An aspartate/insulin receptor chimera mitogenically activates fibroblasts

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• The Escherichia coli aspartate receptor and human insulin receptor are key signaling proteins.
• Chimeric receptors offer insights into protein function and signaling pathway interactions.

Purpose of the Study:

• To construct and express a chimeric receptor (AIR) combining bacterial and mammalian signaling domains.
• To investigate the functional expression and cellular localization of the AIR in mammalian cells.
• To assess the signaling capabilities and cellular effects of the chimeric receptor.

Main Methods:

• Gene fusion of the E. coli aspartate receptor ligand-binding domain with the insulin receptor tyrosine kinase domain.
• Stable expression of the chimeric aspartate insulin receptor (AIR) in a murine fibroblast cell line (CA3).
• Immunofluorescence imaging, Western blotting, and in vitro kinase assays to characterize receptor localization and activity.

Main Results:

• The CA3 cell line stably expressed the 70,000 Mr AIR receptor, recognized by specific antisera.
• Isolated AIR demonstrated autophosphorylation and histone H2B phosphorylation on tyrosine residues.
• Immunofluorescence revealed the AIR localized to intracellular membranes, including the endoplasmic reticulum and Golgi apparatus.
• CA3 cells exhibited mitogenic effects, continuing DNA synthesis under serum deprivation.

Conclusions:

• A chimeric receptor with prokaryotic transmembrane sequences can be expressed in eukaryotic cells within intracellular membranes.
• The chimeric aspartate insulin receptor (AIR) is functionally active, exhibiting kinase activity and mediating cellular responses.
• This study demonstrates the potential for inter-kingdom signaling by engineered chimeric receptors.