title: Atrial natriuretic peptide-dependent photolabeling of a regulatory ATP-binding site on the natriuretic peptide receptor-A.
abstract: The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular ligand-binding domain, a transmembrane-spanning domain, a kinase homology domain (KHD) and a guanylyl cyclase domain. Because the presence of ATP or adenylylimidodiphosphate reduces atrial natriuretic peptide (ANP) binding and is required for maximal guanylyl cyclase activity, a direct interaction of ATP with the receptor KHD domain is plausible. Therefore, we investigated whether ATP interacts directly with a binding site on the receptor by analyzing the binding of a photoaffinity analog of ATP to membranes from human embryonic kidney 293 cells expressing the NPR-A receptor lacking the guanylyl cyclase moiety (DeltaGC). We demonstrate that this receptor (NPR-A-DeltaGC) can be directly labeled by 8-azido-3'-biotinyl-ATP and that labeling is highly increased following ANP treatment. The mutant receptor DeltaKC, which does not contain the KHD, is not labeled. Photoaffinity labeling of the NPR-A-DeltaGC is reduced by 50% in the presence of 550 microm ATP, and competition curve fitting studies indicate a Hill slope of 2.2, suggestive of cooperative binding. This approach demonstrates directly that the interaction of ANP with its receptor modulates the binding of ATP to the KHD, probably through a conformational change in the KHD. In turn, this conformational change is essential for maximal activity. In addition, the ATP analog, 8-azido-adenylylimidodiphosphate, inhibits guanylyl cyclase activity but increases ANP binding to the extracellular domain. These results suggest that the KHD regulates ANP binding and guanylyl cyclase activity independently.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Simon
authors.initials: S
authors.lastname: Joubert
authors.forename: Christian
authors.initials: C
authors.lastname: Jossart
authors.forename: Normand
authors.initials: N
authors.lastname: McNicoll
authors.forename: André
authors.initials: A
authors.lastname: De Léan
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D009320:Atrial Natriuretic Factor / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D017461:Receptors, Atrial Natriuretic Factor / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C436521:8-N(3)-3'-biotinyl-ATP; D004791:Enzyme Inhibitors; D001710:Biotin; D009320:Atrial Natriuretic Factor; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A