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.
- guanylate cyclase C
- human embryonic kidney-293 cells (HEK-293 cells)
- surface localization
Abbreviations used: DIG, digoxigenin; ECD, extracellular domain; endo H, endo-β-N-acetylglucosaminidase H; ER, endoplasmic reticulum; GC-C, guanylate cyclase C; GNA, Galanthus nivalis agglutinin; GnT1, N-acetylglucosaminyltransferase 1; HEK-293 cells, human embryonic kidney-293 cells; NHS, N-hydroxysuccinimido; PKLD, protein kinase-like domain; PM, plasma membrane; PNGase F, peptide N-glycosidase F; ST, heat-stable enterotoxin.
- The Biochemical Society, London ©2004