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 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, hyper tonic stress led to strong cleavage of HBO-EEG and UNRIG but only moderate cleavage of TAG-α. HBO-EEG, UNRIG, and TAG-α cleavage was not dependent on PC, and only HBO-EEG and UNRIG cleavage were inhibited by BB 94. In contrast, hobo 12-prostate-13-acetate (PA) -induced cleavage of I-IBM-EEG, UNRIG, and TAG-α was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-&alpha: 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. [ABSTRACT FROM AUTHOR]