The Kinase Homology Domain of Receptor Guanylyl Cyclase C:  ATP Binding and Identification of an Adenine Nucleotide Sensitive Site

Abstract

The role of the kinase homology domain (KHD) in receptor guanylyl cyclases is to regulate the activity of the catalytic guanylyl cyclase domain. The KHD lacks many of the amino acids required for phosphotransfer activity and, therefore, is not expected to possess kinase activity. Guanylyl cyclase activity of the receptor guanylyl cyclase C (GC-C) is modulated by ATP, and computational modeling showed that the KHD can adopt a structure similar to protein kinases, suggesting that the KHD is the site for ATP interaction. A monoclonal antibody, GCC:4D7, raised to the KHD of GC-C, fails to react with GC-C in the presence of ATP and ATP analogues that regulate GC-C catalytic activity, indicating that a conformational change occurs in the KHD on ATP binding. Mapping of the epitope of the antibody through the use of recombinant protein constructs and phage display showed that the epitope for GC-C:4D7 lies immediately C-terminal to a critical lysine residue (Lys516 in GC-C), required for ATP interaction in protein kinases. By employing a novel approach utilizing ATP-agarose affinity chromatography, we demonstrate that the intracellular domain of GC-C and the KHD bind ATP. Mutation of Lys516 to Ala abolishes ATP binding. Thus, this report is the first to show direct ATP binding to the pseudokinase domain of receptor guanylyl cyclase C, as well as to identify dramatic conformational changes that occur in this domain on ATP binding, akin to those seen in catalytically active protein kinases.

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module

“…Western Blot Analysis-Cells after transfection were directly lysed in SDS sample buffer and subjected to Western blot analysis using the GCC:C8 monoclonal antibody to GC-C essentially as described earlier (2). In cells transfected with GC-A encoding plasmids, a polyclonal antibody to GC-A (a kind gift of Dr. Lincoln R. Potter, University of Minnesota) was used.…”

“…The extracellular domain of GC-C is glycosylated, and we have shown the importance of glycosylation in regulating receptor desensitization in colonic cells. We have also identified a critical residue (Lys-516) in the KHD of GC-C as being important for KHD-mediated modulation of the guanylyl cyclase activity (2,3). A sequence of ϳ70 amino acids is found between the KHD and the guanylyl cyclase domain of receptor GCs, which we refer to here as the linker region (13).…”

“…The intracellular domain of receptor GCs contains a region that shares considerable sequence similarity to protein kinases and is referred to as the kinase homology domain (KHD). Binding of ATP to the KHD induces a conformational change that regulates cGMP production by the guanylyl cyclase domain (2). Thus, receptor GCs exemplify the intricate interactions between domains in transducing the signal from an extracellular ligand to the interior of the cell.…”

“…The KHD shows ϳ25-30% conservation in amino acid sequence across receptor GCs, and computational modeling has not only suggested that this region could adopt the overall structure of a protein kinase but also identified specific residues that could interact with ATP (2,3). The catalytic domains of mammalian receptor GCs are more conserved (ϳ80% sequence similarity).…”

“…1 Supported by a senior research fellowship from the Council of Scientific and Industrial Research, Government of India. 2 Supported by a post-doctoral fellowship from the Department of Biotechnology. 3 To whom correspondence should be addressed.…”