The extracellular domain of several membrane-anchored proteins can be released as a soluble fragment by the action of a cell surface endoproteolytic system. This cleavage results in the generation of a soluble and a cell-bound fragment. In the case of proteins with signaling capability, such as tyrosine kinase receptors, the cleavage process may have an effect on the kinase activity of the cell-bound receptor fragment. By using several cell lines that express the TrkA neurotrophin receptor, we show that this receptor tyrosine kinase is cleaved by a proteolytic system that mimics the one that acts at the cell surface. TrkA cleavage is regulated by protein kinase C and several receptor agonists (including the TrkA ligand NGF), occurs at the ectodomain in a membrane-proximal region, and is independent of lysosomal function. TrkA cleavage results in the generation of a cell-associated fragment that is phosphorylated on tyrosine residues. Tyrosine phosphorylation of this fragment is not detected in TrkA mutants devoid of kinase activity, suggesting that phosphorylation requires an intact TrkA kinase domain, and is not due to activation of an intermediate intracellular tyrosine kinase. The increased phosphotyrosine content of the cell-bound fragment may thus reflect higher catalytic activity of the truncated fragment. We postulate that cleavage of receptor tyrosine kinases by this naturally occurring cellular mechanism may represent an additional mean for the regulation of receptor activity.

The ectodomain of proTGF-alpha, a membrane-anchored growth factor, is converted into soluble TGF-alpha by a regulated cellular proteolytic system that recognizes proTGF-alpha via the C-terminal valine of its cytoplasmic tail. In order to define the biochemical components involved in proTGF-alpha cleavage, we have used cells permeabilized with streptolysin O (SLO) that have been extensively washed to remove cytosol. PMA, acting through a Ca(2+)-independent protein kinase C, activates cleavage as efficiently in permeabilized cells as it does in intact cells. ProTGF-alpha cleavage is also stimulated by GTP gamma S through a mechanism whose pharmacological properties suggest the involvement of a heterotrimeric G protein acting upstream of the PMA-sensitive Ca(2+)-independent protein kinase C. Activated proTGF-alpha cleavage is dependent on ATP hydrolysis, appears not to require vesicular traffic, and acts specifically on proTGF-alpha that has reached the cell surface. These results indicate that proTGF-alpha is cleaved from the cell surface by a regulated system whose signaling, recognition, and proteolytic components are retained in cells devoid of cytosol.