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Characterization of the interleukin 3 receptor
S C Murthy1, A L Mui, G Krystal
1Terry Fox Laboratory, B.C. Cancer Research Center, Vancouver, Canada.
Experimental Hematology
|January 1, 1990
Summary
Researchers identified the murine interleukin 3 (mIL-3) receptor as a 140-kd glycoprotein. Upon binding and crosslinking, it converts to a 70-kd surface protein, suggesting a cleavage mechanism.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The murine interleukin 3 (mIL-3) receptor plays a crucial role in cellular signaling and proliferation.
- Understanding the receptor's structure and function is key to deciphering mIL-3 mediated biological processes.
Purpose of the Study:
- To elucidate the molecular nature of the murine interleukin 3 (mIL-3) receptor.
- To investigate the structural changes and potential processing of the mIL-3 receptor upon ligand binding and crosslinking.
Main Methods:
- Homobifunctional crosslinking agents (disuccinimidyl suberate, dithiobis succinimidylpropionate, glutaraldehyde) were used to crosslink radiolabeled [125I]mIL-3 to its receptor on B6SUtA1 cells.
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to analyze the molecular weights of crosslinked species.
- Incubation at 37°C and treatment with inhibitors (sodium azide, methylamine, bacitracin) were used to study receptor conversion.
- N-glycanase digestion was performed to analyze the carbohydrate content of the receptor species.
- Purification and reelectrophoresis of crosslinked species under reducing conditions were conducted.
Main Results:
- Crosslinking revealed two radiolabeled species, p140 and p70, suggesting a single affinity class for the mIL-3 receptor.
- Incubation at 37°C led to a conversion of p140 to p70, indicating a post-binding modification.
- This conversion was inhibited by specific agents and heat treatment, suggesting a protease-mediated process.
- Purification and reducing conditions generated p70 from p140, further supporting a cleavage event.
- Both p140 and p70 contained similar N-linked carbohydrate moieties, indicating they originate from the same precursor.
Conclusions:
- The mIL-3 receptor is proposed to be a 140-kd glycoprotein.
- Upon mIL-3 binding and chemical crosslinking, the receptor undergoes cleavage, resulting in a 70-kd surface protein.
- A protease associated with the plasma membrane and solubilized receptors appears to mediate this conversion.