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Molecular characterization of a CD95 signaling mutant
E J Peterson1, K M Latinis, G A Koretzky
1University of Iowa, Iowa City 52242, USA.
Arthritis and Rheumatism
|June 17, 1998
Summary
A mutated CD95 receptor lacking a functional death domain impairs CD95 signaling in T cells. This discovery sheds light on the intracellular signaling pathways crucial for apoptosis regulation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- CD95 (also known as Fas or APO-1) is a cell surface receptor crucial for initiating apoptosis.
- Intracellular signaling downstream of CD95 ligation is essential for programmed cell death in T cells.
Purpose of the Study:
- To investigate the intracellular signaling events triggered by CD95 receptor activation.
- To characterize a mutant Jurkat T cell line (DD3) with impaired CD95-mediated apoptosis.
Main Methods:
- Analysis of CD95 surface expression and nucleotide sequencing of CD95 in DD3 cells.
- Functional assays to assess apoptosis induction via CD95 ligation and UV stimulation.
- Western blotting to detect wild-type and mutant CD95 protein expression.
- Transfection of mutant CD95 cDNA into parental Jurkat cells.
Main Results:
- DD3 cells showed reduced apoptosis upon CD95 ligation but remained sensitive to UV-induced cell death.
- A mutant CD95 cDNA from DD3 encoded a protein lacking the critical carboxy-terminal death domain.
- Both wild-type and mutant CD95 proteins were present in DD3 cells.
- Introduction of the mutant CD95 cDNA protected parental cells from CD95-mediated apoptosis.
Conclusions:
- A non-functional CD95 death domain can disrupt normal CD95 receptor signaling in T cells.
- The cytoplasmic death domain is essential for transmitting apoptotic signals through CD95.