The Single Subunit Transmembrane E3 Ligase Gene Related to Anergy in Lymphocytes (GRAIL) Captures and Then Ubiquitinates Transmembrane Proteins across the Cell Membrane

Abstract

The ubiquitin E3 ligase gene related to anergy in lymphocytes (GRAIL) (Rnf128) is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger. GRAIL also contains an equally large luminal region consisting primarily of an uncharacterized protease-associated (PA) domain. Using two-hybrid technology to screen for proteins that bound the PA domain we identified CD151, a member of the tetraspanin family of membrane proteins. GRAIL bound to the luminal/extracellular portion of both CD151 and the related tetraspanin CD81 using its PA domain, which promoted ubiquitination of cytosolic lysine residues. GRAIL exhibited specificity for lysines only within the tetraspanin amino terminus even in the presence of other cytosolic lysine residues in the substrate. GRAIL-mediated ubiquitination promoted proteasomal degradation and cell surface downregulation of tetraspanins via Lys-48 linkages. As a result, the juxtaposition of PA and RING finger domains across a lipid bilayer facilitates the capture of transmembrane substrates for subsequent ubiquitination. These findings identify for the first time a single subunit E3 ligase containing a substrate-binding domain spatially restricted by a membrane from its E2 recruitment domain as well as an E3 ligase for members of the tetraspanin family.The ubiquitin proteasome pathway consists of a combination of proteins that determines with meticulous precision the post-translational fate of nearly every cellular protein. The sequential enzymes involved in conjugation of ubiquitin to a substrate, E1 2 activating enzymes, E2 transferases, and E3ligases, provide a mechanism of increasing stringency to ensure the serial transfer of ubiquitin to its intended target (1). The majority of substrate specificity is conferred by a wide array of protein interaction motifs on the E3 ligase that are separate from the domains responsible for recruitment of the ubiquitination machinery (2). After covalent attachment of ubiquitin, other classes of ubiquitin-binding proteins decipher this highly informative post-translational modification by binding, editing, or removing the attached ubiquitin molecules (3-5). E3 ligases are split into distinct families based on their E2 recruiting domains, including HECT, RING, and U box. RING finger proteins are the most numerous and can be further divided into multisubunit complexes (SCF and anaphase-promoting complex) or single subunit proteins with modular domains (Cbl and Mdm2). Multisubunit complexes partition function into individualized protein components with the substrate-binding region contained in the F box for the SCFs (6), whereas single subunit RING finger E3 ligases depend on substrate capture via protein-protein interaction domains particular to each (7). The regions critical for substrate binding among RING finger E3 ligases vary greatly, making prediction and identification of potential substrates difficult. In addition, the limited number of E3 ligases compared with the total number of prot...

The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength

“…The weak interaction of the E3 ligases with R-spondin suggests a model for molecular competition of the PA domain with the Wnt/Lrp/Fz complex. Of note, the PA domains of PLR-1 and GRAIL are also involved in substrate recognition (Lineberry et al 2008;Moffat et al 2014). (Peng et al 2013a).…”

CD81 Promotes Both the Degradation of Transferrin Receptor 2 (TfR2) and the Tfr2-mediated Maintenance of Hepcidin Expression

“…TfR2 is degraded in lysosomes without detectable ubiquitination (23), but those results did not rule out the possibility that a binding partner of TfR2 could be ubiquitinated. CD81 is ubiquitinated by GRAIL (26). To test whether the interaction between CD81 and TfR2 contributed to the down-regulation of TfR2, Hep3B cells were stably transfected with TfR2, TfR2CD/TfR1-f, or TfR2⌬CD.…”

“…Because CD81 promotes TfR2 degradation and CD81 has been shown to be ubiquitinated by a number of E3 ligases including GRAIL (gene related to anergy in lymphocytes) (26), the association of CD81 with TfR2 could be responsible for the degradation of TfR2. GRAIL is expressed in the liver (31) along with CD81 and TfR2.…”

“…It does so by interacting with a variety of signaling molecules. We were interested in CD81 because of its high level of expression in the liver (25) and its regulation by the ubiquitin E3 ligase, GRAIL (gene related to anergy in lymphocytes, Rnf128) (26). GRAIL mRNA is also abundant in the liver (27).…”

E3 ubiquitin ligases in T‐cell tolerance

“…The authors hypothesized that increased RhoGDI levels may sequester Rho molecules in the cytosol, thus preventing Rho signaling pathways involved in T-cell activation, such as cytoskeleton rearrangements and IL-2 production [55]. Furthermore, recent studies have proposed that Grail ubiquitylates different transmembrane proteins, including CD40L and the tetraspanin proteins CD151 and CD81, in a novel ''across membrane'' mechanism, in which the Grail PA domain binds the target protein in the extracellular/luminal compartment while the RING finger domain catalyzes the ubiquitylation process in the cytosol [56,57].As with Cbl-b and Itch, Grail also appears to regulate T reg cells. Grail is highly expressed in natural occurring T reg cells, and its expression levels correlate with the immunosuppressive activity of T reg cells induced in vivo through repetitive exposure to superantigens [58,59].…”