title: Adaptor protein-3: A key player in RBL-2H3 mast cell mediator release.
abstract: Mast cell (MC) secretory granules are Lysosome-Related Organelles (LROs) whose biogenesis is associated with the post-Golgi secretory and endocytic pathways in which the sorting of proteins destined for a specific organelle relies on the recognition of sorting signals by adaptor proteins that direct their incorporation into transport vesicles. The adaptor protein 3 (AP-3) complex mediates protein trafficking between the trans-Golgi network (TGN) and late endosomes, lysosomes, and LROs. AP-3 has a recognized role in LROs biogenesis and regulated secretion in several cell types, including many immune cells such as neutrophils, natural killer cells, and cytotoxic T lymphocytes. However, the relevance of AP-3 for these processes in MCs has not been previously investigated. AP-3 was found to be expressed and distributed in a punctate fashion in rat peritoneal mast cells ex vivo. The rat MC line RBL-2H3 was used as a model system to investigate the role of AP-3 in mast cell secretory granule biogenesis and mediator release. By immunofluorescence and immunoelectron microscopy, AP-3 was localized both to the TGN and early endosomes indicating that AP-3 dependent sorting of proteins to MC secretory granules originates in these organelles. ShRNA mediated depletion of the AP-3 δ subunit was shown to destabilize the AP-3 complex in RBL-2H3 MCs. AP-3 knockdown significantly affected MC regulated secretion of β-hexosaminidase without affecting total cellular enzyme levels. Morphometric evaluation of MC secretory granules by electron microscopy revealed that the area of MC secretory granules in AP-3 knockdown MCs was significantly increased, indicating that AP-3 is involved in MC secretory granule biogenesis. Furthermore, AP-3 knockdown had a selective impact on the secretion of newly formed and newly synthesized mediators. These results show for the first time that AP-3 plays a critical role in secretory granule biogenesis and mediator release in MCs.
authors.affiliation: Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School - University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Elaine Zayas Marcelino
authors.initials: EZ
authors.lastname: da Silva
authors.affiliation: Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School - University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Edismauro Garcia
authors.initials: EG
authors.lastname: Freitas-Filho
authors.affiliation: Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School - University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Devandir Antonio
authors.initials: DA
authors.lastname: de Souza-Júnior
authors.affiliation: Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School - University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Luis Lamberti Pinto
authors.initials: LL
authors.lastname: daSilva
authors.affiliation: Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School - University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Maria Celia
authors.initials: MC
authors.lastname: Jamur
authors.affiliation: Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School - University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Constance
authors.identifier: http://orcid.org/0000-0002-8633-4612
authors.initials: C
authors.lastname: Oliver
mesh_terms: D033963:Adaptor Protein Complex 3 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015415:Biomarkers; D015550:Cell Degranulation / Q000235:genetics; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D004705:Endocytosis; D005260:Female; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D018836:Inflammation Mediators / Q000378:metabolism; D008297:Male; D008407:Mast Cells / Q000276:immunology / Q000378:metabolism*; D055550:Protein Stability; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017455:Receptors, IgE / Q000235:genetics / Q000378:metabolism
chemical_list: D033963:Adaptor Protein Complex 3; D015415:Biomarkers; D016207:Cytokines; D018836:Inflammation Mediators; D034741:RNA, Small Interfering; D017455:Receptors, IgE