REGULATION OF GUANYLYL CYCLASE-B: ROLES OF ATP AND P-SITE INHIBITORS

Abstract

C-type natriuretic peptide (CNP) activates the transmembrane guanylyl cyclase natriuretic peptide receptor-B (NPR-B/GC-B), which stimulates cGMP synthesis and mediates long bone growth, vasorelaxation, and axonal guidance. Genetic mutations that disrupt normal signaling through this pathway lead to human skeletal over- and under-growth. Currently, the role of ATP in receptor activation is controversial. Although ATP is not required for initial activation, it may stabilize the catalytic domain at longer time points and increase its affinity for GTP. Enzymatic time course experiments conducted in crude membranes indicated that 1mM ATP increases guanylyl cyclase activity 2-3 fold when measured at high (1mM) GTP concentrations but increases activity 10-fold at low (0.1mM) GTP concentrations. Moreover, receptor activity declines at a greater rate in the absence of ATP than in its presence at 0.1mM [GTP] versus 1mM [GTP]. We propose that product inhibition, the binding of purine nucleotides and pyrophosphate to the catalytic domain, explains the observed receptor deactivation. Additional experiments are underway to characterize the roles of P-site inhibitors on GC-B activity, which may provide new insights into the catalytic mechanism of guanylyl cyclase receptors.