PROTEOLYTIC RELEASE OF MEMBRANE-PROTEINS - STUDIES ON A MEMBRANE-PROTEIN-SOLUBILIZING ACTIVITY IN CHO CELLS

Diverse membrane proteins are solubilized by a specific proteolytic cl eavage in the stalk sequence adjacent to the membrane anchor, with rel ease of the extracellular domain. Examples are the amyloid precursor p rotein, membrane-bound growth factors and angiotensin-converting enzym e (ACE). The identities and characteristics of the responsible proteas es remain elusive. We have studied this process in Chinese hamster ova ry (CHO) cells stably expressing wild-type ACE (WT-ACE) or juxtamembra ne (stalk) deletion or chimaera mutants. Determination of the C termin i (i.e. the cleavage sites) of released, soluble wild-type and mutant ACE by MALDI-TOF mass spectrometry indicated that the membrane-protein -solubilizing protease (MPSP) in CHO cells is not constrained by a par ticular cleavage site motif or by a specific distance from the membran e, but instead may position itself with respect to the putative proxim al, folded extracellular domain adjacent to the stalk. Nevertheless, k inetic analyses of release rates indicated that a minimum distance fro m the membrane must be preserved. Interestingly, soluble full-length ( anchor-plus) WT-ACE incubated with fractions of, or intact, CHO cells was not cleaved. In all cases, release was stimulated by a media chang e or by the addition of phorbol ester, with rate enhancements of 5- an d 50-fold, respectively, for WT-ACE. The phorbol ester effect was abol ished by staurosporine, a protein kinase C (PKC) inhibitor. We propose that the CHO cell MPSP that solubilizes ACE: (1) only cleaves protein s embedded in a membrane; (2) requires an accessible stalk and cleaves at a minimum distance from both the membrane and proximal extracellul ar domain; (3) positions itself primarily with respect to the proximal extracellular domain and (4) is regulated in part by a PKC-dependent mechanism.