Intracellular delivery of bioactive peptides to RBL-2H3 cells induces beta-hexosaminidase secretion and phospholipase D activation.
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 beta-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 beta-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.
Howl
John
J
Molecular Pharmacology Group, School of Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton, WV1 1SB, UK. j.howl@wlv.ac.uk
Jones
Sarah
S
Farquhar
Michelle
M
D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D020404:Glycerophospholipids / Q000378:metabolism; D008407:Mast Cells / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology*; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000235:genetics; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology*; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism*; D020826:rab3A GTP-Binding Protein / Q000235:genetics
D020404:Glycerophospholipids; D010455:Peptides; D043884:Receptor, Cannabinoid, CB1; D011993:Recombinant Fusion Proteins; C068564:phosphatidylbutanol; D011493:Protein Kinase C; D010739:Phospholipase D; D001619:beta-N-Acetylhexosaminidases; D020826:rab3A GTP-Binding Protein