Expression of the cholera toxin B subunit in the Golgi apparatus of Swiss 3T3 cells inhibits DNA synthesis induced by basic fibroblast growth factor.
We attempted to express the cholera toxin B subunit (CTXB) in the Golgi apparatus of cultured mammalian cells by means of gene transfection. Complementary DNA of CTXB was ligated with the Golgi-retention signal sequence of human beta 1,4 galactosyltransferase cDNA, and the chimeric gene yielded was inserted into a mammalian expression vector. The resultant construct was transfected into COS-1 cells for transient expression and into Swiss 3T3 cells for stable expression. The expression of a fusion protein encoded by the chimeric gene was demonstrated according to the following criteria: first, detection of a protein exhibiting the expected molecular mass on Western blot analysis using an anti-CTXB antibody; second, detection of the protein located in the Golgi area by indirect immunofluoresence microscopy; and third, detection of GM1 binding activity in cell lysates. Stable transformants satisfying the above criteria were subjected to an assay for mitogen-induced DNA synthesis. These transformants exhibited significantly lower DNA synthesis than mock transfection cells on stimulation with basic fibroblast growth factor (bFGF), whereas the two types of cells exhibited similar responses to 10% fetal calf serum and other mitogens, such as epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and platelet-derived growth factor. Analysis of the binding of radio-iodinated bFGF to the cells revealed that the transformants did not exhibit a significant decrease in the binding affinity or the number of high affinity sites. These results suggest that the fusion protein specifically inhibits the bFGF signaling not at the binding step but rather at a later step(s) triggered by the binding.
Hashimoto
Y
Y
Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Medical Science.
Oshima
A
A
Narimatsu
H
H
Suzuki
A
A
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002772:Cholera Toxin / Q000096:biosynthesis* / Q000235:genetics; D004247:DNA / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000235:genetics; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005677:G(M1) Ganglioside / Q000378:metabolism; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000235:genetics; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014162:Transfection
D018076:DNA, Complementary; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2; D005677:G(M1) Ganglioside; D004815:Epidermal Growth Factor; D004247:DNA; D002772:Cholera Toxin; D009237:N-Acetyllactosamine Synthase