Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin.
We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable enterotoxin receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously expresses STa binding sites. Scatchard analysis of displacement binding studies revealed a single STa binding site with an affinity (Ki) of 97 pM in 293-STaR compared with 55 pM in T84 cells. Saturation isotherms of STa binding gave a Kd of 94 pM for the cloned receptor expressed in 293 cells and 166 pM for the receptor present in T84 cells. Kinetic measurements of STa binding to 293-STaR gave an association rate constant, K1, of 2.4 x 10(8) M-1 min-1 and a dissociation rate constant, K2, of 0.016 min-1. The half-time of dissociation was 43 min, and the Kd calculated from the ratio of the kinetic constants was 67 pM. The pH profile of STa binding showed that the number of STa binding sites is increased 3-fold at pH 4.0 compared with pH 7.0, with no effect on binding affinity. A polyclonal antibody directed against the extracellular domain of STaR immunoprecipitated two proteins of approximately 140 and 160 kDa from both 293-STaR and T84 cells. Cross-linking of 125I-STa to 293-STaR cells resulted in the labeling of proteins with a molecular mass of approximately 153, 133, 81, 68, 56, and 49 kDa, the two smallest being the more abundant. Similar results have been reported for the STaR present on rat brush border membranes. These data suggest that the STaR-guanylyl cyclase identified by molecular cloning is the only receptor for STa present in T84 cells.
de Sauvage
F J
FJ
Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080.
Horuk
R
R
Bennett
G
G
Quan
C
C
Burnier
J P
JP
Goeddel
D V
DV
D001427:Bacterial Toxins / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D003106:Colon / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006162:Guanylate Cyclase; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C034151:heat stable toxin (E coli); D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled