tyrosine kinase inhibitor AG1478 reduced ERK2 activation by 2-fold at early times. Transactivation probably occurs at or close to the plasma membrane, as it was observed within minutes of stimulation when NK 1 R is mostly at the cell surface (16,24). Sustained activation depends on ␤-arrestins, as expression of dominant-negative ␤-arrestin1 V53D inhibited sustained ERK2 activation by 2-fold but did not affect rapid ERK2 activation. The sustained activation probably requires ␤-arrestin-dependent recruitment of NK 1 R and Src to endosomes, as it occurred at a time when the NK 1 R is associated with ␤-arrestins and Src in endosomes (6). We have previously shown that SP induces recruitment of ␤-arrestins, Src, MEK, and ERK1/2 to internalized NK 1 R in endosomes (6). The NK 1 R activates ERK1/2 by EGFR-and ␤-arrestin-dependent mechanisms in colonocytes (43), glioma cells (44), and epithelial cells (6,37). Thus, NK 1 R antagonism at plasma and endosomal membranes is necessary to inhibit SP signaling.

ECE-1 Degrades SP in Endosomes and
Terminates ␤-Arrestin-mediated ERK Activation-Metalloendopeptidases cleave SP at plasma and endosomal membranes to control NK 1 R activation. Neprilysin, which is confined to the plasma membrane, rapidly degrades SP in the extracellular fluid to prevent NK 1 R activation (1,2). Thus, neprilysin can regulate NK 1 R coupling to heterotrimeric G proteins and transactivation of the EGFR. Although ECE-1 isoforms constitutively traffic between the plasma membrane and endosomes, the unusual pH sensitivity of ECE-1 determines whether ECE-1 can degrade neuropeptides at these locations (12,13,30). ECE-1 slowly degrades SP at the neutral pH of extracellular fluid but rapidly degrades SP at the acidic pH of early endosomes. Thus, ECE-1 does not control NK 1 R coupling to heterotrimeric G proteins, and ECE-1 inhibitors did not affect the rapid activation of ERK1/2, which

ECE-1 Regulates ERK Signaling
depends on EGFR transactivation. However, several observations from the present study suggest that ECE-1 degrades SP in endosomes to control ␤-arrestin-dependent ERK1/2 activation. First, the ECE-1 inhibitor SM-19712 caused accumulation of NK 1 R, ␤-arrestins, and Src in purified early endosomes. This finding supports our report that ECE-1 inhibition/knockdown cause sustained colocalization of the NK 1 R and ␤-arrestins in early endosomes, whereas ECE-1 overexpression has the opposite effect, promoting NK 1 R recycling and resensitization and stimulating the return of ␤-arrestins to the cytosol (13). Second, SM-19712 caused markedly sustained ERK2 activation, whereas overexpression of ECE-1c attenuated ERK2 activation. A second ECE-1 inhibitor, PD-069185, also caused prolonged ERK2 activation, although the effect was less pronounced, probably because PD-069185 is not membrane permeant (19), whereas SM-19712 is a membrane permeable inhibitor (29). Third, the H ϩ -ATPase inhibitor bafilomycin A 1 , which prevents endosomal acidification and inhibits ECE-1-dependent SP degradation in endosomes (13), also caused sustained ERK activation. Together, these results support the hypothesis that ECE-1 degrades SP in acidified endosomes to disrupt the SP-NK 1 R-␤-arrestin-Src complex and terminate ERK1/2 activation. The report that an NK 1 R-␤-arrestin fusion protein constitutively activates ERK when expressed in HEK cells (45) supports our suggestion that interaction of NK 1 R and ␤-arrestins, regulated by ECE-1, controls the duration of SP-induced ERK activation.
The ability of ECE-1 to regulate NK 1 R-dependent ERK2 activation requires that the NK 1 R agonist is an ECE-1 substrate and that the NK 1 R shows sustained interactions with ␤-arrestins. Although SMSP is a full and selective agonist of the NK 1 R (33), ECE-1 degraded SMSP more slowly than SP, and SM-19712 had only a small effect on SMSP-stimulated ERK2 activation. Supporting the importance of substrate susceptibility is our observation that ECE-1 does not regulate signaling of angiotensin II, which is not an ECE-1 substrate at acidic pH (12,13). Other mechanisms can regulate the duration of MAPK signals transmitted by the angiotensin AT1A receptor, including the dual specificity phosphatase MKP7, which interacts with ␤-arrestins and dephosphorylates JNK3 bound to ␤-arrestin2 (46). NK 1 R⌬325-407 interacts with ␤-arrestins only transiently (6,32). In contrast to the fulllength NK 1 R, ECE-1 inhibition did not cause retention of NK 1 R⌬325-407 with ␤-arrestins in endosomes and failed to cause sustained NK 1 R⌬325-407-mediated ERK2 activation. Supporting the importance of sustained interactions with ␤-arrestins, ECE-1 does not regulate signaling of the bradykinin B 2 receptor (12), which also transiently interacts with ␤-arrestins (31).
ECE-1 Prevents the Cytotoxic Consequences of SP-induced ERK2 Activation-ECE-1 inhibition caused sustained ERK2 activation in the cytosol and nucleus, suggesting that the ␤-arrestin MAPK signalosome is a hub for ERK2 activation throughout the cell. Although cytosolic ERK2 has many targets (11), the functional relevance of SP-induced activation of cytosolic ERK2 is unclear. Nuclear ERK2 has multiple effects, including phosphorylation of transcription factors and regulation of differentiation, proliferation, and survival (10). SP also controls transcription, proliferation, and survival by ERK-dependent mechanisms, suggesting that ECE-1 may also regulate these components of SP signaling (6,43). Although ERK activation usually promotes differentiation and proliferation, ERK can also mediate cell death in the nervous system (10). We evaluated the role of ECE-1 in controlling SP-induced phosphorylation of the death receptor Nur77, as SP phosphorylates Nur77 and causes cell death by ␤-arrestin-mediated activation of ERK1/2 (37). Nur77 (TR3 or nerve growth factor I-B) is an immediate early gene and an orphan member of the steroidthyroid-retinoid family of nuclear receptors, and Nur77 has been implicated in regulating growth and cell death (47). We FIGURE 9. Proposed mechanism by which endosomal ECE-1 regulates SP-stimulated ERK activation and cell death. 1, SP activation of the NK 1 R induces receptor phosphorylation by G protein receptor kinases (GRK) and membrane translocation of ␤-arrestins (␤-ARR), which mediate NK 1 R desensitization and endocytosis. 2, ␤-arrestins recruit Src, MEK, and ERK1/2 to form a mitogen-activated protein kinase signaling module in endosomes. 3, ECE-1 degrades SP in acidified endosomes, causing disassembly of the SP-NK 1 R-␤-arrestin-Src-MEK-ERK1/2 complex. 4, the NK 1 R, freed of SP and ␤-arrestins, recycles to mediate resensitization. 5, sustained ERK2 activation, which occurs in the absence of ECE-1, causes threonine phosphorylation and activation of Nur77, resulting in cell death.
observed that SP stimulated threonine phosphorylation of Nur77 and that SM-19712 increased this response by Ͼ10-fold. Moreover, whereas brief (10 min) exposure of HEK-NK 1 R cells to SP had no cytotoxic effects in cells with active ECE-1, ECE-1 inhibition revealed marked cytotoxic effects of SP. Thus, ECE-1 is critically important in regulating the actions of SP-NK 1 R signaling in the nucleus and probably also the cytosol. Further studies are required to determine how Nur77 mediates SP-induced cell death, although Nur77 can translocate from the nucleus to mitochondria, where Nur77 interacts with Bcl-2 to activate apoptosis (48).
Roles of Neuronal ECE-1-Proinflammatory and noxious stimuli that excite primary spinal afferent neurons in peripheral tissues stimulate SP release from central projections of these neurons in the dorsal horn of the spinal cord. SP activates the NK 1 R on nociceptive spinal neurons to cause receptor endocytosis (34), ERK1/2 activation (35), and c-Fos expression (41). ERK mediates up-regulation of the NK 1 R and prodynorphin and is responsible for persistent thermal and mechanical hyperalgesia (36). We observed that ECE-1 is coexpressed with the NK 1 R in spinal neurons and is, thus, suitably localized to regulate these effects of SP. Intraplantar injection of capsaicin, which excites primary spinal afferent neurons by activating transient receptor potential vanilloid 1, induced transient ERK activation in the dorsal horn, and intrathecal administration of SM-19712 prolonged this activation. These results suggest that ECE-1 attenuates SP-induced ERK activation in spinal nociceptive neurons and thereby suppresses NK 1 R-mediated inflammatory pain. Additional experiments are required to examine this possibility. It is also possible that ECE-1 regulates c-Fos expression and gene transcription in these neurons, as we observed in HEK cells. Neprilysin also regulates SP signaling in the dorsal horn by degrading SP in the extracellular fluid (49).
The NK 1 R is expressed in myenteric neurons where SP regulates peristalsis (17). We observed that Alexa-SP internalizes in myenteric neurons to colocalize with ECE-1 in endosomes, which are known to contain NK 1 R and ␤-arrestins (24). Thus, ECE-1 is appropriately localized to degrade endocytosed SP in neurons and regulate the stability of SP-NK 1 R-␤-arrestin MAPK signalosome of neurons. Although we were unable to study SP-induced ERK activation in cultured neurons, which did not tolerate serum starvation that is necessary to obtain low basal activity, we did observe that brief exposure to SP resulted in neuronal death in neurons treated with an ECE-1 inhibitor, which supports our findings with HEK-NK 1 R cells. It is not known whether ECE-1 also regulates the neurotoxic actions of SP in the central nervous system (37)(38)(39), and further experimentation is required to define the mechanism of SP toxicity in myenteric neurons. However, SP causes non-apoptotic death of striatal neurons by a mechanism that depends on ␤-arrestin, ERK2, and Nur77 (37), which would be amenable to regulation by ECE-1. ECE-1 knock-out mice lack myenteric neurons in the distal intestine (42). This phenotype mimics that observed in mice lacking the endothelin B receptor and is attributed to defective colonization of the developing intestine by cells of the neural crest. Whether ECE-1 deficiency by promoting sustained ERK2 activation also predisposes neurotoxicity is an intriguing possibility that requires further investigation. How-ever, given that ECE-1 can degrade multiple neuropeptides in acidified endosomes, the mechanism that we describe may be a general one for controlling ERK activation (12)(13)(14)30).