Abstract
Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of approximately 70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand- and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.
Highlights
Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP
Because the highest homology between receptor guanylyl cyclases (GCs) is in the catalytic cyclase domain, we suggest that specific residues in the linker region, through their interactions with the cyclase domain, assist in repressing guanylyl cyclase activity
Based on the phenotypes of the mutations that were observed and a lack of similarity in properties of mutants generated in equivalent positions in a coiled coil, we suggest that this region in guanylyl cyclases may not adopt a classical coiled coil helical structure
Summary
Generation of Mutations in the Linker Region of GC-C and GC-A—Human GC-C cDNA (pBSK-GC-C) (22) was used as template for the generation of mutations in the linker region of GC-C using the single mutagenic oligonucleotide-based protocol described earlier (23). A fragment encoding GC-C was excised from the respective mutant pBSK-GC-C by digestion with XhoI (or SalI in cases where the mutagenic primer introduced an XhoI site) and XbaI and ligated into XhoI-XbaI-digested pcDNA3 to allow expression in mammalian cells. A HindIII fragment from pCMV-GC-A (24) representing the KHD and a part of the linker region was cloned into the HindIII site of pGEM-11Zf(ϩ) (Promega Life Science) to generate the plasmid pGEM-11Zf-GC-AKHD-LR This was subsequently used as template for the generation of L812P, M816P, and Y819P mutations in the linker region of GC-A using the respective mutagenic primer as detailed in supplemental Table 1. In cells transfected with GC-A were used as templates to generate the models of the inactive encoding plasmids, a polyclonal antibody to GC-A Labeled STY72F for 1 h at 37 °C in the presence (nonspecific binding) or absence of 10Ϫ7 M concentrations of unlabeled ST
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