Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.2 (guanylate cyclase)
 8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Expression and purification of the extracellular ligand-binding domain of the atrial natriuretic peptide (ANP) receptor: monovalent binding with ANP induces 2:2 complexes. 988 90

The disulfide bond structure of the extracellular domain of rat atrial natriuretic peptide (ANP) receptor (NPR-ECD) has been determined by mass spectrometry (MS) and Edman sequencing. Recombinant NPR-ECD expressed in COS-1 cells and purified from the culture medium binds ANP with as high affinity as the natural ANP receptor. Reaction with iodoacetic acid yielded no S-carboxymethylcysteine, indicating that all six Cys residues in NPR-ECD are involved in disulfide bonds. Electrospray ionization MS of NPR-ECD deglycosylated by peptide-N-glycosidase F gave a molecular mass of 48377.5+/-1.6 Da, which was consistent with the presence of three disulfide bonds. Liquid chromatography MS analysis of a lysylendopeptidase digest yielded three cystine-containing fragments with disulfide bonds Cys(60)-Cys(86), Cys(164)-Cys(213) and Cys(423)-Cys(432) based on their observed masses. These bonds were confirmed by Edman sequencing of each of the three fragments. No evidence for an inter-molecular disulfide bond was found. The six Cys residues in NPR-ECD, forming a 1-2, 3-4, 5-6 disulfide pairing pattern, are strictly conserved among A-type natriuretic peptide receptors and are similar in B-type receptors. We found that in other families of guanylate cyclase-coupled receptors, the Cys residues involved in 1-2 and 5-6 disulfide pairs are conserved in nearly all, suggesting an important contribution of these disulfide bonds to the receptor's structure and function.
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PMID:Disulfide bond structure of the atrial natriuretic peptide receptor extracellular domain: conserved disulfide bonds among guanylate cyclase-coupled receptors. 1071 72

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 F2 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.
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PMID:Glycosylation sites in the atrial natriuretic peptide receptor: oligosaccharide structures are not required for hormone binding. 2698 Jul 29