Glycosylation of the receptor guanylate cyclase C: role in ligand binding and catalytic activity

• Published in: Biochemical journal 2004-05, Vol.379 (Pt 3), p.653-663
• Main Authors: Ghanekar, Yashoda, Chandrashaker, Akhila, Tatu, Utpal, Visweswariah, Sandhya S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Catalysis; Cell Line; Cell Membrane - metabolism; Endoplasmic Reticulum - metabolism; Galactose - metabolism; Glycosylation; Guanylate Cyclase - chemistry; Guanylate Cyclase - metabolism; Half-Life; Humans; Ligands; Mannose - metabolism; Molecular Sequence Data; Molecular Weight; N-Acetylglucosaminyltransferases - deficiency; N-Acetylneuraminic Acid - metabolism; Protein Conformation; Protein Processing, Post-Translational; Protein Transport; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide - chemistry; Receptors, Peptide - metabolism; Trypsin - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj20040001

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 degrees 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.