Disulfide Linkages and a Three-Dimensional Structure Model of the Extracellular Ligand-binding Domain of Guanylyl Cyclase C Makoto Hasegawa, 1,5 Yoshiko Matsumoto-Ishikawa, 2 Atsushi Hijikata, 3,4 Yuji Hidaka, 2 Mitiko Go, 1 and Yasutsugu Shimonishi 1 Guanylyl cyclase C (GC-C) is a single-transmembrane receptor that is specifically activated by endogenous ligands, including guanylin, and the exogenous ligand, heat-stable enterotoxin. Using combined HPLC separation and MS analysis techniques the positions of the disulfide linkages in the extracellular ligand-binding domain (ECD) of GC-C were determined to be between Cys 7 –Cys 94 , Cys 72 –Cys 77 , Cys 101 –Cys 128 and Cys 179 –Cys 226 . Furthermore, a three- dimensional structural model of the ECD was constructed by homology modeling, using the structure of the ECD of GC-A as a template (van den Akker et al., 2000, Nature, 406: 101–104) and the information of the disulfide linkages. Although the GC-C model was similar to the known structure of GC-A, importantly its ligand-binding site appears to be located on the quite different region from that in GC-A. KEY WORDS: Binding region; disulfide linkages; guanylyl cyclase C; receptor; three-dimensional structure. 1. INTRODUCTION Guanylyl cyclase (GC) 6 -C is a transmembrane pro- tein that is specifically localized on intestinal brush border membranes (Schulz et al., 1990). The enzyme serves as a receptor protein for the polypeptide ligand, guanylin (Currie et al., 1992), and related endogenous ligands, as well as heat-stable entero- toxin (STa), an exogenous ligand produced by enteric bacteria such as enterotoxigenic Escherichia coli (Smith et al., 1970). The binding of a ligand to GC-C results in an elevation of the second messen- ger, cGMP, in intestinal cells and activates a cystic fibrosis transmembrane conductance regulator via interaction with cGMP-dependent protein kinase in apical membranes. This process ultimately results in the secretion of chloride and water from the inside of cells to the extracellular space (Pfeifer et al., 1996). Thus, GC-C is involved in the regulation of chloride transport in electrolytes, that is, the homeostasis of intestinal fluid and electrolytes in 1 Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan. 2 Division of Protein Organic Chemistry, Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan 3 Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Aichi, 464-8602, Japan. 4 Current address: Laboratory for Immunogenomics, Research Center for Allergy and Immunology (RCAI), The Institute of Physical and Chemical Research (RIKEN), Yokohama, Kanag- awa, 230-0045, Japan. 5 To whom correspondence should be addressed. Nagahama Institute of Bio-Science and Technology, Tamura-cho 1266, Nagahama, Shiga 526-0829, Japan. Tel.: +81-749-64 8110, Fax: +81-749-8138, E-mail: m_hasegawa@nagahama-i-bio-ac.jp Abbreviations: GC, guanylyl cyclase; ECD, extracellular domain; STa, heat-stable enterotoxin produced by enterotoxigenic Escheri- chia coli; STp(4-17), porcine STa containing the amino acid sequence from position 4 to 17; ANB, 5-azido-2-nitrobezyl; PNGase-F, peptide-N-glycosidase F; LC, liquid chromatography. 315 1572-3887/05/0700-0315/0 Ó 2005 Springer Science+Business Media, Inc. The Protein Journal, Vol. 24, No. 5, July 2005 (Ó 2005) DOI: 10.1007/s 10930-005-6752-x