Expression and purification of the extracellular ligand-binding domain of the atrial natriuretic peptide (ANP) receptor: monovalent binding with ANP induces 2:2 complexes.
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.
Misono
K S
KS
Department of Molecular Cardiology, The Lerner Research Institute of The Cleveland Clinic Foundation, Ohio 44195, USA.misonok@cesmtp.ccf.org
Sivasubramanian
N
N
Berkner
K
K
Zhang
X
X
D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D019556:COS Cells; D005110:Extracellular Space / Q000378:metabolism*; D008024:Ligands; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014162:Transfection
D008024:Ligands; D046911:Macromolecular Substances; D010446:Peptide Fragments; D009320:Atrial Natriuretic Factor; D017461:Receptors, Atrial Natriuretic Factor