Impaired Trafficking and Activation of Tumor Necrosis Factor-α-converting Enzyme in Cell Mutants Defective in Protein Ectodomain Shedding

G protein-coupled receptors can trigger metalloproteinase-dependent shedding of proteins from the cell surface. We now report that G protein-coupled receptors can themselves undergo regulated metalloproteinasedependent shedding. The N-terminal exodomain of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, displayed regulated shedding in endothelial cells, which normally express this receptor. Cleavage occurred at a site predicted to render the receptor unresponsive to thrombin. A chimeric protein in which the N-terminal exodomain of PAR1 was fused to an unrelated transmembrane segment was shed as efficiently as PAR1, shedding of both proteins was stimulated by phorbol ester and by a PAR1 agonist. TNF␣ protease inhibitor-2 (TAPI-2), phenanthroline, and tissue inhibitor of metalloproteinase-3 (TIMP-3) but not TIMP-1 or -2 inhibited such shedding. These and other data suggest that the information that specifies PAR1 shedding resides within its N-terminal exodomain rather than its heptahelical segment, that activation of protein kinase C or of PAR1 itself can stimulate PAR1 shedding in trans, and that ADAM17/TACE or a metalloproteinase with similar properties mediates PAR1 shedding. Regulated shedding reduced the amount of cell surface PAR1 available for productive cleavage by thrombin by half or more, but thus far we have been unable to demonstrate an effect of PAR1 shedding on cellular responsiveness to thrombin. Nonetheless, regulated shedding of G protein-coupled receptors represents a new mechanism by which signaling by this important class of receptors might be modulated.

Section: Resultsmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Regulated Shedding of PAR1 N-terminal Exodomain from Endothelial Cells

Ludeman

Zheng

Ishii

et al. 2004

“…2B). The stimulated shedding of numerous proteins, including several TACE substrates, in response to agents such as phorbol esters is absent in the M2 CHO cell line (5,10,70,72). We therefore assessed the ability of this cell line to shed p75 NTR when stimulated.…”

Section: Resultsmentioning

confidence: 99%

“…Initially, we used wild-type CHO cells and the previously described M2 mutant CHO cells, which are defective in the metalloprotease-mediated shedding of several transmembrane proteins (5). The mutation in the M2 cells inactivates the TNF␣ convertase (TACE, ADAM17) 2 and abolishes the stimulated shedding of all TACE substrates examined to date (69,70). Cells transiently transfected with either a control vector or a plasmid encoding for rat p75 NTR were pulse-labeled.…”

Section: Resultsmentioning

confidence: 99%

Evidence for a Critical Role of the Tumor Necrosis Factor α Convertase (TACE) in Ectodomain Shedding of the p75 Neurotrophin Receptor (p75NTR)

Weskamp

Schlöndorff

Lum

et al. 2004

Self Cite

138

114

Protein ectodomain shedding, the proteolytic release of the extracellullar domain of membrane-tethered proteins, can dramatically affect the function of cell surface receptors, growth factors, cytokines, and other proteins. In this study, we evaluated the activities involved in ectodomain shedding of p75 NTR , a neurotrophin receptor with critical roles in neuronal differentiation and survival. p75 NTR is shed in a variety of cell types, including dorsal root ganglia cells and PC12 cells. In Chinese hamster ovary cells, inhibitors of the MEK/ERK and p38 MAP kinase pathways uncovered distinct signaling pathways required for the constitutive and stimulated shedding of p75 NTR . Stimulated p75 NTR shedding is abrogated in M2 mutant Chinese hamster ovary cells that lack functional tumor necrosis factor-␣ converting enzyme (TACE, also referred to as ADAM17) and in cells isolated from adam17؊/؊ mice, but not in cells from adam9/12/15؊/؊ or adam10؊/؊ mice. Stimulated p75 NTR shedding is strongly reduced by deletion of 15 amino acid residues in its extracellular membrane-proximal stalk domain. However, similar to other shed proteins, point mutations and overlapping shorter deletions within this region have little or no effect on shedding. Because ectodomain shedding of p75 NTR releases a soluble ectodomain and could also be a prerequisite for its regulated intramembrane proteolysis, these findings may have important implications for the functional regulation of p75 NTR .Protein ectodomain shedding is emerging as an important post-translational mechanism for regulating the function of membrane-anchored proteins. Shedding involves the proteolytic processing of membrane-tethered proteins leading to the release of their extracellular-or ectodomain (reviewed in Refs. 1-4). About 2-4% of the proteins on the cell surface are released by metalloproteases in response to phorbol ester stimulation (5). These molecules comprise a variety of structurally and functionally distinct proteins, including epidermal growth factor receptor ligands such as TGF␣ 1 and HB-EGF, TNF family members, and other cytokines, receptors such as the p55 and p75 TNF receptors and the interleukin-6 receptor, and several other proteins, including the amyloid precursor protein, Notch, and Delta (see Ref. 3 and references therein). Ectodomain shedding has been shown to be essential for proper signaling via epidermal growth factor receptor ligands (6 -15), Notch-mediated lateral inhibition (16 -23), limiting TNFR-mediated inflammatory reactions (24), and regulating axonal guidance (25)(26)(27). Even if the functional consequences of ectodomain release remain to be evaluated for most shed proteins, it seems likely that ectodomain shedding will affect the function of the majority of its substrate proteins.Despite the growing number of proteins that are known to undergo ectodomain shedding, much remains to be learned about the mechanisms underlying this process and the responsible proteases. The TNF␣ convertase (TACE) is one of the first proteases shown to be in...

“…It has been shown recently that this mutant cell line has a specific defect that prevents the activation of TACE, but not other metalloproteases (45). Therefore, to determine whether active TACE participates in the shedding of betaglycan induced by pervanadate, we analyze the effect of pervanadate on the M1 mutant and on the wild type CHO cells that were stably transfected with HA-tagged betaglycan (HABG construct, see Fig.…”

Section: Pervanadate Activates the Shedding Of Betaglycan-ectodo-mentioning

confidence: 99%

The Shedding of Betaglycan Is Regulated by Pervanadate and Mediated by Membrane Type Matrix Metalloprotease-1

Velasco-Loyden

Arribas

López‐Casillas

2004

Self Cite

125

Betaglycan is a membrane-anchored proteoglycan that binds transforming growth factor-␤ (TGF-␤) via its core protein. A soluble form of betaglycan can be released by proteolytic cleavage (also known as shedding) of the membrane-bound form, yielding soluble betaglycan. The mechanism leading to the generation of soluble betaglycan is poorly understood. Because the membrane and soluble forms of betaglycan have opposite effects regulating the availability of TGF-␤, it is important to characterize the shedding of betaglycan further. Here we present evidence showing that in certain cell types, pervanadate, a general tyrosine phosphatase inhibitor, induces the release of the previously described fragment that encompasses almost the entire extracellular domain of betaglycan (sBG-120). In addition, treatment with pervanadate unveils the existence of a novel 90-kDa fragment (sBG-90). Noticeably, the cleavage that generates sBG-90 is mediated by a tissue inhibitor of metalloprotease-2-sensitive protease. Overexpression of all membrane type matrix metalloproteases (MT-MMPs) described to date indicates that MT1-MMP and MT3-MMP are endowed with ability to generate sBG-90. Furthermore, the patterns of expression of different MTMMPs in the cell lines used in this study suggest that MT1-MMP is the protease involved in the shedding of sBG-90. Overexpression of MT1-MMP in COS-1 cells, which do not express detectable levels of this metalloprotease, confirms the feasibility of this hypothesis. Unexpectedly, during the course of these experiments, we observed that MT2-MMP decreases the levels of MT1-MMP and betaglycan. Finally, binding competition experiments indicate that, similar to the wild type membrane betaglycan, sBG-90 binds the TGF-␤2 isoform with greater affinity than TGF-␤1, suggesting that once released, it could affect the cellular availability of TGF-␤.