Roosterman Dirk, Kempkes Cordula, Cottrell Graeme S, Padilla Benjamin E, Bunnett Nigel W, Turck Christoph W, Steinhoff Martin
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.
Endocrinology. 2008 May;149(5):2200-7. doi: 10.1210/en.2007-1628. Epub 2008 Feb 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.
激动剂诱导的生长抑素受体（ssts）内化决定了细胞随后对肽类激动剂的反应性，并影响sst受体闪烁扫描。为了研究sst2A的转运，将带有表位标签的大鼠sst2A在人胚肾细胞中表达，并通过抗体标记进行追踪。共聚焦显微镜分析显示，用生长抑素和奥曲肽刺激可诱导sst2A内化。内化的sst2A仍被隔离在早期内体中，刺激后60分钟，内化的sst2A仍与β-抑制蛋白1-增强型绿色荧光蛋白（EGFP）、内皮素转化酶-1（ECE-1）和rab5a共定位。内化的（125）I-Tyr（11）-SST-14被内体肽酶迅速水解，放射性代谢产物从细胞中释放出来。内化的（125）I-Tyr（1）-奥曲肽作为完整肽积累，并作为完整肽配体从细胞中释放。我们已确定ECE-1是负责使内化的SST-14失活的肽酶之一。ECE-1介导的SST-14裂解被特异性ECE-1抑制剂SM-19712以及通过使用巴弗洛霉素A（1）防止内体酸化所抑制。ECE-1在酸性环境中裂解SST-14但不裂解奥曲肽。金属肽酶血管紧张素-1转化酶和ECE-2不水解SST-14或奥曲肽。我们的结果首次表明，用SST-14和奥曲肽刺激可诱导sst2A隔离到早期内体中，并且内吞的SST-14被位于早期内体中的肽酶降解。此外，奥曲肽不被内体肽酶降解，并作为完整肽释放。这种机制可能解释了sst2A刺激后奥曲肽和SST-14之间的功能差异。此外，对肽酶调节的神经肽转运的进一步研究可能会在癌症诊断中产生神经肽受体失活方面的新概念。
