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

详细信息    查看全文

• 作者：Rashna Bhandari ; N. Srinivasan ; Mahaboobi ; Yashoda Ghanekar ; K. Suguna ; and Sandhya S. Visweswariah
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：August 7, 2001
• 年：2001
• 卷：40
• 期：31
• 页码：9196 - 9206
• 全文大小：548K
• 年卷期：v.40,no.31(August 7, 2001)
• ISSN：1520-4995

文摘

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 guanylylcyclase domain. ATP regulates the activity of guanylyl cyclase C (GC-C), the receptor for the guanylinand 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 tyrosinekinases, and sequence comparison with other protein kinases suggested that lysine₅₁₆ was positioned inthe KHD to interact with ATP. A monoclonal antibody GCC:4D7, raised to the KHD of GC-C, did notrecognize ATP-bound GC-C, and its epitope mapped to a region in the KHD of residues 491-568 ofGC-C. Mutation of lysine₅₁₆ to an alanine in full-length GC-C (GC-C_K516A) dramatically reduced theligand-stimulated activity of mutant GC-C, altered the ATP-mediated effects observed with wild-typeGC-C, and failed to react with the GCC:4D7 monoclonal antibody. ATP interaction with wild-type GC-Cconverted a high-molecular weight oligomer of GC-C to a smaller sized oligomer. In contrast, GC-C_K516Adid not exhibit an alteration in its oligomeric status on incubation with ATP. We therefore suggest thatthe KHD in receptor guanylyl cyclases provides a critical structural link between the extracellular domainand the catalytic domain in regulation of activity in this family of receptors, and the presence of K₅₁₆ iscritical for the possible proper orientation of ATP in this domain.