title: Determination of the binding site on the extracellular domain of guanylyl cyclase C to heat-stable enterotoxin.
abstract: Guanylyl cyclase C, one of the family of membrane-bound guanylyl cyclases, consists of an extracellular domain and an intracellular domain, which are connected by a single transmembrane polypeptide. The extracellular domain binds unique small polypeptides with high specificity, which include the endogenous peptide hormones, guanylin and uroguanylin, as well as an exogenous enterotoxigenic peptide, heat-stable enterotoxin, secreted by pathogenic Escherichia coli. Information on this specific binding is propagated into the intracellular domain, followed by the synthesis of cGMP, a second messenger that regulates a variety of intracellular physiological processes. This study reports the design of a photoaffinity labeled analog of heat-stable enterotoxin (biotinyl-(AC(5))(2)-[Gly(4), Pap(11)]STp(4-17)), which incorporates a Pap residue (p-azidophenylalanine) at position 11 and a biotin moiety at the N terminus, and the use of this analog to determine the ligand-binding region of the extracellular domain of guanylyl cyclase C. The endoproteinase Lys-C digestion of the extracellular domain, which was covalently labeled by this ligand, and mass spectrometric analyses of the digest revealed that the ligand specifically binds to the region (residue 387 to residue 393) of guanylyl cyclase C. This region is localized close to the transmembrane portion of guanylyl cyclase C on the external cellular surface. This result was further confirmed by characterization of site-directed mutants of guanylyl cyclase C in which each amino acid residue was substituted by an Ala residue instead of residues normally located in the region. This experiment provides the first direct demonstration of the ligand-binding site of guanylyl cyclase C and will contribute toward an understanding of the receptor recognition of a ligand and the modeling of the interaction of the receptor and its ligand at the molecular level.
authors.affiliation: Division of Protein Organic Chemistry, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Hasegawa
authors.forename: Y
authors.initials: Y
authors.lastname: Hidaka
authors.forename: Y
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: T
authors.initials: T
authors.lastname: Sanni
authors.forename: Y
authors.initials: Y
authors.lastname: Shimonishi
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D019921:Biotinylation; D016764:Cell Polarity; D006152:Cyclic GMP / Q000378:metabolism; D004768:Enterotoxins / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D019920:Photoaffinity Labels; D011485:Protein Binding; D017433:Protein Structure, Secondary; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D008024:Ligands; D019920:Photoaffinity Labels; D018000:Receptors, Peptide; C034151:heat stable toxin (E coli); D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D006152:Cyclic GMP