The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGF␣, neuregulin 1␤ (NRG), and heparinbinding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substratespecific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGF␣ and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKC enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.Metalloprotease-mediated ectodomain cleavage of transmembrane proteins such as EGF ligand precursors is involved in the regulation of many physiological signaling pathways, and its dysregulation can cause kidney disease, heart disease, and cancer (1, 2). For example, in the kidney, many of the deleterious effects of chronic exposure to the G-protein-coupled receptor (GPCR) 2 agonist angiotensin II are mediated by metalloproteinase (ADAM17)-dependent release of the EGF ligand TGF␣ and subsequent epidermal growth factor receptor activation. Angiotensin II is the main effector of the renin-angiotensin system and has important roles in the regulation of blood pressure and aldosterone secretion (3).Although several ADAM gene disruptions are lethal in the mouse, metalloprotease inhibitors have shown therapeutic potential; however, not unexpectedly, the essential role of ADAMs in normal physiology as well as the broad spectrum nature of currently available inhibitors are probably the cause for a number of their side effects. In addition, concerns have been raised that some metalloproteases act as tumor suppressors (4,5). A more precise inhibiti...