title: Endothelin-converting enzyme-1 degrades internalized somatostatin-14.
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.
authors.affiliation: Department of Dermatology, Interdisziplinäres Zentrum für Klinische Forschung Münster, and Ludwig Bolzmann Institute for Cell and Immunobiology of the Skin, University Münster, D-48149 Münster, Germany. roosterman@gmx.net
authors.forename: Dirk
authors.initials: D
authors.lastname: Roosterman
authors.forename: Cordula
authors.initials: C
authors.lastname: Kempkes
authors.forename: Graeme S
authors.initials: GS
authors.lastname: Cottrell
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Padilla
authors.forename: Nigel W
authors.initials: NW
authors.lastname: Bunnett
authors.forename: Christoph W
authors.initials: CW
authors.lastname: Turck
authors.forename: Martin
authors.initials: M
authors.lastname: Steinhoff
mesh_terms: D000595:Amino Acid Sequence; D019390:Arrestins / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D002478:Cells, Cultured; D004705:Endocytosis; D010450:Endopeptidases / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D006801:Humans; D007457:Iodine Radioisotopes / Q000493:pharmacokinetics; D008666:Metalloendopeptidases / Q000378:metabolism / Q000502:physiology*; D015282:Octreotide / Q000493:pharmacokinetics; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D017481:Receptors, Somatostatin / Q000819:agonists / Q000378:metabolism; D013004:Somatostatin / Q000493:pharmacokinetics*; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D007457:Iodine Radioisotopes; D017481:Receptors, Somatostatin; D000071557:beta-Arrestins; C106140:somatostatin receptor sst2A; D013004:Somatostatin; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; C000606924:ECE2 protein, human; D000072396:Endothelin-Converting Enzymes; D015282:Octreotide