Regulation of Guanylyl Cyclase-B: Characterizing the Roles of Gö6976, ATP, and P-site Inhibitors

Abstract

Guanylyl cyclases (GC) play important roles in a wide range of organisms. Three transmembrane GC receptors bind natriuretic peptides with varying affinities. C-type natriuretic peptide (CNP) activates guanylyl cyclase-B (GC-B/NPR-B), which stimulates cGMP synthesis. Although the physiological effects of GC receptors have been well characterized, their regulatory mechanisms are not well understood. The inhibitory effects of the indolocarbazole, Gö6976, on GC-B activity were characterized, and data indicated that Gö6976 potently inhibits the GC activity of GC-B via a process that does not require changes in known phosphorylation sites, inactivation of all catalytic sites, or intact cell architecture. As an ATP analog, Gö6976 may inhibit GC-B by blocking the ability of ATP to activate and/or stabilize the receptor. Maximum activation of GClinked natriuretic peptide receptors in broken cell preparations requires natriuretic peptide binding to the extracellular domain and ATP binding to an unknown intracellular region. Additionally, natriuretic peptide activation of GC-B requires that the intracellular domain be phosphorylated on multiple serine and threonine residues. The dual role of ATP as an allosteric activator of GC-B and a substrate for the kinase that phosphorylates GC-B has led to confusion in the field. Enzymatic timecourse experiments conducted in crude membranes at low and high GTP concentrations support the role of ATP as an allosteric regulator of GTP. Previous studies indicated that dephosphorylation explains long-term inactivation of GCs. However, the deactivation and downregulation of a constitutively phosphorylated version of GC-B were identical to those of the wild type receptor, which indicates that these processes are phosphorylation-independent. One explanation for the time-dependent inactivation of GC-B is the binding of product compounds to the GC domain, similarly to how purine site (P-site) inhibitors inhibit adenylyl cyclase. Preliminary studies characterized the effect of 2’d3’GMP and PPi on GC-B activity in order to gain insight into the catalytic mechanisms of GC receptors.