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

Abstract

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.

Brain-derived neurotrophic factor receptor TrkB exists as a preformed dimer in living cells

“…These receptors are the aspartate receptor Tar [17], epidermal growth factor receptor (EGFR) family [12,18-21], erythropoietin receptor [22], growth hormone receptor (GHR) [23,24], Toll-like receptor-9 [25], natriuretic peptide receptor A (NPRA) [26], and nerve growth factor receptor TrkA [8]. In the cases of EGFR, GHR, NPRA and Tar, it has been proposed that ligand binding is likely to induce the rotation/twist of the transmembrane domains of the preformed receptor dimers, resulting in rearrangement of the cytoplasmic domains for activation [18,24,26,27]. Therefore, further study of structures of the Trk receptor family will shed light on molecular mechanisms underlying the receptor activation.…”

Normal Activation of Discoidin Domain Receptor 1 Mutants with Disulfide Cross-links, Insertions, or Deletions in the Extracellular Juxtamembrane Region

“…4 and 8). In sharp contrast, replacing JM residues with cysteine yielded constitutively active mutants for Erb-B2, the erythropoietin receptor, the p75 neurotrophin receptor and the natriuretic peptide receptor A (29, 30, 32–34) and common gain-of-function mutations in other RTKs result in unpaired cysteines that form intermolecular disulfide bonds (44–47). In the EGF receptor, extracellular insertion of a flexible sequence close to the cell membrane resulted in increased basal activation (28), whereas DDR1 activation was unaffected by deletions from the JM region and by insertions of flexible glycine-serine sequences (Figs.…”

“…Refs. 29–32). Using this method, covalent dimerization by disulfide bridges is enforced at various positions, which allows conclusions to be drawn about the relative geometry of receptor protomers within signaling dimers.…”

“…Using this method, covalent dimerization by disulfide bridges is enforced at various positions, which allows conclusions to be drawn about the relative geometry of receptor protomers within signaling dimers. For some receptors, cysteine substitution mutagenesis leads to constitutively active mutants (29, 30, 33, 34), whereas for other receptors regions that are involved in conformational changes can be identified (32). We found that the 10 extracellular JM residues of DDR1 closest to the TM domain could be cross-linked with very high efficiency.…”

Receptor Guanylyl Cyclases in Sensory Processing

“…ANP binding to GC-A induces a 40° rotation (or twist) of the receptor’s TMD parallel to the plane of the membrane (141). The subunits of GCD dimers also rotate to form a catalytically active structure as described above (136).…”