Differential protein-protein interactions of full length human FasL and FasL fragments generated by proteolysis

Abstract

Fas ligand (FasL) is a death factor of the tumor necrosis factor superfamily. Like other members of this family of type II transmembrane proteins, FasL is subject to ectodomain shedding by a disintegrin and metalloproteinases (ADAMs) liberating soluble FasL and leaving membrane-integral N-terminal fragments (NTFs). These NTFs are further processed by intramembrane proteolysis through signal peptide peptidase-like 2a (SPPL2a), releasing intracellular domains (ICDs) which might translocate to the nucleus to regulate transcription. Previous work established that the proline-rich domain within the cytosolic N-terminus of FasL is required for protein-protein interactions with different Src homology 3 (SH3) or WW domain proteins. Distinct binding partners regulate FasL storage and surface appearance or are involved in other aspects of FasL biology. Given the large number of FasL interactors, we asked whether proteolytically processed FasL fragments associate with the same or distinct sets of SH3 domain proteins. To address this, we performed co-precipitation experiments using a monoclonal antibody directed against the FasL N-terminus for subsequent protein detection of full length FasL and NTFs/ICDs in Western blots. We demonstrate that members of the sorting nexin (SNX) family bind full length FasL and its N-terminal fragments whereas members of the Pombe Cdc15 homology (PCH) protein family bind full length FasL, but fail to associate with processed FasL. Thus, we provide first evidence that full length FasL and FasL fragments display selectivity regarding their association with intracellular binding partners. The differential binding most likely governs the fate and function of the intracellular FasL fragments.

Keywords: (F-)BAR; (Fer/CIP4 homology-)Bin–Amphiphysin–Rvs; ADAM; ESCRT; Fas ligand; FasL; I-CLiP; ICD; Intramembrane proteolysis; Monoclonal antibody; N-terminal fragment; NTF; PCH; PCH proteins; PRD; Pombe Cdc15 homology; Protein–protein interaction; Proteolysis; RIP; SH3; SNX; SPPL2a; Signal transduction; Sorting nexins; Src homology 3; T lymphocytes; TNF; a disintegrin and metalloproteinase; endosomal sorting complexes required for transport; intracellular domain; intramembrane-cleaving protease; mab; monoclonal antibody; proline-rich domain; regulated intramembrane proteolysis; signal peptide peptidase like 2a; sorting nexin; tumor necrosis factor.