Heregulin was shown to promote the proteolytic cleavage of its receptor, ErbB-4, in several cell lines. The growth factor also rapidly promoted the transient translocation of ErbB-4 to a detergent-insoluble fraction, in which the receptor was hyper-tyrosine-phosphorylated compared with the receptor present in the detergentsoluble pool. However, an 80-kDa proteolytic fragment of ErbB-4 was found in the detergent-soluble fraction, but not in the detergent-insoluble fraction. Although the heregulin-induced cleavage of ErbB-4 produced a fragment of ErbB-4 very similar to that induced by 12-Otetradecanoylphorbol-13-acetate or pervanadate (each of which is blocked by metalloprotease inhibitors), the growth factor-induced cleavage was not sensitive to these inhibitors under the same conditions. The heregulin-induced cleavage of ErbB-4 could be blocked by conditions that prevent clathrin-coated pit formation, suggesting that heregulin-mediated ErbB-4 cleavage occurs subsequent to internalization. When reagents that prevent acidification of endosomes were employed, heregulin-induced ErbB-4 cleavage was sensitive to metalloprotease inhibitors. The results imply that during liganddependent receptor trafficking, activated ErbB-4 receptors are subject to proteolytic cleavage involving an intracellular metalloprotease.ErbB-4 is a member of the epidermal growth factor family of receptor tyrosine kinases (1) and is expressed in many normal adult and fetal tissues, particularly nerve and heart (2). However, its expression in carcinoma cells is limited compared with other ErbB receptors (2-4). Mice lacking ErbB-4 die during mid-embryogenesis from aborted development of heart ventricle myocardial trabeculae and are also deficient in axon guidance during development of the central nervous system (5, 6). Although all the EGF 1 receptor family members are able to stimulate cell proliferation, ErbB-4 is also implicated as a positive regulator of the differentiation of certain epithelial and neuronal tissues (7-9). Ligands for the ErbB-4 receptor include various heregulin isoforms, which also bind to ErbB-3 (10, 11), and betacellulin (10, 12), which also binds to the EGF receptor (12). Following ligand stimulation, the ErbB-4 receptor undergoes homodimerization and/or heterodimerization with other receptors of the ErbB family and initiates cellular responses (13,14).Desensitization and down-regulation of activated growth factor receptors have an important role in regulating cellular events triggered by ligand binding and determine signaling duration and/or potency. A common pathway for down-regulation of many activated growth factor receptors is clathrincoated pit-mediated endocytosis (15). In the case of the EGF receptor, the activated receptor rapidly enters clathrin-coated pits and is internalized and subsequently sorted from endosomes to lysosomes, where the receptor and its ligand are degraded (15, 16). However, studies with NIH 3T3 cells expressing wild-type ErbB receptors or chimeric receptors and some ErbB-expressing human carc...