Functional inactivation of the human guanylyl cyclase C receptor: modeling and mutation of the protein kinase-like domain.
Receptor guanylyl cyclases possess an extracellular ligand-binding domain, a single transmembrane region, a region with sequence similar to that of protein kinases, and a C-terminal guanylyl cyclase domain. ATP regulates the activity of guanylyl cyclase C (GC-C), the receptor for the guanylin and stable toxin family of peptides, presumably as a result of binding to the kinase homology domain (KHD). Modeling of the KHD of GC-C indicated that it could adopt a structure similar to that of tyrosine kinases, and sequence comparison with other protein kinases suggested that lysine(516) was positioned in the KHD to interact with ATP. A monoclonal antibody GCC:4D7, raised to the KHD of GC-C, did not recognize ATP-bound GC-C, and its epitope mapped to a region in the KHD of residues 491--568 of GC-C. Mutation of lysine(516) to an alanine in full-length GC-C (GC-C(K516A)) dramatically reduced the ligand-stimulated activity of mutant GC-C, altered the ATP-mediated effects observed with wild-type GC-C, and failed to react with the GCC:4D7 monoclonal antibody. ATP interaction with wild-type GC-C converted a high-molecular weight oligomer of GC-C to a smaller sized oligomer. In contrast, GC-C(K516A) did not exhibit an alteration in its oligomeric status on incubation with ATP. We therefore suggest that the KHD in receptor guanylyl cyclases provides a critical structural link between the extracellular domain and the catalytic domain in regulation of activity in this family of receptors, and the presence of K(516) is critical for the possible proper orientation of ATP in this domain.
Bhandari
R
R
Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science, Bangalore 560012, India.
Srinivasan
N
N
Mahaboobi
M
M
Ghanekar
Y
Y
Suguna
K
K
Visweswariah
S S
SS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation / Q000235:genetics; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D008239:Lysine; D000409:Alanine