Ligand binding-dependent limited proteolysis of the atrial natriuretic peptide receptor: juxtamembrane hinge structure essential for transmembrane signal transduction.
The atrial natriuretic peptide (ANP) receptor is a 130-kDa transmembrane protein containing an extracellular ANP-binding domain, a single transmembrane sequence, an intracellular kinase-homologous domain, and a guanylate cyclase (GCase) domain. We observed that the receptor, when bound with ANP, was rapidly cleaved by endogenous or exogenously added protease to yield a 65-kDa ANP-binding fragment. No cleavage occurred without bound ANP. This ligand-induced cleavage abolished GCase activation by ANP. Cleavage occurred in an extracellular, juxtamembrane region containing six closely spaced Pro residues and a disulfide bond. Such structural features are shared among the A-type and B-type ANP receptors but not by ANP clearance receptors. The potential role of the hinge structure was examined by mutagenesis experiments. Mutation of Pro(417), but not other Pro residues, to Ala abolished GCase activation by ANP. Elimination of the disulfide bond by Cys to Ser mutations yielded a constitutively active receptor. Pro(417), and Cys(423) and Cys(432) forming the disulfide bond are strictly conserved among GCase-coupled receptors, while other residues are largely variable. The conserved Pro(417) and the disulfide bond may represent a consensus signaling motif in the juxtamembrane hinge structure that undergoes a marked conformational change upon ligand binding and apparently mediates transmembrane signal transduction.
Huo
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Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.
Abe
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Misono
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D000302:Adrenal Cortex / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection
D008024:Ligands; D011392:Proline; D010450:Endopeptidases; D017461:Receptors, Atrial Natriuretic Factor; D003545:Cysteine