Role of Extracellular Domain Dimerization in Agonist-Induced Activation of Natriuretic Peptide Receptor A

Abstract

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 ,␤-cyclohexylAla 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 dosedependent 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.

Allosteric Modification, the Primary ATP Activation Mechanism of Atrial Natriuretic Factor Receptor Guanylate Cyclase

“…In solution they existed as monomers as determined by FPLC (Experimental Procedures). It is in agreement with the existing biochemical, crystalographical and molecular modeling data showing that dimeric contact points between two ANF-RGC monomers are within the extracellular domain (34, 35, 61, 62) and the catalytic domain (25). …”

Role of Juxtamembrane and Transmembrane Domains in the Mechanism of Natriuretic Peptide Receptor A Activation

“…Constitutive NPRA Activation by Relative Subunit Rotation. Previous studies in our laboratory demonstrated that dimerization of NPRA is not sufficient for activation (19). These results have been confirmed in this study.…”

Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation pattern

“…The GC‐A receptor consists of an extracellular ligand‐binding domain of approximately 441 amino acids (aa), a short membrane‐spanning region (21 aa) and an intracellular portion (567 aa), containing a kinase homology (KH) domain, the dimerization domain and the C‐terminal catalytic GC domain [1,7]. In the absence of ligand, GC‐A forms homodimers or homotetramers, the KH domain is highly phosphorylated and the catalytic activity is tightly repressed [8–10]. On ANP binding, there is no change in the oligomeric state, but apparently a conformational change occurs which activates the cyclase domain [11].…”