Functional inactivation of the human guanylyl cyclase C receptor: Modelingand 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 pr otein kinases, and a C-terminal guanylyl cyclase domain. ATP regulates the activity of guanylyl cyclase C (GC-C), the receptor for the guanylin and st able toxin family of peptides, presumably as a result of binding to the kin ase homology domain (KHD). Modeling of the KHD of GC-C indicated that it co uld adopt a structure similar to that of tyrosine kinases, and sequence com parison with other protein kinases suggested that lysine(516) was positione d 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 li gand-stimulated activity of mutant GC-C, altered the ATP-mediated effects o bserved with wild-type GC-C, and failed to react with the GCC:4D7 monoclona l 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-CK516A did not exhibit an alteration in its oligomeric status on incubation with ATP. We therefore suggest that the KHD in receptor guanylyl cyclases provid es a critical structural link between the extracellular domain and the cata lytic 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 i n this domain.