Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase

Abstract

Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G(s)α to C2 and the ensuing 7° rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.

Figure 1

Amino acid sequences of ANP, BNP and CNP from rat. Two Cys residues in each peptide form an intra-molecular disulfide-bond, which is essential for the activity [90]. Conserved residues are shaded.

Figure 2

The molecular topology of the NPRA. The NPRA occurs as a pre-formed homodimer. Each monomer contains an extracellular ANP-binding domain (ECD), a transmembrane domain, and an intracellular domain (ICD) consisting of a protein kinase-like domain (PKLD) and a GC domain (GCD). The ECD contains a highly conserved chloride binding site [33, 17, 18] and a juxtamembrane GC-signature motif [20]. Bound chloride (cyan ball) is essential for ANP binding [36]. The juxtamembrane GC-signature motif plays a critical role in transmembrane signal transduction. The PKLD binds the positive allosteric effector ATP [21, 22] and is phosphorylated at multiple sites [23].

Figure 3

(a) Diagram illustrating the covalent structure of the ECD. The ECD contains five N-linked oligosaccharides (boxes) [31] and three disulfide-bonds (orange lines) [30]. The glycosylated Asn residues and disulfide-bonded Cys residues are indicated. No free Cys residue is present in the ECD.

Figure 4

(a,b) Crystal structures of apo ECD dimer (PDB: 1DP4) and ANP-ECD complex (PDB: 1T34) [33, 17]. ANP is shown in green. Protein-bound chloride atoms are shown by magenta balls. (c) Close-up view of ANP binding interactions. Major interactions are shown circled. (d) Close-up view of the chloride binding site in the apo ECD [33, 18]. Chloride is hydrogen bonded to the hydroxyl-group of Ser53, and the backbone NH moieties of Gly85 and Cys86. The binding site also contains the only cis-peptide bond in the ECD (green arrow head) and the Cys60-Cys-86 disulfide bond. The van der Waals radius of the chloride atom is represented by a green dotted ball.

Figure 5

(a) Schematic illustration of ANP-induced change in the ECD dimer structure. ANP binding causes a twisting motion of the two ECD monomers from the apo position (blue) to the complex position (orange) [17, 35]. (b) Viewed toward the membrane, the juxtamembrane domains in the apo form (blue circles) translate to the complex position (orange circles) with essentially no change in the inter-domain distance. The arrows depict parallel translocation. This motion causes a change in the angular relationship between the two domains equivalent to rotating each domain by 24° counter-clockwise. Because the dimerized receptor is free to spin or move about in the cell membrane, this rotation motion occurring in the juxtamembrane domains would be the only structural change that is “recognized” by the receptor upon ANP binding. (c) ANP-induced conformational change observed by single-particle electron microscopy [72]. Reconstruction of the apo ECD dimer (blue mesh) is superimposed onto that of the ANP-ECD complex (gold surface). The reconstructions are rendered at 70% of the correct molecular volume for clarity.

Figure 6

Rotation mechanism proposed for transmembrane signaling by the NPRA. Taken from a textbook, Biochemistry by Garrett and Grisham, 4th edition, 2009 [73] (drawing adapted from [35]). The details are in the text.

Figure 7

(a) Structure of Cyg12 GCD dimer (PDB: 3ET6), which is an open inactive conformation [78]. The arrows show the surface grooves in the GCD that correspond to the G_sα binding site in AC C1 domain [80]. The N- and C-terminal ends of each monomer are labeled. The 2-fold symmetry axis in the dimer runs perpendicular to the plane of the page. The dimer structure is seen from the C-terminal end. (b) Model for GC activation [78]. The GCD monomer (yellow) was aligned to the C1 domain of the activated G_sα-AC complex [82] (PDB: 1CJU) and overlaid onto the open-inactive GCD dimer (cyan, PDB: 3ET6). (c) Model of the closed active GCD dimer conformation (yellow) overlaid onto the open inactive GCD dimer (cyan). The rotation of each of the two domains (around each own axis) leads to the closed and active conformation (arrows).