Substrate specificity for the E3 ligase GRAIL depends on regional ubiquitination of transmembrane targets

T cell activation is a finely coordinated process that requires the execution of precise events for clearance of a foreign pathogen. Anergy, a long-lived state devoid of T cell activation and cytokine production, results from sub-optimal stimulatory conditions and correlates with expression of the E3 ligases Itch, Cbl-b, and GRAIL. The G&barbelow;ene R&barbelow;elated to A&barbelow;nergy i&barbelow;n L&barbelow;ymphocytes ( GRAIL), originally cloned due to its differential upregulation in anergized T cell clones, inhibits T cell activation and cytokine production by its E3 ligase activity when overexpressed in naive T cells and T cell hybridomas. While substrates of both Itch and Cbl-b have been identified, no substrates have yet been found for the predominantly endosomal localized GRAIL. In an effort to better understand how GRAIL inhibits lymphocyte activation, we focused on identifying novel protein-protein interactions between GRAIL and other transmembrane proteins that could potentially serve as targets for ubiquitination. Studies using cell surface biotinylation and total internal reflection microscopy (TIRF-M) found that the largest glycosylated form of GRAIL localized to the plasma membrane in concentrated populations with heterogeneous dynamics. A bacterial two-hybrid screen using the GRAIL amino terminus as bait identified the tetraspanin CD151, resident to both endosomes and the plasma membrane, as a potential transmembrane substrate. In vitro studies demonstrated that GRAIL utilized the previously uncharacterized PA domain to bind the large extracellular loop (LEL) of CD151 and mediated cytosolic tetraspanin ubiquitination. In spite of little sequence homology among the LEL regions within the tetraspanin family, GRAIL could bind via its PA domain and ubiquitinate every tetraspanin tested. GRAIL overexpression downregulated the cell surface pool of endogenous tetraspanins in a proteasome dependent manner using lysine 48-conjugated polyubiquitin chains. Mutagenesis of potential tetraspanin target lysine residues demonstrated that GRAIL utilized regional specificity for polyubiquitination of lysines only within the tetraspanin amino terminus. Subsequent identification of CD40L as another substrate for GRAIL ubiquitination supports a novel model where GRAIL utilizes a split substrate binding and ubiquitination mechanism for regulation of endosomal or plasma membrane transmembrane proteins with small, non-catalytic cytosolic residues that play a role in T cell activation and signaling.