Identification of ligand recognition sites in heat-stable enterotoxin receptor, membrane-associated guanylyl cyclase C by site-directed mutational analysis.
Guanylyl cyclase C (STaR), a receptor protein for heat-stable enterotoxin (STa) elaborated by Escherichia coli, is associated with and spans the plasma membrane of mammalian intestinal cells. The extracellular domain functions in the binding of STa and the association of each domain to an oligomeric form. Two amino acid residues, Arg-136 and Asp-347, were identified as the residues binding to STa in the extracellular domain of pig STaR by site-directed mutagenesis and analysis of expression on 293T cells. Replacement of these residues by other amino acid residues resulted in the loss of binding of pig STaR to STa, and as a result, STa-induced guanylyl cyclase activity was eliminated. Furthermore, mutation in a region (from Asp-347 to Val-401) which is close to the transmembrane domain caused a significant reduction in both STa-binding activity and guanylyl cyclase catalytic activity. These results suggest that the region adjacent to the transmembrane domain plays an important role in facilitating a favorable conformation of STaR for STa binding.
Wada
A
A
Institute for Protein Research, Osaka University, Suita, Japan.
Hirayama
T
T
Kitaura
H
H
Fujisawa
J
J
Hasegawa
M
M
Hidaka
Y
Y
Shimonishi
Y
Y
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000382:microbiology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000235:genetics* / Q000378:metabolism
D004768:Enterotoxins; D018000:Receptors, Peptide; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled