Diverse Cell Surface Protein Ectodomains Are Shed by a System Sensitive to Metalloprotease Inhibitors

Abstract

The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membraneanchored growth factor transforming growth factor ␣ precursor (proTGF-␣) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having the same recessive phenotype and belonging to the same genetic complementation group. Furthermore, two structurally distinct agents, TAPI-2 and 1,10-phenanthroline, which are known to inhibit metalloproteases, block PMA-activated shedding of proTGF-␣, cell adhesion receptor L-selectin, interleukin 6 receptor ␣ subunit, ␤-amyloid precursor protein, and an entire set of anonymous Chinese hamster ovary cell surface proteins. Certain serine protease inhibitors prevent release of these proteins by interfering with their maturation and transport to the cell surface but do not inhibit ectodomain shedding from the cell surface. The results suggest the existence of a common system for membrane protein ectodomain shedding involving one or several proteolytic activities sensitive to metalloprotease inhibitors, whose ability to act can be disrupted by recessive mutations in a single gene.

Stable expression of chimeric anti-CD3 receptors on mammalian cells for stimulation of antitumor immunity

“…It seems likely that the introduction of oligosaccharide chains into the chimeric receptors hindered access to proteases since many integral membrane proteins are shed by proteolytic cleavage. [34][35][36] Our results clearly demonstrate that the level of surface expression correlated with the degree of linker glycosylation (Fig 2c). Control of surface shedding by variation of glycosylation may be useful for tuning the retention of chimeric proteins on cells.…”

Syndecans: multifunctional cell-surface co-receptors

“…A large number of transmembrane proteins, including membrane-anchored growth factors, celladhesion molecules, cytokine receptors and some enzymes, are proteolytically released from the plasma membrane [61]. This process has been shown to be stimulated by phorbol esters, although the target proteins themselves are not subject to protein kinase C-dependent phosphorylation.…”

“…Mutant cell lines have been isolated that are defective in the shedding of a diverse set of proteins. Independently isolated mutant cell lines have been shown to belong to the same genetic complementation group [61]. Moreover, the shedding of these proteins can be inhibited by treatment of cells with inhibitors of metalloproteinase activity.…”

Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites

“…Most or all of the established physiological functions of ACE can be ascribed to the membrane-anchored form [1,18]. However, shedding of ACE is likely to be tightly regulated, as has been shown for other shed membrane proteins, such as TNF-α, L-selectin and the amyloid precursor protein [19]. Testis ACE is released after cleavage at an Arg-Ser bond 24 residues proximal to the TM domain [20,21].…”