Benjamin E Padilla

University of Münster, Muenster, North Rhine-Westphalia, Germany

Are you Benjamin E Padilla?

Claim your profile

Publications (14)95.4 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    AJP Cell Physiology 07/2011; 301(4):C780-91. · 3.71 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Repair of incisional hernias in renal transplant recipients is compromised because of immunosuppressive therapy. Retrospective review. University tertiary care institution. Forty-two recipients of renal transplants or combined renal-pancreas transplants who underwent incisional herniorrhaphy were included in our study. Incisional herniorrhaphy. Postoperative complications and recurrence of incisional hernia. Forty-two patients (mean age, 49.6 years) underwent incisional herniorrhaphy (mean area, 99.9 cm(2)) following renal transplantation (26 cadaveric donor renal, 12 combined renal-pancreas, and 4 living related donor renal) from January 1, 1995, to December 31, 2005. Using various techniques, hernia repairs were performed on average 36.4 months following transplantation. Diabetes mellitus was a frequent cause of end-stage renal disease (16 patients), followed by polycystic kidney disease (6 patients), focal segmental glomerular sclerosis (3 patients), hypertension (2 patients), Alport syndrome (2 patients), and IgA nephropathy (2 patients), with 11 patients having lupus or glomerulonephritis. Four patients developed cellulitis, 2 patients required mesh removal, and 1 patient was admitted for abscess drainage and intravenous antibiotics. Fourteen patients had recurrence of incisional hernias, with 3 patients experiencing 2 recurrences and 1 patient experiencing 4 recurrences. To our knowledge, this is the largest series of incisional herniorrhaphies performed among patients following renal transplantation. Although smoking history, the presence of diabetes, and immunosuppressive therapy were not associated with the initial development of an incisional hernia, they were associated with complications. Component separation performed by transplant and plastic and reconstructive surgeons should be considered in the setting of recurrent hernias and large defects.
    Archives of surgery (Chicago, Ill.: 1960) 01/2011; 146(1):21-5. · 4.32 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Proceedings of the National Academy of Sciences 10/2009; 106(42):17615-22. · 9.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and beta-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. beta-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, beta-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 beta-arrestin-dependent signaling of endocytosed GPCRs.
    Journal of Biological Chemistry 07/2009; 284(33):22411-25. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We sought to examine insulin-sensitive food intake behavior and neuroendocrine and metabolic variables of rats that had undergone a duodenal-jejunal bypass (DJB). A DJB that circumvents the duodenum and proximal jejunum while leaving the stomach unperturbed rapidly improves insulin sensitivity in type 2 diabetic rats. This segment of proximal small intestine is innervated by the gastroduodenal branch of the vagus nerve, the transection of which influences food intake choices in streptozotocin-diabetic rats. Rats were first placed on a choice of chow and lard for 7 days and additionally provided with an enriched liquid diet for another 7 days before surgery and were allowed only the liquid diet for 7 days after either a sham or DJB operation. After surgery, DJB-operated rats initially consumed less than the sham-operated counterparts. When the rats were subsequently provided with the choice of chow and lard for 7 days, there were no differences in intake between the DJB and sham-operated groups. Similarly, the majority of metabolic and neuroendocrine variables measured were unchanged. However, DJB-operated rats exhibited greater mesenteric white adipose tissue weight, fecal output, arcuate nucleus neuropeptide Y mRNA expression, plasma corticosterone, and glucagon levels together with reduced plasma leptin concentrations. DJB surgery does not produce significant differences in food intake choices after a period of recovery; however, there are enduring metabolic and neuroendocrine changes, which are collectively important to understanding the beneficial outcomes of the operation.
    Annals of surgery 03/2009; 249(2):269-76. · 7.90 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Endocrinology 06/2008; 149(5):2200-7. · 4.72 Impact Factor
  • Gastroenterology 01/2008; 134(4). · 12.82 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to pep- tide agonists and influences sst receptor scintigraphy. To in- vestigate 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 internaliza- tion of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, inter- nalized sst2A still colocalized with -arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized 125I-Tyr11-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Inter- nalized 125I-Tyr1-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 respon- sible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhib- itor, SM-19712, and by preventing acidification of endosomes usingbafilomycinA1.ECE-1cleavedSST-14butnotoctreotide in an acidic environment. The metallopeptidases angioten- sin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimu- lation 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. Fur- thermore, octreotide was not degraded by endosomal pepti- dases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investiga- tion of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inac- tivation in cancer diagnosis. (Endocrinology 149: 2200-2207, 2008)
    Endocrinology 01/2008; 149(5):2200-2207. · 4.72 Impact Factor
  • Gastroenterology 01/2008; 134(4). · 12.82 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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), beta-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-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 B(2) receptor, which transiently interacts with beta-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/beta-arrestin complex, freeing internalized receptors from beta-arrestins and promoting recycling and resensitization.
    The Journal of Cell Biology 01/2008; 179(5):981-97. · 10.82 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with beta-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide-receptor-arrestin complex is sequestered into endosomes. Although dissociation of beta-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 beta-arrestins followed by trafficking of the SP-NK1R-beta-arrestin complex to early endosomes containing ECE-1a-d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; beta-arrestins returned to the cytosol, and the NK1R, freed from beta-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 beta-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.
    Proceedings of the National Academy of Sciences 08/2007; 104(28):11838-43. · 9.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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 [Ca(2+)](i). Cycloheximide did not affect resensitization, but bafilomycin A(1), 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 approximately 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.
    Journal of Biological Chemistry 04/2007; 282(16):12260-71. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). 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 (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (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 NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.
    Journal of Biological Chemistry 10/2006; 281(38):27773-83. · 4.65 Impact Factor
  • Source