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A high molecular weight component of the human tumor necrosis factor receptor is associated with cytotoxicity
Abstract:
We compared the molecular structure of the receptor to human recombinant tumor necrosis factor (HurTNF) on cells of different tissue origin that differ in their response to one of the known activities of TNF. We studied tumor cell lines that respond to the cytotoxic action of TNF and resistant variants that bind TNF, normal cell lines that are stimulated to proliferate by TNF and those that are not affected by TNF, and peripheral blood granulocytes whose activation is also augmented by TNF. Using 125I-labeled HurTNF, we found that it bound mainly to four cellular polypeptides (138, 90, 75, and 54 kDa), three of which were found in every cell type examined and one (138 kDa) that was observed only in a human breast carcinoma cell line (MCF-7) that is highly responsive to the cytotoxic action of TNF. The 138-kDa polypeptide was not found in resistant variants of MCF-7 that bind TNF. In contrast to the other polypeptides, the 138-kDa protein was detected 30 min after incubation at 4 degrees C, as compared to 5 min. Scatchard analysis and cross-linking data suggest a model for the TNF receptor structure whereby the receptor is composed of noncovalently linked membrane-bound polypeptides that bind TNF with high affinity (Kd, 0.05-0.8 X 10(-9) M) with the 138-kDa protein being the least abundant and/or even absent in most cells.
Insights
Tumor necrosis factor (TNF) receptors were analyzed in various human cells. A unique 138 kDa polypeptide, potentially key to TNF
Area of Science:
- Molecular biology
- Cell biology
- Immunology
Background:
- Tumor necrosis factor (TNF) mediates diverse cellular responses.
- Understanding TNF receptor structure is crucial for its biological functions.
- Cellular responses to TNF vary based on tissue origin and receptor expression.
Purpose of the Study:
- To compare the molecular structure of TNF receptors across different human cell types.
- To identify specific polypeptides involved in TNF binding and cellular response.
- To elucidate the structural basis of TNF sensitivity and resistance.
Main Methods:
- Radiolabeling of human recombinant TNF (HurTNF) with 125I.
- Binding studies using various human cell lines (tumor and normal) and granulocytes.
- Analysis of bound polypeptides using SDS-PAGE and Scatchard analysis.
- Cross-linking studies to investigate receptor complex formation.
Main Results:
- HurTNF bound to four main cellular polypeptides: 138, 90, 75, and 54 kDa.
- Three polypeptides (90, 75, 54 kDa) were ubiquitously expressed.
- A unique 138 kDa polypeptide was found exclusively in a TNF-sensitive breast carcinoma cell line (MCF-7) and absent in resistant variants.
- The 138 kDa protein exhibited slower binding kinetics compared to other polypeptides.
- Scatchard and cross-linking data suggested a receptor model of noncovalently linked polypeptides with high affinity binding.
Conclusions:
- The TNF receptor complex comprises multiple noncovalently associated polypeptides.
- The 138 kDa polypeptide is a distinct component potentially involved in mediating TNF's cytotoxic effects.
- Differential expression of the 138 kDa polypeptide may explain varying cellular sensitivity to TNF.