title: Glycosylation of the receptor guanylate cyclase C: role in ligand binding and catalytic activity.
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 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.
authors.affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Yashoda
authors.initials: Y
authors.lastname: Ghanekar
authors.forename: Akhila
authors.initials: A
authors.lastname: Chandrashaker
authors.forename: Utpal
authors.initials: U
authors.lastname: Tatu
authors.forename: Sandhya S
authors.initials: SS
authors.lastname: Visweswariah
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D006031:Glycosylation; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D008024:Ligands; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017351:N-Acetylglucosaminyltransferases / Q000172:deficiency; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000737:chemistry* / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D008024:Ligands; D018000:Receptors, Peptide; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D014357:Trypsin; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D019158:N-Acetylneuraminic Acid; D008358:Mannose; D005690:Galactose