Shedding of the Transferrin Receptor Is Mediated Constitutively by an Integral Membrane Metalloprotease Sensitive to Tumor Necrosis Factor α Protease Inhibitor-2

Abstract

The transferrin receptor (TfR) is a transmembrane protein that mediates cellular uptake of iron. Although the serum concentration of the soluble TfR (sTfR) is altered in several diseases and used for diagnostic purposes, the identity and regulation of the shedding protease is unknown. In this study we quantified sTfR release from microsomal membranes and leukocytic cell lines in the presence of numerous protease inhibitors and cell activating compounds. We show that sTfR release is mediated by an integral membrane metalloprotease and can be inhibited by matrix metalloproteinase inhibitor 2 and tumor necrosis factor ␣ protease inhibitor-2 (TAPI-2). Cleavage is also inhibited by a specific furin inhibitor, indicating that the protease is activated by a furin-like proprotein convertase. Whereas stimulation of the cells by the ectodomain shedding activator phorbol 12-N-myristate 13-acetate did not alter sTfR release significantly, the phosphatase inhibitor pervanadate led to an increase of TfR shedding in several leukocytic cell lines. Our results suggest that TfR shedding is constitutively mediated by a member of the metalloprotease family known as ADAM (for a disintegrin and metalloprotease).The transferrin receptor (TfR) 1 is a type II transmembrane protein that mediates uptake of iron by binding the iron carrier protein transferrin (Tf). Following internalization of the complex, iron is released in the acidic endosomes and the TfR⅐Tf complex recycles back to the cell surface where apotransferrin is released at neutral pH. The TfR is composed of two homologous peptide chains of 760 amino acids linked by two disulfide bonds (Cys-89 and Cys-98) close to the transmembrane domain. Each polypeptide is divided into a large C-terminal extracellular domain of 672 amino acids, a transmembrane domain of 21 amino acids, and an N-terminal cytoplasmic domain of 67 amino acids. The extracellular domain that binds Tf is kept by a juxtamembrane stalk at 2.9 nm from the plasma membrane (1). Within the stalk Thr-104 is O-glycosylated (2, 3), but detailed structure and function of the stalk remain unknown.Although the appearance of the soluble form of the transferrin receptor (sTfR) in human serum has been known for quite a long time (4) and its concentration is accepted as diagnostic marker for erythropoietic activity (5, 6), little is known about the molecular basis of the shedding process, in particular the nature of the shedding protease involved. In diseases accompanied by enhanced erythropoiesis or TfR expression, like iron deficiency anemia, increased serum sTfR levels are observed (7-10), whereas lower levels of serum sTfR were determined in patients with aplastic anemia (8). The sTfR level remains unchanged in anemia arising from chronic disease, so that it is a reliable marker to distinguish iron deficiency anemia from the anemia of chronic inflammation and liver disease (11). It was shown that a human red blood cell fraction does not have an own TfR-specific proteolytic activity, whereas a white blood cell fraction doe...

Redox regulation of CD21 shedding involves signaling via PKC and indicates the formation of a juxtamembrane stalk

“…This supports previous experiments where we induced CD21 shedding by BCR/CD40 crosslinking and by PMA/CaI stimulation (Masilamani et al, 2003a). Recent reports have shown that shedding of several other proteins such as pro-EGF (Le Gall et al, 2003), the transferrin receptor (Kaup et al, 2002) or betaglycan (Velasco-Loyden et al, 2004) are also activated by pervanadate.…”

Stroke-Induced Subventricular Zone Proliferation is Promoted by Tumor Necrosis Factor-α-Converting Enzyme Protease Activity

“…TAPI-2 has been employed as a potent inhibitor of TACE proteolysis in vivo and in vitro (Kaup et al, 2002;Lomniczi et al, 2006). However, ADAM10 is capable of analogous a-cleavage of APP and TNFa, and like TACE, activates many EGFR proligands as well (Blobel, 2005;Kaup et al, 2002;Sahin et al, 2004). Here, we found ADAM10 transcription was increased several-fold in the SVZ following stroke.…”

Transferrin receptor 2 is a component of the erythropoietin receptor complex and is required for efficient erythropoiesis

“…24 Alternatively, the lower molecular weight form could be the result of proteolytic events that only occur in some cell types, such as erythroblasts in the case of TfR1. 25,26 The association between EpoR and TfR2 was found to be constitutive, although association of TfR2 with the cell surface form of EpoR was always slightly elevated in Epo-stimulated cells (Figure 2A), suggesting that Epo binding could strengthen the association between both proteins. EpoR western blots further revealed 2 bands that have been already thoroughly characterized and that differ in size due to glycosylation.…”