Related Experiment Videos
Identification and characterization of cellular receptors for the growth regulator, oncostatin M
P S Linsley1, M Bolton-Hanson, D Horn
1Oncogen, Seattle, Washington 98121.
Abstract:
Oncostatin M is a polypeptide growth regulator produced by activated T cells and phorbol ester-treated U937 cells. To identify specific cellular receptors for this factor, we have characterized the binding of 125I-labeled oncostatin M to a variety of normal and malignant mammalian cells. Recombinant oncostatin M was labeled with 125I with full retention of growth inhibitory activity on A375 melanoma cells. 125I-Oncostatin M bound to sensitive cells in a time- and temperature-dependent fashion. Binding was specifically inhibited by unlabeled native or recombinant oncostatin M, but not by other polypeptide growth factors tested. Binding to human leukemic and normal blood cells was generally less than to nonhematopoietic cells. With four different cell lines, maximal growth inhibition by oncostatin M was achieved at less than maximal binding site occupancy. Scatchard graphs of direct binding data were curvilinear and indicated that 125I-oncostatin M bound with higher apparent affinity at lower 125I-oncostatin M concentrations. Using a two binding site model, affinity constants of Kd1 = 11 +/- 11 pM and Kd2 = 1000 +/- 380 pM were extrapolated from binding data with A375 cells, and values of Kd1 = 3 +/- 2 pM and Kd2 = 400 +/- 44 pM from A549 cells. The major 125I-oncostatin M binding species in a number of mammalian cell lines was identified by chemical cross-linking as a specific protein(s) of Mr = 150,000-160,000. 125I-Oncostatin M was internalized (t1/2 = 30 min) and degraded subsequent to binding to a responsive cell line.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Researchers identified specific cellular receptors for Oncostatin M, a polypeptide growth regulator. They found it binds to a 150-160 kDa protein on mammalian cells, with high affinity and internalization, suggesting a key role in cell signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Oncostatin M (OSM) is a polypeptide growth regulator produced by activated T cells and U937 cells.
- Understanding OSM's cellular receptors is crucial for elucidating its biological functions.
Purpose of the Study:
- To characterize the binding of radioiodinated Oncostatin M (125I-OSM) to mammalian cells.
- To identify the specific cellular receptors responsible for OSM binding and activity.
Main Methods:
- Radiolabeling of recombinant OSM with 125I while preserving biological activity.
- Incubation of 125I-OSM with various normal and malignant mammalian cells to assess binding kinetics.
- Chemical cross-linking to identify the molecular weight of OSM binding proteins.
- Analysis of binding data using Scatchard plots and a two-binding-site model.
Main Results:
- 125I-OSM binding was time- and temperature-dependent and specific to OSM.
- Binding affinity varied across cell types, with higher affinity observed at lower OSM concentrations (Kd1 in picomolar range).
- A specific protein of Mr = 150,000-160,000 was identified as the major OSM binding species.
- OSM was internalized and degraded by responsive cells following receptor binding.
Conclusions:
- Mammalian cells possess specific high-affinity receptors for Oncostatin M.
- The identified 150-160 kDa protein is likely the primary receptor for OSM.
- OSM binding leads to internalization and degradation, indicating active cellular uptake and signaling pathways.