Natriuretic peptide receptor A (NPRA) is a noncovalent homodimeric receptor, composed of an extracellular domain (ECD) with a ligand-binding site, a single transmembrane domain (TM), and an intracellular domain (ICD) exhibiting guanylyl cyclase activity. NPRA activation by atrial natriuretic peptide (ANP) leads to cGMP production, which plays important roles in cardiovascular homeostasis. Initial studies have shown that activation of NPRA involves a conformational change in the juxtamembrane domain (JM). However, crystallographic study of the soluble ECD of NPRA has failed to document JM structure, and the conformational change involved in transmembrane signal transduction is still unknown. To analyze this conformational change, we first sequentially substituted nine amino acids of the JM with a cysteine residue. By studying the mutant's capacity to form ANP-induced or constitutive covalent disulfide dimers, we evaluated the relative proximity of JM residues, before and after NPRA activation. These results obtained with the full-length receptor demonstrate a high proximity of specific JM residues and are in disagreement with crystallography data. We also tested the hypothesis that signal transduction involves a TM rotation mechanism leading to ICD activation. By introducing one to five alanine residues into the TM alpha-helix, we show that a TM rotation of 40 degrees leads to constitutive NPRA activation. We finally studied the role of the TM in NPRA dimerization. By using the ToxR system, we demonstrate that the last JM residues are required to stabilize the TM dimer. Using these experimental data, we generated a new molecular model illustrating the active conformation of NPRA, where the JM and TM are depicted.