Details

Autor(en) / Beteiligte

• PENNICA, D
• LAM, V. T
• MIZE, N. K
• WEBER, R. F
• LEWIS, M
• FENDLY, B. M
• LIPARI, M. T
• GOEDDEL, D. V

Titel

Biochemical properties of the 75-kDa tumor necrosis factor receptor. Characterization of ligand binding, internalization, and receptor phosphorylation

Ist Teil von

• The Journal of biological chemistry, 1992-10, Vol.267 (29), p.21172-21178

Ort / Verlag

Bethesda, MD: American Society for Biochemistry and Molecular Biology

Erscheinungsjahr

1992

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• An expression plasmid encoding the human 75-kDa tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable human cell line (293/TNF-R2) overexpressing TNF-R2. Ligand binding analysis revealed high affinity binding (Kd = 0.2 nM) with approximately 94,000 +/- 7,500 sites/cell for 125I-TNF-alpha and approximately 5-fold lower affinity for TNF-beta (Kd = 1.1 nM) with 264,000 +/- 2,000 sites/cell. Cross-linking of 125I-TNF-alpha and 125I-TNF-beta to 293/TNF-R2 cells yielded predominant complexes with apparent molecular weights of 211,000 for TNF-alpha and 205,000 and 244,000 for TNF-beta, suggesting these complexes contain two or three TNF-R2 molecules. Immunoprecipitation of TNF-R2 from 32P-labeled 293/TNF-R2 cells demonstrated that the receptor is phosphorylated. The majority (97%) of 32Pi incorporation was found in serine residues with a very low level of incorporation (3%) in threonine residues. TNF-alpha treatment of 293/TNF-R2 cells did not significantly affect the degree or pattern of phosphorylation. Cell surface-bound 125I-TNF-alpha was slowly internalized by the 293/TNF-R2 cell line with a t1/2 = 25 min. Shedding of the extracellular domain of TNF-R2 was induced by 4 beta-phorbol 12-myristate 13-acetate but not by TNF-alpha or TNF-beta.