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Ectodomain cleavage of the EGF ligands HB‐EGF, neuregulin1‐β, and TGF‐α is specifically triggered by different stimuli and involves different PKC isoenzymes
Author(s) -
Herrlich Andreas,
Klinman Eva,
Fu Jonathan,
Sadegh Cameron,
Lodish Harvey
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.08-113852
Subject(s) - ectodomain , epidermal growth factor , protein kinase c , microbiology and biotechnology , cleavage (geology) , chemistry , lysophosphatidic acid , tgf alpha , adam10 , signal transduction , heparin binding egf like growth factor , metalloproteinase , biology , receptor , biochemistry , matrix metalloproteinase , disintegrin , paleontology , fracture (geology)
Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparinbinding EGF‐like growth factor (HB‐EGF), neuregulin (NRG), and transforming growth factor‐alpha (TGF‐α), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage— hypertonic stress, phorbol ester, or activation of G‐ protein‐coupled receptors [ e.g ., by lysophosphatidic acid (LPA)]—are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence‐activated cell sorting (FACS) ‐based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB‐EGF and NRG but only moderate cleavage of TGF‐α. HB‐EGF, NRG, and TGF‐α cleavage was not dependent on PKC, and only HB‐EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12‐myristate‐13‐acetate (TPA) ‐induced cleavage of HB‐EGF, NRG, and TGF‐α was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF‐α and was inhibited by BB94;only LPA‐induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacyl‐ glycerol (DAG) and calcium] significantly enhanced TPA‐induced NRG cleavage. Employed in a high‐ throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different extracellular stimuli into ectodomain cleavage.— Herrlich, A., Klinman, E., Fu, J., Sadegh, C., Lodish, H. Ectodomain cleavage of the EGF ligands HB‐EGF, neuregulin1‐β, and TGF‐α is specifically triggered by different stimuli and involves different PKC isoenzymes. FASEB J. 22, 4281–4295 (2008)