Characterization and partial purification of the human receptor for the heat‐stable enterotoxin

Abstract

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 radioligand (the C-terminal tyrosine residue is replaced by phenylalanine in the mutant), a single class of high-affinity receptor sites was detected in T84 cells, with a Kd of 0.1 nM, similar in affinity to the receptor described in human intestinal tissue. The receptor was solubilised from T84 cell membranes and affinity cross-linking of the solubilised preparation indicated that a single species of M, 160000 served as the receptor. Freshly solubilised preparations of the receptor retained heat-stable enterotoxin-activable guanylyl cyclase activity. Purification of the receptor was achieved through sequential affinity chromatography on GTP-epoxy-Sepharose and wheat-germ-agglutinin columns resulting in purification of the receptor by 3000 fold. The heat-stable enterotoxin-binding characteristics of the receptor were unchanged during the purification and silver staining of the purified receptor preparation indicated a band of M, 160000, which was specifically cross-linked to the lZ51-labeled mutant peptide. The purified receptor retained 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 cyclase/receptor.The heat-stable enterotoxins (ST) are a family of lowmolecular-mass peptide toxins, and are one of the major causes of watery secretory diarrhoea all over the world [1, 21. Various forms of the toxins, differing in amino acid sequence, are produced by a number of pathogenic bacteria [3, 41, and all these peptides contain a cysteine-rich core essential for full biological activity [5, 61. ST peptides bind to a receptor on intestinal cells and activate membrane-bound guanylyl cyclase [7, 81. Increased levels of cyclic GMP (cGMP) within the cell are hypothesised to lead to enhanced C1-secretion from the intestinal cell by as yet undefined mechanisms, resulting in fluid loss and subsequent diarrhoea Early biochemical studies had postulated that in rat intestinal membranes the ST-binding and guanylyl cyclase activities were located on separate molecules [lo]. However, the cloning and expression of the rat and human intestinal ST receptor [ll-131 suggested that the ST receptor was in fact a high-molecular-mass protein and a member of the guanylyl cyclase family of receptors, described earlier for atrial natriuretic factor and the sea urchin egg peptides [14]. These observations implied that the ST-binding and guanylyl cyclase activities were present in the same receptor molecule. How- ever, there is no biochemical evidence using purified receptor preparations to confirm that the ST receptor does in fact exist in the cell as suggested for the recombinant protein. Hugues et al. reported the purification of the rat ST receptor from intestinal membranes using ST-ligand af...

Hyaluronan Anchoring and Regulation on the Surface of Vascular Endothelial Cells Is Mediated through the Functionally Active Form of CD44

“…BW5147 cells were grown in suspension, washed twice with chilled PBS, and resuspended in lysis buffer. The cell lysates were sonicated briefly, and membranes were prepared by centrifugation at 100,000 ϫ g (57). Pellets were resuspended in non-reducing sample buffer and resolved on a 10% polyacrylamide gel containing 0.1% SDS, after which proteins were transferred to nitrocellulose (Amersham Pharmacia Biotech).…”

Functional Inactivation of the Human Guanylyl Cyclase C Receptor: Modeling and Mutation of the Protein Kinase-like Domain

“…Membranes were prepared from this cell line as described previously (42), and the amount of protein was estimated using a modification of the Bradford protein assay (43). GC-C content in the membrane was monitored by performing receptor binding analysis with a radiolabeled ST peptide as described previously (31). Binding data were analyzed with GraphPad PRISM (San Diego, CA) to determine the K d and B max .…”

“…T84 (CCL 247) and COS7 cell lines were obtained from ATCC (Rockville, MD). Stable toxin of the human variety (STh) and a mutant form of the STh peptide (ST Y72F ) were purified from strains hyperexpressing the peptide, as described previously (30,31).…”

“…STh (10 -7 M) was then added to the cells and incubation continued for 1 h, after which the cells were lysed and the cGMP content was measured by radioimmunoassay (46). GC-C K516A content in membranes prepared from cells was monitored as described previously (31).…”

Biochemical Characterization of the Intracellular Domain of the Human Guanylyl Cyclase C Receptor Provides Evidence for a Catalytically Active Homotrimer

“…For kinetic analysis, varying concentrations of MgGTP were used, maintaining the ratio of Mg 2+ /GTP as 10:1. Tubes were incubated at 37°C for 10 min, the reaction was terminated by the addition of 400 µL of 50 mM sodium acetate buffer, pH 4.5, and the sample was boiled for 5 min and centrifuged, and supernatant was taken for cGMP radioimmunoassay as described earlier (27). Some assays were performed by replacing MgCl 2 with 4 mM MnCl 2 .…”