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Intracellular Delivery of Bioactive Peptides to RBL‐2H3 Cells Induces β‐Hexosaminidase Secretion and Phospholipase D Activation
Author(s) -
Howl John,
Jones Sarah,
Farquhar Michelle
Publication year - 2003
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200300694
Subject(s) - protein kinase c , secretion , exocytosis , microbiology and biotechnology , phospholipase c , phospholipase d , intracellular , biology , gene isoform , secretory pathway , biochemistry , signal transduction , golgi apparatus , cell , gene
This investigation compared the secretory efficacies of a series of peptides delivered to the cytoplasm of RBL‐2H3 mast cells. Mimetic peptides, designed to target intracellular proteins that regulate cell signalling and membrane fusion, were synthesised as transportan 10 (TP10) chimeras for efficient plasma membrane translocation. Exocytosis of β‐hexosaminidase, a secretory lysosomal marker, indicated that peptides presenting sequences derived from protein kinase C (PKC; C1 H‐CRRLSVEIWDWDL‐NH 2 ) and the CB 1 cannabinoid receptor (C3 H‐RSKDLRHAFRSMFPSCE‐NH 2 ) induced β‐hexosaminidase secretion. Other peptide cargoes, including a Rab3A‐derived sequence and a homologue of C3, were inactive in similar assays. Translocated C1 also activated phospholipase D (PLD), an enzyme intimately involved in the regulated secretory response of RBL‐2H3 cells, but C1‐induced secretion was not dependent upon phosphatidate synthesis. Neither down‐regulation of Ca 2+ ‐sensitive isoforms of PKC nor the application of a selective PKC inhibitor attenuated the secretory efficacy of C1. These observations indicate that the molecular target of C1 is a protein involved in the regulated secretory pathway that is upstream of PLD but is not a PKC isoform. This study also confirmed that TP10 is a relatively inert cell‐penetrating vector and is, therefore, widely suitable for studies in cells that are sensitive to peptidyl secretagogues.