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Phosphorylation of the transferrin receptor in isolated sheep reticulocyte plasma membranes
Abstract:
The transferrin receptor of sheep reticulocyte plasma membranes undergoes phosphorylation with [gamma-32P]ATP in isolated plasma membranes. The phosphorylation is stimulated by Mn2+, Co2+, and Mg2+, but not by Ca2+, Ba2+, Zn2+, Fe2+, or Cu2+. There is no detectable effect of cyclic nucleotides on the phosphorylation of the receptor. Transferrin and a monoclonal antibody against the transferrin receptor have no apparent effect on receptor phosphorylation in intact cells or isolated membranes. Immunoprecipitates of the receptor retain ability to phosphorylate the receptor. The phosphorylation appears to be at a serine residue which turns over with a half time of 20-30 min. ATP appears to be the best, but not the only substrate for receptor phosphorylation.
Insights
Sheep reticulocyte transferrin receptors are phosphorylated by ATP, with activity enhanced by specific divalent cations. This phosphorylation occurs at serine residues and is independent of transferrin binding.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- The transferrin receptor (TfR) plays a crucial role in cellular iron uptake.
- Understanding the regulation of TfR function, including post-translational modifications like phosphorylation, is essential.
Purpose of the Study:
- To investigate the phosphorylation of the sheep reticulocyte transferrin receptor.
- To identify factors influencing TfR phosphorylation and its characteristics.
Main Methods:
- Isolation of sheep reticulocyte plasma membranes.
- In vitro phosphorylation assays using [gamma-32P]ATP.
- Analysis of phosphorylation sites and substrate requirements.
- Testing the effects of various cations, cyclic nucleotides, transferrin, and antibodies.
Main Results:
- Sheep reticulocyte TfR undergoes phosphorylation in isolated plasma membranes.
- Phosphorylation is stimulated by Mn2+, Co2+, and Mg2+, but not by Ca2+, Ba2+, Zn2+, Fe2+, or Cu2+.
- Cyclic nucleotides, transferrin, and anti-TfR antibodies do not affect phosphorylation.
- Immunoprecipitated TfR retains phosphorylation activity, indicating intrinsic kinase activity or associated kinase.
- Phosphorylation occurs at a serine residue with a turnover half-time of 20-30 minutes.
- ATP is the preferred substrate, though not exclusively.
Conclusions:
- Sheep reticulocyte TfR is a phosphoprotein, likely regulated by specific divalent cations.
- Phosphorylation occurs on a serine residue and is an intrinsic property or associated with the receptor.
- This finding provides insights into the post-translational regulation of transferrin receptor function.