The atrial natriuretic peptide (ANP) hormone is secreted by the heart in response to an increase in blood pressure. ANP exhibits several potent anti-hypertensive actions in the kidney, adrenal gland and vascular system. These actions are induced by hormone binding extracellularly to the ANP receptor, thereby activating its intracellular guanylyl cyclase domain for the production of cyclic GMP. Here we present the crystal structure of the glycosylated dimerized hormone-binding domain of the ANP receptor at 2.0-A resolution. The monomer comprises two interconnected subdomains, each encompassing a central beta-sheet flanked by alpha-helices, and exhibits the type I periplasmic binding protein fold. Dimerization is mediated by the juxtaposition of four parallel helices, arranged two by two, which brings the two protruding carboxy termini into close relative proximity. From affinity labelling and mutagenesis studies, the ANP-binding site maps to the side of the dimer crevice and extends to near the dimer interface. A conserved chloride-binding site is located in the membrane distal domain, and we found that hormone binding is chloride dependent. These studies suggest mechanisms for hormone activation and the allostery of the ANP receptor.

antiviral mechanism ͉ autoinhibition ͉ NMR ͉ peptide activator ͉ protein kinase

The receptor for atrial natriuretic peptide (ANP) is a type-I transmembrane protein containing an extracellular ligand-binding domain, a single transmembrane sequence, an intracellular kinase-homologous domain, and a guanylate cyclase (GCase) domain. Binding of ANP to the extracellular domain causes activation of the GCase domain by an as yet unknown mechanism. To facilitate studies of the receptor structure and signaling mechanism, we have expressed the extracellular ANP-binding domain of rat ANP receptor (NPR-ECD) in a water-soluble form. NPR-ECD was purified to homogeneity by ANP-affinity chromatography. SDS-PAGE gave a single 61-kDa band, which coincided with a radioactive band obtained by photoaffinity-labeling with N4alpha-azidobenzoyl-125I-ANP(4-28). Edman degradation gave a single amino-terminal sequence expected for the mature protein. Both trifluoromethanesulfonic acid and peptide-N-glycosidase F treatments yielded a 50-kDa band, indicating N-glycosylation. The molecular mass of 57 725 Da determined by mass spectrometry indicates the carbohydrate content at 16%. NPR-ECD bound ANP with an affinity comparable to that of the full-length receptor. The ligand selectivity of NPR-ECD (in the order ANP > brain natriuretic peptide >> C-type natriuretic peptide) was also similar to that of the full-length receptor. HPLC gel filtration of NPR-ECD gave a peak with an apparent mass of 74 kDa. Preincubation with ANP generated a new 150-kDa peak with a concomitant decrease of the 74-kDa peak. This shift in peak positions was ANP concentration-dependent and was complete at the NPR-ECD-to-ANP molar ratio of 1:1, indicating equimolar binding. The change in the apparent native molecular weight from 74 to 150 kDa suggests that binding causes dimerization of the NPR-ECD:ANP complex to yield an [NPR-ECD:ANP]2 complex.

Atrial natriuretic peptide (ANP) is a hormone involved in cardiovascular homeostasis through its natriuretic and vasodilator actions. The ANP receptor that mediates these actions is a glycosylated transmembrane protein coupled to guanylate cyclase. The role of glycosylation in receptor signaling remains unresolved. In this study, we determined, by a combination of HPLC/MS and Edman sequencing, the glycosylation sites in the extracellular domain of ANP receptor (NPR-ECD) from rat expressed in COS-1 cells. HPLC/MS analysis of a tryptic digest of NPR-ECD identified five glycosylated peptide fragments, which were then sequenced by Edman degradation to determine the glycosylation sites. The data revealed Asn-linked glycosylation at five of six potential sites. The type of oligosaccharide structure attached at each site was deduced from the observed masses of the glycosylated peptides as follows: Asn13 (high-mannose), Asn180 (complex), Asn306 (complex), Asn347 (complex), and Asn395 (high-mannose and hybrid types). Glycosylation at Asn180 and Asn347 was partial. The role of glycosyl moieties in ANP binding was examined by enzymatic deglycosylation of NPR-ECD followed by binding assay. NPR-ECD deglycosylated with endoglycosidase F 2 and endoglycosidase H retained ANP-binding activity and showed an affinity for ANP similar to that of untreated NPR-ECD. Endoglycosidase treatment of the full-length ANP receptor expressed in COS-1 cells also had no detectable effect on ANP binding. These results suggest that, although glycosylation may be required for folding and transport of the newly synthesized ANP receptor to the cell surface, the oligosaccharide moieties themselves are not involved in hormone binding.Keywords: glycosylation; ligand binding; mass spectrometry; natriuretic peptide; receptor.Atrial natriuretic peptide (ANP) is a peptide hormone secreted from the atrium of the heart in response to blood volume expansion. It stimulates renal salt excretion, dilates blood vessels, and suppresses aldosterone and renin secretion, thus lowering blood pressure and volume [1]. These ANP actions are mediated by a family of transmembrane receptors coupled to guanylate cyclase, which include A-type and B-type natriuretic peptide receptor (NPR-A and NPR-B, respectively). NPR-A mediates most of the known actions of ANP and brain natriuretic peptide. NPR-B is thought to mediate the action of C-type natriuretic peptide in the central nervous system [2]. Both NPR-A and NPR-B receptors consist of a single polypeptide that contains an extracellular ANP-binding domain, a single transmembrane sequence, and an intracellular domain consisting of a kinase-homologous domain and a guanylate cyclase domain. Binding of the hormone to the extracellular domain activates the intracellular guanylate cyclase domain, generating cGMP as the intracellular second messenger. The mechanism of this guanylate cyclase activation by hormone binding remains largely unknown.Both NPR-A and NPR-B receptors occur as a glycoprotein containing Asn-linked oligosaccharide...

Importance Cancer is the main cause of death by disease in children. Children experience the highest incidence of cancer in the first year of life. However, there is no comprehensive registration system for children with tumors in China. Objective To summarize the diagnosis and treatment of infant cancer and analyze the status of standardized diagnosis and management among several treatment centers in Beijing, China, thereby providing evidence to guide further clinical research. Methods From January 1, 2010 to December 31, 2019, patients with newly diagnosed infantile malignant solid tumors were admitted to six large tertiary pediatric solid tumor diagnosis and treatment centers in Beijing. The epidemiology, clinical features, and therapeutic effects of tumors in these patients were analyzed retrospectively. All patients were followed up until March 31, 2020. Results In total, 938 patients were enrolled in this study. There were 530 boys (56.5%) and 408 girls (43.5%); the median age was 6.0 months (range, 0–12.0 months). The three most common tumors were retinoblastoma in 366 patients (39.0%), neuroblastoma in 266 patients (28.4%), hepatoblastoma in 133 patients (14.2%), and central nervous system tumors in 52 patients (5.5%). The estimated 5‐year overall survival rate was 81.3% ± 1.8%, and the 5‐year event‐free survival rate was 71.8% ± 2.9%. The 5‐year overall survival rates of non‐rhabdomyosarcoma soft tissue sarcoma, neuroblastoma, and retinoblastoma were 100%, 88% ± 2.2%, and 86.9% ±2.1%, respectively. The 5‐year event‐free survival rates were 81.1% ± 2.7% for neuroblastoma, 81.6% ± 9.8% for non‐rhabdomyosarcoma soft tissue sarcoma, and 72.7% ± 14.1% for extracranial malignant germ cell tumors. Interpretation The three most common infantile malignant solid tumors were retinoblastoma, neuroblastoma, and hepatoblastoma. Multidisciplinary combined diagnosis and treatment is needed for infantile tumors.