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* Institute of Immunology, Tsinghua University School of Medicine, Beijing, People’s Republic of China; and
Institute of Immunology and National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai, People’s Republic of China
Cytokines produced by immune cells play pivotal roles in the regulation of both innate and adaptive immunity. However, the mechanisms controlling secretion of cytokines have not been fully elucidated. Secretory carrier membrane proteins (SCAMPs) are widely distributed integral membrane molecules implicated in regulating vesicular transport. In this study, we report the functional characterization of human SCAMP5 (hSCAMP5), a novel SCAMP protein that is widely expressed by a variety of neuronal and nonneuronal tissues and cells. By measuring the cytokine secretion (RANTES/CCL5 and IL-1β) as an exocytotic model, we show that hSCAMP5 can promote the calcium-regulated signal peptide-containing cytokine (CCL5 but not IL-1β) secretion in human epithelial cancer cells, human monocytes, and mouse macrophages. By using subcellular fractionation, immunofluorescence confocal microscopy, and membrane vesicle immunoisolation methods, we find that hSCAMP5 is mainly localized in the Golgi-associated compartments, and the calcium ionophore ionomycin can trigger a rapid translocation of hSCAMP5 from Golgi apparatus to plasma membrane along the classical exocytosis pathway. During the translocation of hSCAMP5 from Golgi apparatus to plasma membrane, hSCAMP5 can codistribute and complex with local soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) molecules. We further demonstrate that hSCAMP5 can directly interact with the calcium sensor synaptotagmins via the cytosolic C-terminal tail of hSCAMP5, thus providing a potential molecular mechanism linking SCAMPs with the SNARE molecules. Our findings suggest that hSCAMP5, in cooperation with the SNARE machinery, is involved in calcium-regulated exocytosis of signal peptide-containing cytokines.
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1 This work was supported by grants from the Foundation for the Author of Excellent Doctoral Dissertation of China (200775), National Natural Science Foundation of China (30572122, 30771118, 30721091), National Key Basic Research Program of China (2007CB512403), and National Specific Program of New Drug Development and Shanghai Committee of Science and Technology (07QA14067).
2 These authors contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Xuetao Cao, Institute of Immunology, Tsinghua University School of Medicine, Beijing, People’s Republic of China. E-mail address: caoxt{at}public3.sta.net.cn
4 Abbreviations used in this paper: NSF, N-ethylmaleimide sensitive factor; SNARE, soluble NSF attachment protein receptor; v-SNARE, vesicular SNARE; t-SNARE, target SNARE; VAMP, vesicle-associated membrane protein; ER, endoplasmic reticulum; TGN, trans-Golgi network; EE, early endosome; PM, plasma membrane; SCAMP, secretory carrier membrane protein; EGF, epidermal growth factor; TMR, transmembrane region; hSCAMP5, human SCAMP5; BMSC, bone marrow stromal cell; siRNA, small interfering RNA; HDM, high density microsome; LDM, low density microsome; GDV, Golgi-derived vesicles; NPF, asparagine-proline-phenylalanine; SNAP-23, synaptosomal-associated protein of 23 kDa.
5 The online version of this article contains supplementary material.
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