Cellular refractoriness to the heat-stable enterotoxin peptide is associated with alterations in levels of the differentially glycosylated forms of guanylyl cyclase C.
The heat-stable enterotoxin peptides (ST) produced by enterotoxigenic Escherichia coli are one of the major causes of transitory diarrhea in the developing world. Toxin binding to its receptor, guanylyl cyclase C (GC-C), results in receptor activation and the production of high intracellular levels of cGMP. GC-C is expressed in two differentially glycosylated forms in intestinal epithelial cells. Prolonged exposure of human colonic cell lines to ST peptides induces cellular refractoriness to the ST peptide, in terms of intracellular cGMP accumulation. We have investigated the mechanism of cellular desensitization in human colonic Caco2 cells, and observe that exposure of cells to ST leads to a time and dose-dependent inability of cells to respond to the peptide in terms of GC-C stimulation, both in whole cells and membranes prepared from desensitized cells. This is concomitant with a 50% reduction in ST-binding activity in desensitized cells. Desensitization was correlated with a loss of the plasma membrane-associated, hyperglycosylated 145 kDa form of GC-C, while the predominant 130 kDa form, localized both on the plasma membrane and the endoplasmic reticulum, continued to be present in ST-treated cells. Desensitized cells recovered ST-responsiveness on removal of the ST peptide, which was correlated with a reappearance of the 145 kDa form on the cell surface, following processing of the endoplasmic reticulum-associated pool of the 130 kDa form. Selective internalization of the 145 kDa form of the receptor was required for cellular desensitization, as ST-treatment of cells at 4 degrees C did not lead to refractoriness. We therefore show a novel means of regulation of cellular responsiveness to the ST peptide, whereby altering cellular levels of the differentially glycosylated forms of GC-C can lead to differential ligand-mediated activation of the receptor.
Ghanekar
Yashoda
Y
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.
Chandrashaker
Akhila
A
Visweswariah
Sandhya S
SS
D001427:Bacterial Toxins / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity*; D015153:Blotting, Western; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004768:Enterotoxins / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity*; D004847:Epithelial Cells / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006031:Glycosylation; D006162:Guanylate Cyclase / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D008970:Molecular Weight; D011233:Precipitin Tests; D011485:Protein Binding; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D018000:Receptors, Peptide; C034151:heat stable toxin (E coli); D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D006152:Cyclic GMP