ACTIVATION-INDUCED AGGREGATION AND PROCESSING OF THE HUMAN FAS ANTIGEN - DETECTION WITH CYTOPLASMIC DOMAIN-SPECIFIC ANTIBODIES

Fas (APO1/CD95) is a type 1 transmembrane protein critically involved in receptor-mediated apoptosis. Previous studies have shown that Pas e xists in monomeric form in resting cells and aggregates upon cross-lin king to form a complex that serves to recruit additional signaling mol ecules to the cell membrane, To study the molecular fate of the Pas an tigen following receptor activation, a monoclonal antibody specific fo r the cell death domain of Fas has been generated, This monoclonal ant ibody (3D5) could be used in Western blot analysis using total cell ly sates to identify different forms of Fas antigens without immunoprecip itation. High molecular mass (>200 kDa), SDS- and beta-mercaptoethanol -resistant Fas aggregates were formed immediately following receptor c ross-linking, and a 97-kDa band (p97) was detected about 2 h later, p9 7 could be detected by antibodies against either the death domain or t he C terminus, However, p97 could not be precipitated by antiextracell ular domain antibodies. Thus, p97 most likely represents a processed f orm of the high molecular weight Fas aggregates, Although p97 generati on followed a similar time course as CPP32 activation and poly(ADP-rib ose) polymerase cleavage, it could not be inhibited by cysteine protea se, calpain, or proteasome inhibitors.