Role of extracellular domain dimerization in agonist-induced activation of natriuretic peptide receptor A

Natriuretic peptide receptor (NPR) A is composed of an extracellular domain (ECD) with a ligand binding site, a single transmembrane region, a kinase homology domain, and a guanylyl cyclase domain. The natural agonists atrial and brain natriuretic peptides (ANP, BNP) bind and activate NPRA, leading to cyclic GMP production, which is responsible for their role in cardiovascular homeostasis. Previous studies suggested that stabilization of a dimeric form of NPRA by agonist is essential for receptor activation. However, ligand specificity and sequential steps of this dimerization process have not been investigated. We used radioligand binding, fluorescence resonance energy transfer homoquenching, and molecular modeling to characterize the interaction of human NPRA-ECD with ANP, BNP, the superagonist (Arg(10),Leu(12),Ser(17),Leu(18))-rANP-(1-28), the minimized analog mini-ANP and the antagonist (Arg(6),beta-cyclohexyl-Ala(8),d-Tic(16),Arg(17),Cys(18))-rANP-(6-18)-amide (A71915). ANP binds to preformed ECD dimers and spontaneous dimerization is the rate-limiting step of the ligand binding process. All the studied peptides, including A71915 antagonist, induce a dose-dependent fluorescence homoquenching, specific to dimerization, with potencies highly correlated with their binding affinities. A71915 induced more quenching than other peptides, suggesting stabilization by the antagonist of ECD dimer in a distinct inactive conformation. In summary, these results indicate that the ligand-induced dimerization process of NPRA is different from that for cytokine receptor model. Agonists or antagonists bind to preformed dimeric ECD, leading to dimer stabilization in an active or inactive conformation, respectively. Furthermore, the highly sensitive fluorescence assay designed to assess dimerization could serve as a powerful tool for further detailing the kinetic steps involved in natriuretic peptide receptor binding and activation.