CHARACTERIZATION AND PARTIAL-PURIFICATION OF THE HUMAN RECEPTOR FOR THE HEAT-STABLE ENTEROTOXIN

The receptor for the Escherichia coli heat-stable enterotoxin has been characterized and partially purified from the T84 human colonic cell line. Using a novel mutant heat-stable enterotoxin peptide as a radiol igand (the C-terminal tyrosine residue is replaced by phenylalanine in the mutant), a single class of high-affinity receptor sites was detec ted in T84 cells, with a K-d, of 0.1 nM, similar in affinity to the re ceptor described in human intestinal tissue. The receptor was solubili sed from T84 cell membranes and affinity cross-linking of the solubili sed preparation indicated that a single species of M(r) 160000 served as the receptor. Freshly solubilised preparations of the receptor reta ined heat-stable enterotoxin-activable guanylyl cyclase activity. Puri fication of the receptor was achieved through sequential affinity chro matography on GTP-epoxy-Sepharose and wheat-germ-agglutinin columns re sulting in purification of the receptor by 3000 fold. The heat-stable enterotoxin-binding characteristics of the receptor were unchanged dur ing the purification and silver staining of the purified receptor prep aration indicated a band of M(r) 160000, which was specifically cross- linked to the I-125-labeled mutant peptide. The purified receptor reta ined guanylyl cyclase activity, but the activity was not stimulated on addition of human heat-stable enterotoxin, suggesting that accessory structural factors may be involved in the activation of the guanylyl c yclase/receptor.