Intracellular delivery of bioactive peptides to RBL-2H3 cells induces β-hexosaminidase secretion and phospholipase D activation

被引:18
作者
Howl, J [1 ]
Jones, S [1 ]
Farquhar, M [1 ]
机构
[1] Wolverhampton Univ, Sch Appl Sci, Mol Pharmacol Grp, Wolverhampton WV1 1SB, England
关键词
enzymes; mast cells; peptidomimetics; secretion; transportan;
D O I
10.1002/cbic.200300694
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 trasportan 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 CRRLSVEIWDWL-NH) and the CB1 cannabinoid receptor (C3 H RSKDLRHAFRSMFPOSCE-NH) induced beta-hexosamindase secretion. Other peptide cargoes, including a Rab3A-derived sequence and a homologue of C3, were inactive in similar assay. 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 Ca2+-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 nut 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.
引用
收藏
页码:1312 / 1316
页数:5
相关论文
共 35 条
[1]   Restricted tissue expression pattern of a novel human rasGAP-related gene and its murine ortholog [J].
Allen, M ;
Chu, S ;
Brill, S ;
Stotler, C ;
Buckler, A .
GENE, 1998, 218 (1-2) :17-25
[2]   ACTIVATION OF EXOCYTOSIS BY THE HETEROTRIMERIC-G PROTEIN-G(I3) [J].
ARIDOR, M ;
RAJMILEVICH, G ;
BEAVEN, MA ;
SAGIEISENBERG, R .
SCIENCE, 1993, 262 (5139) :1569-1572
[3]   Phospholipase D1 localises to secretory granules and lysosomes and is plasma-membrane translocated on cellular stimulation [J].
Brown, FD ;
Thompson, N ;
Saqib, KM ;
Clark, JM ;
Powner, D ;
Thompson, NT ;
Solari, R ;
Wakelam, MJO .
CURRENT BIOLOGY, 1998, 8 (14) :835-838
[4]   Mastoparan selectively activates phospholipase D2 in cell membranes [J].
Chahdi, A ;
Choi, WS ;
Kim, YM ;
Beaven, MA .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (14) :12039-12045
[5]   EVIDENCE THAT THE RAB3A-BINDING PROTEIN, RABPHILIN3A, ENHANCES REGULATED SECRETION - STUDIES IN ADRENAL CHROMAFFIN CELLS [J].
CHUNG, SH ;
TAKAI, Y ;
HOLZ, RW .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (28) :16714-16718
[6]   Novel mastoparan analogs induce differential secretion from mast cells [J].
Farquhar, M ;
Soomets, U ;
Bates, RL ;
Martin, A ;
Langel, Ü ;
Howl, J .
CHEMISTRY & BIOLOGY, 2002, 9 (01) :63-70
[7]  
FARQUHAR M, UNPUB
[8]  
FARQUHAR M, 2002, THESIS U WOLVERHAMPT
[9]  
HOWL J, 1993, METHODS NEUROSCI, V13, P281
[10]   Characterization of CB1 cannabinoid receptors using receptor peptide fragments and site-directed antibodies [J].
Howlett, AC ;
Song, C ;
Berglund, BA ;
Wilken, GH ;
Pigg, JJ .
MOLECULAR PHARMACOLOGY, 1998, 53 (03) :504-510