An aspartate/insulin receptor chimera mitogenically activates fibroblasts.
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.
Biemann
H P
HP
Department of Cell Biology, Genzyme Corporation, Cambridge, Massachusetts 02139, USA.
Harmer
S L
SL
Koshland
D E
DE
D016475:3T3 Cells; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D016895:Culture Media, Serum-Free; D004247:DNA / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008322:Mammals; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D017459:Receptors, Amino Acid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism
D016895:Culture Media, Serum-Free; D017459:Receptors, Amino Acid; D011993:Recombinant Fusion Proteins; C031959:aspartic acid receptor; D001224:Aspartic Acid; D004247:DNA; D011972:Receptor, Insulin; D013936:Thymidine