Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin.

F.J De Sauvage,R Horuk,G Bennett,C Quan,J.P Burnier,D.V Goeddel

doi:10.1016/s0021-9258(19)50452-5

Abstract

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.

Highlights

9).This imbalance of ions is accompanied by a massive accumulation of water in the gut that gives rise to thediarrhea
It has been suggested that the stable enterotoxins (STa) binding site and the guanylyl cyclase activity are on separate proteins [10, 11]
We report here the molecular characterization of a latter contains a protein kinase-like domain followedby a recombinant cell line (293-STaR) expressing the heat-COOH-terminal guanylyl cyclase catalytic domain

Summary

Introduction

9).This imbalance of ions is accompanied by a massive accumulation of water in the gut that gives rise to thediarrhea. It has been suggested that the STa binding site and the guanylyl cyclase activity are on separate proteins [10, 11].

Results

Conclusion