Transforming Growth Factor-t and 13-Amyloid Precursor Protein Share a Secretory Mechanism

Cleavage and release of membrane protein ectodomains, a regulated process that affects many cell surface proteins, remains largely uncharacterized. To investigate whether cell surface proteins are cleaved through a shared mechanism or through multiple independent mechanisms, we mutagenized Chinese hamster ovary (CHO) cells and selected clones that were unable to cleave membrane-anchored transforming growth factor o~ (TGF-a). The defect in TGF-a cleavage in these clones is most apparent upon cell treatment with the protein kinase C (PKC) activator PMA, which stimulates TGF-ct cleavage in wild-type cells. The mutant clones do not have defects in TFG-ct expression, transport to the cell surface or turnover. Concomitant with the loss of TGF-ot cleavage, these clones have lost the ability to cleave many structurally unrelated membrane proteins in response to PMA. These proteins include ~-amyloid precursor protein (/3-APP), whose cleavage into a secreted form avoids conversion into the amyloidogenic peptide A/~, and a group of cell surface proteins whose release into the medium is stimulated by PMA in wild type CHO cells but not in mutants. The mutations prevent cleavage by PKC-dependent as well as PKC-independent mechanisms, and thus affect an essential component that functions downstream of these various signaling mechanisms. We propose that regulated cleavage and secretion of membrane protein ectodomains is mediated by a common system whose components respond to multiple activators and act on susceptible proteins of diverse structure and function. large group of membrane proteins undergo cleavage with release of their ectodomain into the extracelular medium (Ehlers and Riordan, 1991). This group includes membrane-anchored growth factors (Massagu6 and Pandiella, 1993), growth factor receptors, cell adhesion molecules, ectoenzymes and molecules of unknown function such as the ~-amyloid precursor protein (Ehlers and Riordan, 1991; Massagu6 and Pandiella, 1993). Membraneanchored growth factors and ectoenzymes provide localized concentrations of these activities, and their cleavage yields soluble forms that can diffuse and act at a distance from the site of production (Ehlers and Riordan, 1991; Massagu6 and Pandiella, 1993). The membrane-anchored forms of these proteins may be biologically more effective than the soluble forms, as in the case of kit ligand/stem cell growth factor whose soluble form cannot substitute for its membraneanchored form in critical aspects of mouse development (Flanagan et al., 1991). Cleavage switches the role of growth factor receptors and cell adhesion molecules by converting them into soluble antagonists of the factors they bind (Fermindez-Botran, 1991; Gordon, 1991). Cleavage of~-amyloid precursor protein (/~-APP) 1 releases a large soluble form (APPs) that escapes the intracellular degradative process that yields AB (Esch et al., 1990; Sisodia et al., 1990; Golde et al., 1992; Haass and Selkoe, 1993). Deposits of insoluble A/3 are invariably associated with Alzheimer's disease, suggesting that defects in ~-APP release may play a role in the pathogenesis of this disease (Haass and Selkoe, 1993). Thus, cleavage and secretion of membrane protein ectodomains fulfills different biological roles depending on the protein, and alterations in this process may lead to disease. Cleavage of membrane protein ectodomains is often a regulated process. The extracellular domains of proTGF-ot (Pandiella and Massagu6, 1991a), kit ligand (Huang et al., 1992), colony-stimulating factor 1 (CSF-1) (Stein and Rettenmier, 1991), CSF-1 receptor (Downing et al., 1989), tumor necrosis factor ct (TNF-ot) receptors (Porteu and Nathan, 1990; Porteu et al., 1991), IL-6 receptor (Serra-Pages et al., 1994), LAR transmembrane protein tyrosine phosphatase (Miillberg et al., 1992), ~APP protein (Buxbaum et Address all correspondence to J. Massague, Memorial Sloan-Kettering Cancer Center, Box 116, 1275 York Ave., New York, NY 10021. Tel (212) 639-8975. Fax: (212) 717-3298. 1. Abbreviations used in this paper: ~-APP, ~-amyloid precursor protein; CHO, Chinese hamster ovary; CSF, colony-stimulating factor; HA, hemagglutinin epitope; PKC, protein kinase C. © The Rockefeller University Press, 0021-9525/95/02/433/9 $2.00 The Journal of Cell Biology, Volume 128, Number 3, February 1995 433-441 433 on F ebuary 3, 2013 jcb.rress. org D ow nladed fom Published February 1, 1995