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T-cell activation molecule 4-1BB binds to extracellular matrix proteins
N J Chalupny1, R Peach, D Hollenbaugh
1Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121.
Abstract:
The recently isolated 4-1BB cDNA clone encodes a cell surface protein expressed by activated T cells. Its extracellular domain is homologous to members of the nerve growth factor receptor super family and its cytoplasmic domain contains a sequence homologous to the binding site for the T-cell-specific tyrosine kinase p56lck found in the cytoplasmic domains of CD4 and CD8 alpha. At present the function of 4-1BB is not known. We prepared a 4-1BB-immunoglobulin fusion protein (4-1BB Rg). This protein was used in immunohistochemical studies to identify tissues that express the 4-1BB ligand. 4-1BB Rg bound to virtually all tissues examined, suggesting that extracellular components might function as its ligands. To explore this possibility, 4-1BB was expressed in COS cells and found to mediate the binding of fibronectin, vitronectin, laminin, and collagen VI but not of collagen I. The binding of extracellular matrix proteins to 4-1BB was not mediated by Arg-Gly-Asp (RGD) or CS-1 amino acid sequences. Experiments with overlapping proteolytic fragments of fibronectin showed that 4-1BB interacts with multiple regions of fibronectin. The interaction between extracellular matrix proteins and 4-1BB was completely blocked by the anionic carbohydrate polymer fucoidan and was partially blocked by the anionic carbohydrate polymer dextran sulfate and the glycosaminoglycan heparin sulfate but was unaffected by desulfated heparin. These results suggest that carbohydrates may play a role in mediating the 4-1BB-extracellular matrix protein adhesion.
Insights
The 4-1BB protein binds to extracellular matrix proteins like fibronectin, suggesting a role in cell adhesion. Carbohydrates appear to mediate this interaction, offering new insights into T-cell function.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- 4-1BB is a cell surface protein found on activated T cells.
- Its function remains largely unknown, despite homology to the nerve growth factor receptor superfamily.
Purpose of the Study:
- To investigate the function of 4-1BB.
- To identify ligands for 4-1BB, particularly extracellular matrix (ECM) components.
- To explore the role of carbohydrates in 4-1BB-ECM interactions.
Main Methods:
- Preparation of a 4-1BB-immunoglobulin fusion protein (4-1BB Rg) for binding studies.
- Immunohistochemical analysis to detect 4-1BB ligand expression.
- Expression of 4-1BB in COS cells to test binding to various ECM proteins.
- Inhibition assays using carbohydrate polymers (fucoidan, dextran sulfate, heparin sulfate) to elucidate binding mechanisms.
Main Results:
- 4-1BB Rg bound to numerous tissues, indicating widespread ligand expression.
- 4-1BB directly bound fibronectin, vitronectin, laminin, and collagen VI, but not collagen I.
- Binding was independent of RGD and CS-1 sequences but involved multiple fibronectin regions.
- Fucoidan completely blocked 4-1BB-ECM binding, while dextran sulfate and heparin sulfate partially blocked it.
Conclusions:
- 4-1BB interacts with multiple extracellular matrix proteins.
- Carbohydrate moieties, particularly sulfated polysaccharides, play a significant role in mediating 4-1BB-ECM adhesion.
- These findings suggest a novel mechanism for T-cell adhesion and interaction with the extracellular environment.