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 rece ptor, is composed of an extracellular domain (ECD) with a ligand binding si te, a transmembrane spanning, a kinase homology domain (KHD), and a guanyly l cyclase domain. Atrial natriuretic peptide (ANP) and brain natriuretic pe ptide (BNP), the natural agonists, bind and activate the receptor leading t o cyclic GMP production. This receptor has been reported to be spontaneousl y dimeric or oligomeric. In response to agonists, the KI-ID-mediated guanyl ate 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 interch ain disulfide bridge and to favor the dimeric interactions. Here we show th at NPR-A(C423S) mutant, expressed mainly as a covalent dimer, increases the affinity of pBNP for this receptor by enhancing a high affinity binding co mponent, Dimerization primarily depends on ECD since a secreted NPB-A C423S soluble ectodomain (EGD(C423S)) also documents a covalent dimer, ANP bindi ng 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 bin ding affinity. Furthermore, C423S mutation leads to cellular constitutive a ctivation (20-40-fold) of basal catalytic production of cyclic GMP by the f ull-length mutant. In vitro particulate guanylyl cyclase assays demonstrate that NPR-A(C423S) 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 concl ude the following: I) dimers are functional units of NPR-A guanylyl cyclase activation; and 2) agonists are inducing dimeric contact of the juxtamembr anous region leading to the removal of the KHD-mediated guanylyl cyclase re pression, hence allowing catalytic activation.