Functional Inactivation of the Human Guanylyl Cyclase C Receptor: Modeling and Mutation of the Protein Kinase-like Domain

Bhandari, Rashna and Srinivasan, N and Mahaboobi, * and Ghanekar, Yashoda and Suguna, K and Visweswariah, Sandhya S (2001) Functional Inactivation of the Human Guanylyl Cyclase C Receptor: Modeling and Mutation of the Protein Kinase-like Domain. In: Biochemistry, 40 (31). 9196 -9206.

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Abstract

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 $(GC-CK_{516A})$ 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, $GC-CK_{516A}$ 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 $K_5_1_6$ is critical for the possible proper orientation of ATP in this domain.

Item Type:	Journal Article
Publication:	Biochemistry
Publisher:	American Chemical Society
Additional Information:	Copyright of this article belongs to American Chemical Society.
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit Division of Biological Sciences > Molecular Reproduction, Development & Genetics
Date Deposited:	30 Mar 2007
Last Modified:	19 Sep 2010 04:36
URI:	http://eprints.iisc.ac.in/id/eprint/10542

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