Activated release of membrane-anchored TGF-α in the absence of cytosol

Abstract

The ectodomain of proTGF-α, a membraneanchored growth factor, is converted into soluble TGF-α by a regulated cellular proteolytic system that recognizes proTGF-α via the C-terminal valine of its cytoplasmic tail. In order to define the biochemical components involved in proTGF-α cleavage, we have used cells permeabilized with streptolysin O (SLO) that have been extensively washed to remove cytosol. PMA, acting through a Ca2+-independent protein kinase C, activates cleavage as efficiently in permeabilized cells as it does in intact cells. ProTGF-α cleavage is also stimulated by GTPγS through a mechanism whose pharmacological properties suggest the involvement of a heterotrimeric G protein acting upstream of the PMA-sensitive Ca2+-independent protein kinase C. Activated proTGF-α cleavage is dependent on ATP hydrolysis, appears not to require vesicular traffic, and acts specifically on proTGF-α that has reached the cell surface. These results indicate that proTGF-α is cleaved from the cell surface by a regulated system whose signaling, recognition, and proteolytic components are retained in cells devoid of cytosol.