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Differential modulation of two interferon-alpha binding proteins on a human lymphoblastoid cell line
C R Faltynek1, G L Princler, M Schwabe
1Biological Carcinogenesis and Development Program, Program Resources, Inc./DynCorp, Frederick, MD.
Insights
This study reveals that human interferon (IFN)-alpha binds to two distinct receptor proteins, identified as 115-kDa and 135-kDa complexes. The 135-kDa complex exhibits higher affinity binding, suggesting a more complex IFN-alpha receptor interaction than previously understood.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Interferon (IFN)-alpha and IFN-beta interactions with their receptors were initially thought to involve a single high-affinity receptor class.
- Recent findings suggest a more complex interaction, indicated by biphasic Scatchard plots and multiple IFN-alpha receptor cross-linked complexes.
Purpose of the Study:
- To investigate the molecular basis of IFN-alpha receptor interactions using the Daudi B lymphoblastoid cell line.
- To identify and characterize the specific protein components involved in IFN-alpha binding and signal transduction.
Main Methods:
- Utilized cross-linking techniques with radiolabeled IFN-alpha (125I-rIFN-alpha A) to identify receptor complexes.
- Employed ligand-blotting assays to detect IFN-alpha binding proteins.
- Performed Scatchard analysis and receptor down-regulation experiments to assess binding affinities and dynamics.
Main Results:
- Identified two primary IFN-alpha receptor cross-linked complexes with apparent molecular masses of 115 kDa and 135 kDa.
- Ligand-blotting confirmed the presence of two distinct IFN-alpha binding proteins corresponding to these complexes.
- Observed that the 135-kDa complex showed greater signal intensity at low IFN-alpha concentrations and was preferentially down-regulated, indicating higher affinity binding.
Conclusions:
- The 135-kDa complex represents a higher-affinity IFN-alpha binding protein, while the 115-kDa complex represents a lower-affinity binding protein.
- These findings indicate a more intricate IFN-alpha receptor system than previously proposed, involving at least two distinct protein interactions.
- The differential affinity and regulation of these complexes provide new insights into IFN-alpha signal initiation and modulation.
Abstract:
The interaction between human interferon (IFN)-alpha or IFN-beta with its receptor was originally described as the binding to a single class of high-affinity receptors. However, more recently, biphasic Scatchard plots as well as multiple IFN-alpha receptor cross-linked complexes have been reported. In this study using the Daudi B lymphoblastoid cell line, two primary IFN-alpha receptor cross-linked complexes with apparent Mr of 115 and 135 kilodaltons (kDa) were obtained. Both complexes were observed under a variety of cross-linking conditions, including the addition of a mixture of protease inhibitors throughout the binding reaction and solubilization of the cells. These two complexes appear to be caused by the binding and cross-linking of 125I-rIFN-alpha A to two separate proteins because we also observed two IFN-alpha binding proteins using a ligand-blotting technique. At low concentrations of 125I-rIFN-alpha A, it was found that the intensity of the signal in the 135-kDa cross-linked complex was greater than that of the 115-kDa complex. Addition of increasing concentrations of unlabeled rIFN-alpha A to a 4 degrees C binding reaction reversed the ratio in intensities of the two complexes. Moreover, after pretreatment of the cells at 37 degrees C with low concentrations of unlabeled rIFN-alpha A, there was preferential down-regulation of both the 135-kDa complex and the higher affinity binding component of the biphasic Scatchard plot. These results suggest that the 135-kDa complex represents the binding of 125I-rIFN-alpha A to a protein having higher affinity for IFN than the protein that gives rise to the 115-kDa complex.(ABSTRACT TRUNCATED AT 250 WORDS)