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Interactions of cellular polypeptides with the cytoplasmic domain of the mouse Fas antigen
1Department of Cell Biology and Anatomy, Hematology/Oncology Division, Cornell University Medical College, New York, New York 10021, USA.
Abstract:
The mouse Fas/APO-1 antigen represents a 45-kilodalton transmembrane receptor that initiates apoptosis by a poorly defined signaling mechanism. The cytoplasmic domain of Fas does not display any known enzymatic activities but is capable of interacting with a number of proteins that were identified recently using the yeast interactive cloning method. To investigate direct biochemical interactions from cellular lysates prepared from Fas-responsive cells, a series of recombinant glutathione S-transferase-mouse Fas fusion proteins representing different regions of the mouse Fas cytoplasmic domain was used. Polypeptides of 25, 50, and 70 kilodaltons were found to associate with the Fas intracellular domain, and this binding was stable in the presence of 1 M NaCl. These interactions were also detected using a mouse Fas fusion protein containing an Ile to Asn mutation, which is responsible for a lymphoproliferative disorder in certain strains of mice (lprcg). Furthermore, the binding of cellular proteins to Fas could be blocked upon incubation with a polyclonal antibody directed against the cytoplasmic domain of Fas. The strong association of cellular proteins with the cytoplasmic region implies that constitutive interactions may exist to regulate apoptotic signaling through the Fas antigen.
Insights
Researchers identified proteins that bind to the Fas receptor
Area of Science:
- Cell Biology
- Immunology
Background:
- The Fas/APO-1 antigen is a transmembrane receptor crucial for initiating apoptosis.
- Its cytoplasmic domain lacks enzymatic activity but interacts with other proteins.
- The signaling mechanism of Fas-mediated apoptosis is not fully understood.
Purpose of the Study:
- To investigate direct biochemical interactions involving the cytoplasmic domain of the mouse Fas receptor.
- To identify cellular proteins that bind to Fas intracellularly.
Main Methods:
- Used recombinant glutathione S-transferase-mouse Fas fusion proteins representing different regions of the Fas cytoplasmic domain.
- Analyzed interactions using cellular lysates from Fas-responsive cells.
- Employed a Fas fusion protein with an Ile to Asn mutation (lprcg) and a polyclonal antibody against the Fas cytoplasmic domain.
Main Results:
- Identified polypeptides of 25, 50, and 70 kilodaltons that associate with the Fas intracellular domain.
- These interactions remained stable in 1 M NaCl.
- Binding was observed even with a mutated Fas protein linked to lymphoproliferative disorder.
- Binding could be inhibited by an antibody targeting the Fas cytoplasmic domain.
Conclusions:
- Cellular proteins strongly associate with the Fas cytoplasmic region.
- These constitutive interactions likely play a role in regulating Fas-mediated apoptotic signaling.