Epitope conservation and immunohistochemical localization of the guanylin/stable toxin peptide receptor, guanylyl cyclase C.
The heat-stable enterotoxins (ST) are a family of cysteine-rich low-molecular weight peptides produced by pathogenic bacteria, and are one of the major causes of watery diarrhea all over the world. These toxins mediate their action by binding to an intestinal cell surface receptor that is a membrane-associated guanylyl cyclase (GCC). This receptor also serves as the receptor for the recently characterised endogenous ligand, guanylin. We have expressed various domains of the receptor in Escherichia coli and used purified proteins for the generation of both polyclonal and monoclonal antibodies. While polyclonal antibodies were able to partially inhibit ST binding to the native receptor present in the T84 human colonic cell line, GCC:B10 monoclonal antibody did not interfere with ligand binding. Western blot analysis, using membranes prepared from human colonic T84 cells, detected two bands of size 160 and 140 kDa, representing alternately glycosylated forms of the receptor. Using the recombinant proteins, we could map the epitope of GCC:B10 monoclonal antibody to the intracellular domain of the receptor. We used the antibody to localize the receptor throughout the rat intestine, and in the porcine and bonnet monkey colon. We could detect receptor expression in the villus and the crypts of the duodenum, jejunum, ileum, and caecum, and in the crypts of the colon. Receptor expression was observed in cells that had earlier been shown to express cGMP-dependent kinase, but not the cystic fibrosis transmembrane regulator, a known downstream target of cGMP/G-kinase, which suggests that GCC/ cGMP could regulate additional cellular signal transduction machinery.
Nandi
A
A
Center for Reproductive Biology and Molecular Endocrinology, Indian Institute of Science, Bangalore, India.
Bhandari
R
R
Visweswariah
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000917:Antibody Formation; D001717:Birds; D003106:Colon / Q000737:chemistry; D017124:Conserved Sequence / Q000276:immunology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006031:Glycosylation; D006162:Guanylate Cyclase / Q000032:analysis* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D000882:Haplorhini; D006801:Humans; D007150:Immunohistochemistry; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013045:Species Specificity; D013552:Swine; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008024:Ligands; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled