Mary Gerhart

TRAIL‐R2: a novel apoptosis‐mediating receptor for TRAIL

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL‐R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL‐R2, by ligand‐based affinity purification and subsequent molecular cloning. TRAIL‐R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL‐R2 contains two extracellular cysteine‐rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell‐surface‐expressed TRAIL‐R2, and TRAIL‐induced apoptosis is inhibited by a TRAIL‐R2–Fc fusion protein. TRAIL‐R2 mRNA is widely expressed and the gene encoding TRAIL‐R2 is located on human chromosome 8p22‐21. Like TRAIL‐R1, TRAIL‐R2 engages a caspase‐dependent apoptotic pathway but, in contrast to TRAIL‐R1, TRAIL‐R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family.

Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability.

We previously implicated tumor necrosis factor-α converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-α (TGF-α), pro-TGF-α. Here we examined TGF-α processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-α as compared with wild-type cultures and that TGF-α cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-α and TACE cDNAs increased shedding of mature TGF-α with concomitant conversion of cell-associated pro-TGF-α to a processed form. Purified TACE accurately cleaved pro-TGF-α in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-α containing only the ectodomain at the C-terminal site. In vitro, TACE accurately cleaved peptides corresponding to cleavage sites of several epidermal growth factor (EGF) family members, and transfection of TACE into TACE-deficient cells increased the shedding of amphiregulin and heparin-binding EGF (HB-EGF) proteins. Consistent with the hypothesis that TACE regulates EGF receptor (EGFR) ligand availability in vivo, mice heterozygous for Tace and homozygous for an impaired EGFR allele (wa-2) were born with open eyes significantly more often thanTace +/+ Egfr wa-2 / wa-2 counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.

T1/ST2 Signaling Establishes It as a Member of an Expanding Interleukin-1 Receptor Family

Through data base searches, we have discovered new proteins that share homology with the signaling domain of the type I interleukin-1 receptor (IL-1RI): human “randomly sequenced cDNA 786” (rsc786), murine MyD88, and two partial Drosophila open reading frames, MstProx and STSDm2245. Comparisons between these new proteins and known IL-1RI homologous proteins such as Toll, 18-Wheeler, and T1/ST2 revealed six clusters of amino acid similarity. We tested the hypothesis that sequence similarity between the signaling domain of IL-1RI and the three mammalian family members might indicate functional similarity. Chimeric IL-1RI receptors expressing the putative signaling domains of T1/ST2, MyD88, and rsc786 were assayed by three separate IL-1 responsive assays, NF-κB, phosphorylation of an epidermal growth factor receptor peptide, and an interleukin 8 promoter-controlled reporter construct, for their ability to transduce an IL-1-stimulated signal. All three assays were positive in response to the T1/ST2 chimera, while the MyD88 and rsc786 chimeras failed to respond. These data indicate that the sequence homology between IL-1RI and T1/ST2 indicates a functional homology as well.

TACE/ADAM17 Processing of EGFR Ligands Indicates a Role as a Physiological Convertase

Abstract: EGF family growth factors, including transforming growth factor‐alpha (TGFα), amphiregulin (AR), and heparin‐binding EGF (HB‐EGF), are invariably expressed as transmembrane precursors that are cleaved at one or more sites in the extracellular domain to release soluble growth factor. Considerable attention has focused on the identification of proteases responsible for these processing events. We previously implicated tumor necrosis factor‐alpha converting enzyme (TACE/ADAM17) in the generation of soluble TGFα from its transmembrane precursor, proTGFα. Here, we review our findings that primary keratinocytes from TaceΔZn/ΔZn mice, which express a nonfunctional TACE, released dramatically lower levels of soluble TGFα compared to their normal counterparts, even though TGFα mRNA and cell‐associated protein levels were similar in the two cell populations. Restoration of TACE activity in TaceΔZn/ΔZn cells increased shedding of TGFα species, including the mature, 6‐kDa protein. Further, exogenous TACE enzyme accurately cleaved the N‐terminal processing site of proTGFα in cell lysates, as well as both physiologic sites of a soluble proTGFα ectodomain. TACE also accurately cleaved peptide substrates corresponding to the processing sites of several additional EGF family members, and restoration of TACE activity enhanced the shedding of soluble AR and HB‐EGF proteins from TaceΔZn/ΔZn cells. Finally, reduction of functional TACE gene dosage greatly exacerbated the open‐eye defect of Egfrwa‐2/wa‐2 newborns, which is regulated by redundant actions of several EGF family ligands. The implications of these results for the biology of the EGF family and TACE are discussed.

γ-Secretase-mediated growth hormone receptor proteolysis: Mapping of the intramembranous cleavage site

GH receptor (GHR) undergoes regulated proteolysis by both metalloprotease (α-secretase) and γ-secretase activities. α-Secretase activity regulates GHR availability and sensitivity and generates circulating GH binding protein. The function of γ-secretase cleavage is yet uncertain. We investigated GHR determinants that affect inducible sequential α- and γ-secretase cleavage and thus remnant and stub generation, respectively. Purification and N-terminal sequencing of the stub revealed that γ-secretase cleavage occurs at an ε-site in GHRs transmembrane domain four residues from the intracellular domain. Mutagenesis revealed that deletion of the proximal two transmembrane residues prevented both α- and γ-secretase-mediated proteolysis and deletion of four residues around the ε-site precluded surface GHR expression and proteolysis. However, point mutations in and around the ε-site affected neither α- or γ-secretase cleavage. We conclude that both cleavages likely occur at the cell surface and sequentially (α-secretase followed by γ-secretase) and that ε-site cleavage by γ-secretase does not require a consensus sequence.