An Aspartate/Insulin Receptor Chimera Mitogenically Activates Fibroblasts

Hans-Peter Biemann,Stacey L Harmer,Daniel E Koshland

doi:10.1074/jbc.271.44.27927

Abstract

A gene encoding the ligand-binding domain of the Escherichia coli aspartate receptor fused to the cytoplasmic domain of the insulin receptor tyrosine kinase to produce the chimeric aspartate insulin receptor (AIR) was expressed in mammalian cells. A murine fibroblast transfectant line designated CA3 was generated that stably expressed the AIR receptor. This 70,000 Mr receptor containing the tyrosine kinase of the insulin receptor was recognized by aspartate receptor-specific antisera. When isolated in cellular membrane preparations, AIR was found to be capable of autophosphorylation and phosphorylation of histone H2B on tyrosine. The receptor was found to be predominately cytoplasmic and to be situated in the endoplasmic reticulum and Golgi membranes by immunofluorescence imaging of CA3 cells. Mitogenic effects of AIR were observed; CA3 cells continued DNA synthesis under serum deprivation conditions that prevented parental cells from cycling. These results demonstrate that a chimeric receptor containing procaryotic transmembrane sequences is expressed by a eucaryotic cell in intracellular membranes and functionally couples to cellular signaling pathways.

Highlights

A gene encoding the ligand-binding domain of the Escherichia coli aspartate receptor fused to the cytoplasmic domain of the insulin receptor tyrosine kinase to produce the chimeric aspartate insulin receptor (AIR) was expressed in mammalian cells
The cytoplasmic domain of the human insulin receptor is fused to the cytoplasmic end of the second aspartate receptor (AR) transmembrane domain, resulting in a receptor with an extracellular aspartate binding domain and an intracellular kinase domain
We report here that the hybrid AIR can be expressed in mouse 3T3 cells and enters the endoplasmic reticulum where it is apparently retained

Summary

Introduction

A gene encoding the ligand-binding domain of the Escherichia coli aspartate receptor fused to the cytoplasmic domain of the insulin receptor tyrosine kinase to produce the chimeric aspartate insulin receptor (AIR) was expressed in mammalian cells. Mitogenic effects of AIR were observed; CA3 cells continued DNA synthesis under serum deprivation conditions that prevented parental cells from cycling These results demonstrate that a chimeric receptor containing procaryotic transmembrane sequences is expressed by a eucaryotic cell in intracellular membranes and functionally couples to cellular signaling pathways. Two different hybrids derived from the procaryotic E. coli AR extracellular domain and the human insulin receptor kinase domain have been expressed and studied in E. coli [4, 5], one of which was regulated by aspartate. Each ␣␤ molecule has an insulin binding site, and the ␣2␤2 receptor exhibits negatively cooperative insulin binding [10, 11] Both receptors transmit signals across the membrane by conformational changes because receptor clustering mechanisms have been ruled out [6, 9]. Intersubunit autophosphorylation precedes activation of the exogenous kinase activity of the IR [14]

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Conclusion