Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer.
Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn(2+), at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent cross-linking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.
Vijayachandra
K
K
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Guruprasad
M
M
Bhandari
R
R
Manjunath
U H
UH
Somesh
B P
BP
Srinivasan
N
N
Suguna
K
K
Visweswariah
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary / Q000235:genetics; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000235:genetics; D013388:Succinimides / Q000737:chemistry
D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D013388:Succinimides; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; C019358:disuccinimidyl suberate