Glycosylation of the receptor guanylate cyclase C: role in ligand binding and catalytic activity
- 1 May 2004
- journal article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 379 (3), 653-663
- https://doi.org/10.1042/bj20040001
Abstract
GC-C (guanylate cyclase C) is the receptor for heat-stable enterotoxins, guanylin and uroguanylin peptides. Ligand binding to the extracellular domain of GC-C activates the guanylate cyclase domain leading to accumulation of cGMP. GC-C is expressed as differentially glycosylated forms in HEK-293 cells (human embryonic kidney-293 cells). In the present study, we show that the 145 kDa form of GC-C contains sialic acid and galactose residues and is present on the PM (plasma membrane) of cells, whereas the 130 kDa form is a high mannose form that is resident in the endoplasmic reticulum and serves as the precursor for the PM-associated form. Ligand-binding affinities of the differentially glycosylated forms are similar, indicating that glycosylation of GC-C does not play a role in direct ligand interaction. However, ligand-stimulated guanylate cyclase activity was observed only for the fully mature form of the receptor present on the PM, suggesting that glycosylation had a role to play in imparting a conformation to the receptor that allows ligand stimulation. Treatment of cells at 20 °C led to intracellular accumulation of a mature glycosylated form of GC-C that now showed ligand-stimulated guanylate cyclase activity, indicating that localization of GC-C was not critical for its catalytic activity. To determine if complex glycosylation was required for ligand-stimulated activation of GC-C, the receptor was expressed in HEK-293 cells that were deficient in N-acetylglucosaminyltransferase 1. This minimally glycosylated form of the receptor was expressed on the cell surface and could bind a ligand with an affinity comparable with the 145 kDa form of the receptor. However, this form of the receptor was poorly activated by the ligand. Therefore our studies indicate a novel role for glycosidic modification of GC-C during its biosynthesis, in imparting subtle conformational changes in the receptor that allow for ligand-mediated activation and perhaps regulation of basal activity.Keywords
This publication has 42 references indexed in Scilit:
- Determination of the Binding Site on the Extracellular Domain of Guanylyl Cyclase C to Heat-stable EnterotoxinJournal of Biological Chemistry, 1999
- Dual regulation of heat‐stable enterotoxin‐mediated cGMP accumulation in T84 cells by receptor desensitization and increased phosphodiesterase activityFEBS Letters, 1997
- Identification of ligand recognition sites in heat-stable enterotoxin receptor, membrane-associated guanylyl cyclase C by site-directed mutational analysisInfection and Immunity, 1996
- Glycosylation is critical for natriuretic peptide receptor-B functionMolecular and Cellular Biochemistry, 1996
- Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase CMolecular and Cellular Biochemistry, 1996
- Structure, glycosylation, and localization of rat intestinal guanylyl cyclase C: modulation by fastingAmerican Journal of Physiology-Gastrointestinal and Liver Physiology, 1996
- Guanylyl cyclase C is up-regulated by nonparenchymal cells and hepatocytes in regenerating rat liver.1996
- Expression of the Extracellular Domain of the Human Heat-Stable Enterotoxin Receptor inEscherichia coliand Generation of Neutralizing AntibodiesProtein Expression and Purification, 1996
- The significance of Ser1029 of the heat‐stable enterotoxin receptor (STaR): Relation of STa‐mediated guanylyl cyclase activation and signaling by phorbol myristate acetateFEBS Letters, 1996
- Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.1994