Expression of a truncated guanylate cyclase (GC-C), a receptor for heat-stable enterotoxin of enterotoxigenic Escherichia coli, and its dimer formation in COS-7 cells.
A fragment of guanylate cyclase C (GC-C) of about 1.7 k bp corresponding to amino acids 1-553 spanning the extracellular and transmembrane domains and a portion of the intracellular region was amplified using template cDNA prepared from rat intestinal cells by the polymerase chain reaction method. The cloned 1.7 k bp fragment was inserted into the mammalian expression vector pCGUT and the truncated GC-C expressed on the surface of COS-7 cells was demonstrated to bind heat-stable enterotoxin by photo affinity labeling with 125I-N-5-azidonitrobenzoyl-STh[5-19]. Analysis by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis showed that the truncated GC-C formed dimers on the surface of COS-7 cells. The intracellular region of GC-C was found not to be necessary for dimer formation by the GC-C. Comparison of the molecular weights of the truncated GC-C expressed in COS-7 cells and Escherichia coli suggested that the truncated GC-C was glycosylated in the mammalian expression system.
Hirayama
T
T
Institute of Medical Science, University of Tokyo, Japan.
Wada
A
A
Hidaka
Y
Y
Fujisawa
J
J
Takeda
Y
Y
Shimonishi
Y
Y
D000345:Affinity Labels; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D000345:Affinity Labels; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C034151:heat stable toxin (E coli); D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled