Protein-fragment complementation assays to analyze the CD95 complex

CD95/Fas belongs to the tumor necrosis factor (TNF) receptor superfamily and plays a pivotal role in immune homeostasis. Its ligand, CD95L (FasL or CD178) belongs to the TNF superfamily. CD95 contains three cysteine-rich domains (CRDs) in its extracellular (EC) region numbered 1-3 from the N-terminal region to the plasma membrane. While CRD2 and CRD3 interact with CD95L, CRD1 has been designed the pre-ligand assembly domain (PLAD) since it promotes the receptor self-aggregation in a ligand-independent manner. Upon CD95L binding, CD95 recruits Fas-associated death domain (FADD) at the level of the CD95 death domain (DD). FADD in turn aggregates caspase-8 to form the "death-inducing signalling complex" (DISC). Herein, we describe a method, called protein-fragment complementation assay (PCA) to identify PPIs and use it to validate interaction of CD95 with itself and with FADD. PCA is performed with the reporter enzyme Renilla reniformis luciferase (Rluc). RLuc has been divided in two inactive fragments, fused to different proteins of interest (POIs). If the two POIs interact, they can bring closer the two unfolded RLuc fragments fostering their folding and the quantification of luciferase activity.