TY - JOUR
T1 - Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-β, and TGF-α is specifically triggered by different stimuli and involves different PKC isoenzymes
AU - Herrlich, Andreas
AU - Klinman, Eva
AU - Fu, Jonathan
AU - Sadegh, Cameron
AU - Lodish, Harvey
PY - 2008/12
Y1 - 2008/12
N2 - Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding 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 batimas-tat (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 diacylglycerol (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.
AB - Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding 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 batimas-tat (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 diacylglycerol (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.
KW - EGF receptor
KW - G-protein
KW - Osmotic stress
UR - http://www.scopus.com/inward/record.url?scp=57349158779&partnerID=8YFLogxK
U2 - 10.1096/fj.08-113852
DO - 10.1096/fj.08-113852
M3 - Article
C2 - 18757500
AN - SCOPUS:57349158779
SN - 0892-6638
VL - 22
SP - 4281
EP - 4295
JO - FASEB Journal
JF - FASEB Journal
IS - 12
ER -