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Fas (APO-1/CD95), which is a member of the tumor necrosis factor receptor superfamily, is a cell surface receptor that induces apoptosis. A protein tyrosine phosphatase, Fas-associated phosphatase-1 (FAP-1), that was previously identified as a Fas binding protein interacts with the C-terminal 15 amino acids of the regulatory domain of the Fas receptor. To identify the minimal region of the Fas C-terminal necessary for binding to FAP-1, we employed an in vitro inhibition assay of Fas/FAP-1 binding using a series of synthetic peptides as well as a screen of random peptide libraries by the yeast two-hybrid system. The results showed that the C-terminal three amino acids (SLV) of human Fas were necessary and sufficient for its interaction with the third PDZ (GLGF) domain of FAP-1. Furthermore, the direct cytoplasmic microinjection of this tripeptide (Ac-SLV) resulted in the induction of Fas-mediated apoptosis in a colon cancer cell line that expresses both Fas and FAP-1. Since t(S/T)X(V/L/I) motifs in the C termini of several other receptors have been shown to interact with PDZ domain in signal transducing molecules, this may represent a general motif for protein-protein interactions with important biological functions.

Expansion of trinucleotide repeats (CAG)n and (CGG)n is found in genes responsible for certain human hereditary neurodegenerative diseases. By gel-mobility shift assay, we detected a single-stranded (AGC)n repeat-binding activity primarily in mouse brain extracts and very low or undetectable activity in other tissue extracts. Two (AGC)n-repeat binding proteins, with apparent molecular weights of 44 and 40 kDa, have been purified from mouse adult brain by a DNA affinity column and fast protein liquid chromatography. UV-cross linking of radiolabeled (AGC)n repeats with crude brain extracts and with purified two proteins of 44 and 40 kDa produced identical doublet bands, indicating that these proteins are in fact responsible for the (AGC)n-binding activity in brain extracts. We designated these two proteins TRIP-1 for the 44 kDa protein and TRIP-2 for the 40 kDa protein, where TRIP represents trinucleotide repeat-binding protein. TRIP-1 and TRIP-2 bind to a specific subset of trinucleotide repeat sequences including (AGC)n, (AGT)n, (GGC)n, and (GGT)n repeats but not to various other trinucleotide repeats. A minimum of eight (AGC) trinucleotide repeating units is required for TRIP-1 and -2 recognition and binding. The (AGC)n repeat-binding activity increases in the brain after birth and reaches a plateau within 3 weeks. In the brain, TRIP-1 and TRIP-2 may alter the function of the genes containing the expanded-trinucleotide repeats.