A disulfide-bridged mutant of natriuretic peptide receptor-A displays constitutive activity. Role of receptor dimerization in signal transduction

Natriuretic peptide receptor-A (NPR-A), a particulate guanylyl cyclase receptor, is composed of an extracellular domain (ECD) with a ligand binding site, a transmembrane spanning, a kinase homology domain (KHD), and a guanylyl cyclase domain. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), the natural agonists, bind and activate the receptor leading to cyclic GMP production. This receptor has been reported to be spontaneously dimeric or oligomeric. In response to agonists, the KHD-mediated guanylate cyclase repression is removed, and it is assumed that ATP binds to the KHD. Since NPR-A displays a pair of juxtamembrane cysteines separated by 8 residues, we hypothesized that the removal of one of those cysteines would leave the other unpaired and reactive, thus susceptible to form an interchain disulfide bridge and to favor the dimeric interactions. Here we show that NPR-AC423S mutant, expressed mainly as a covalent dimer, increases the affinity of pBNP for this receptor by enhancing a high affinity binding component. Dimerization primarily depends on ECD since a secreted NPR-A C423S soluble ectodomain (ECDC423S) also documents a covalent dimer. ANP binding to the unmutated ECD yields up to 80-fold affinity loss as compared with the membrane receptor. However, the ECD C423S mutation restores a high binding affinity. Furthermore, C423S mutation leads to cellular constitutive activation (20-40-fold) of basal catalytic production of cyclic GMP by the full-length mutant. In vitro particulate guanylyl cyclase assays demonstrate that NPR-AC423S displays an increased sensitivity to ATP treatment alone and that the effect of ANP + ATP joint treatment is cumulative instead of synergistic. Finally, the cellular and particulate guanylyl cyclase assays indicate that the receptor is desensitized to agonist stimulation. We conclude the following: 1) dimers are functional units of NPR-A guanylyl cyclase activation; and 2) agonists are inducing dimeric contact of the juxtamembranous region leading to the removal of the KHD-mediated guanylyl cyclase repression, hence allowing catalytic activation.