Benjamin E. Padilla

Endosomes: A legitimate platform for the signaling train

Although long regarded as a conduit for the degradation or recycling of cell surface receptors, the endosomal system is also an essential site of signal transduction. Activated receptors accumulate in endosomes, and certain signaling components are exclusively localized to endosomes. Receptors can continue to transmit signals from endosomes that are different from those that arise from the plasma membrane, resulting in distinct physiological responses. Endosomal signaling is widespread in metazoans and plants, where it transmits signals for diverse receptor families that regulate essential processes including growth, differentiation and survival. Receptor signaling at endosomal membranes is tightly regulated by mechanisms that control agonist availability, receptor coupling to signaling machinery, and the subcellular localization of signaling components. Drugs that target mechanisms that initiate and terminate receptor signaling at the plasma membrane are widespread and effective treatments for disease. Selective disruption of receptor signaling in endosomes, which can be accomplished by targeting endosomal-specific signaling pathways or by selective delivery of drugs to the endosomal network, may provide novel therapies for disease.

Endothelin-converting enzyme-1 regulates endosomal sorting of calcitonin receptor-like receptor and β-arrestins

Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a–d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), β-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and β-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and β-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B2 receptor, which transiently interacts with β-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/β-arrestin complex, freeing internalized receptors from β-arrestins and promoting recycling and resensitization.

Endothelin-converting enzyme 1 degrades neuropeptides in endosomes to control receptor recycling

Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with β-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide–receptor–arrestin complex is sequestered into endosomes. Although dissociation of β-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase endothelin-converting enzyme 1 (ECE-1), determines stability of the peptide–receptor–arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK1R) induced membrane translocation of β-arrestins followed by trafficking of the SP–NK1R–β-arrestin complex to early endosomes containing ECE-1a–d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; β-arrestins returned to the cytosol, and the NK1R, freed from β-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK1R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK3R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide–receptor–arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control β-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.

Post-endocytic Sorting of Calcitonin Receptor-like Receptor and Receptor Activity-modifying Protein 1

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10-7 m CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca2+]i. Cycloheximide did not affect resensitization, but bafilomycin A1, an inhibitor of vacuolar H+-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10-7 m CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded ∼4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.

Endosomal Endothelin-converting Enzyme-1 A REGULATOR OF β-ARRESTIN-DEPENDENT ERK SIGNALING

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.

Ubiquitin-dependent Down-regulation of the Neurokinin-1 Receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK1R). The effects of sustained stimulation by high concentrations of SP on NK1R trafficking and Ca2+ signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca2+ signaling by wild-type NK1R (NK1Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK1RΔ5K/R, C-terminal tail lysines; and NK1RΔ10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca2+ ions. SP desensitized NK1Rwt, NK1RΔ5K/R, and NK1RΔ10K/R. However, NK1RΔ5K/R and NK1RΔ10K/R resensitized 4–8-fold faster than NK1Rwt by cycloheximide-independent mechanisms. NK1RΔ325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK1Rwt, NK1RΔ5K/R, and NK1RΔ10K/R. After 4 h in SP-free medium, NK1RΔ5K/R and NK1RΔ10K/R recycled to the plasma membrane, whereas NK1Rwt remained internalized. SP induced ubiquitination of NK1Rwt and NK1RΔ5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK1RΔ10K/R was not ubiquitinated. Whereas SP induced degradation of NK1Rwt, NK1RΔ5K/R and NK1RΔ10K/R showed ∼50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK1R, which mediates its degradation and down-regulation.

Endothelin-Converting Enzyme-1 Degrades Internalized Somatostatin-14

Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with beta-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized (125)I-Tyr(11)-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized (125)I-Tyr(1)-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A(1). ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of β-arrestin1 and PP2A with noninternalized NK(1)R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

The use of magnets with single-site umbilical laparoscopic surgery

Single-site umbilical incision laparoscopic surgery (SSULS) is increasingly being used to treat a variety of childhood surgical diseases. Existing SSULS approaches have inefficient triangulation and poor ergonomics. In an effort to overcome these shortcomings, magnet-assisted laparoscopy was developed. Specialized magnetic graspers are introduced through a standard 12-mm port and are controlled by a powerful external magnet. This study is a retrospective analysis of all magnet-assisted laparoscopic operations performed at the Fundacion Hospitalaria Private Childrens Hospital from September 2009 to January 2011. Outcomes include demographics, diagnosis, operative time, intraoperative complications, and conversion rates. Forty-four magnet-assisted laparoscopic operations were performed. The operations included 23 appendectomies, 8 cholecystectomies, 3 Nissen fundoplications, 2 gastrojejunostomies, 2 splenectomies, 2 ovarian tumor/cyst resections, 1 retroperitoneal lymphangioma resection, 1 left adrenalectomy, 1 total abdominal colectomy and 1 pulmonary wedge resection. The mean operative times for the most commonly performed operations were 61 minutes for appendectomy and 93 minutes for cholecystectomy. The operations were classified as follows: Group I, adjunct to conventional laparoscopy (5 operations); Group II, adjunct to multiple-access umbilical laparoscopy (11 operations); and Group III, true single-port laparoscopy (28 operations). Among Group II/III operations, 6 operations required 1 additional port outside the umbilicus. No operations required more that 1 additional port, and no operations were converted to the open technique. There were no intraoperative complications. Magnet-assisted laparoscopic surgery is safe and effective in children. The use of magnetic graspers improves triangulation and ergonomics while reducing the number and size of abdominal incisions.

Initial Experience with Magnet-Assisted Single Trocar Appendectomy in Children

BACKGROUND Single-incision laparoscopic surgery (SILS) is increasingly being used to treat acute appendicitis. Existing SILS techniques suffer from inefficient triangulation and poor ergonomics. In an effort to improve on existing SILS techniques, we developed the magnet-assisted single trocar (MAST) appendectomy. SUBJECTS AND METHODS We retrospectively analyzed all MAST appendectomies performed between March 2010 and February 2011. Outcomes included demographics, diagnosis, operative time, hospital stay, and complications. RESULTS Twenty-three MAST appendectomies were performed in 10 boys and 13 girls. The mean age at operation was 12.22 years (range, 5-19 years), and the mean weight was 46.5 kg (range, 25-82 kg). At presentation the mean white blood cell count was 15,000 with 74% polymorphonuclear neutrophils. The mean operative time was 61 minutes (range, 20-105 minutes), and length of stay was 3.6 days (range, 1-7 days). In total, 4 operations (17%) required one additional 5-mm trocar to complete the operation, and none was converted to an open operation. There were no intraoperative complications, nor were there any wound infections. CONCLUSIONS MAST appendectomy is safe and effective in children. Magnetic instruments provide excellent triangulation and improve ergonomics. This technique uses a single 12-mm trocar and can be performed without the aid of a surgical assistant.