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Atrial natriuretic peptide‐dependent photolabeling of a regulatory ATP‐binding site on the natriuretic peptide receptor‐A
Author(s) -
Joubert Simon,
Jossart Christian,
McNicoll Normand,
De Léan André
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04952.x
Subject(s) - npr2 , npr1 , receptor , chemistry , gucy2d , atrial natriuretic peptide , biochemistry , photoaffinity labeling , binding site , transmembrane domain , cyclase , microbiology and biotechnology , biophysics , guanylate cyclase 2c , natriuretic peptide , biology , medicine , endocrinology , heart failure
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 (ΔGC). We demonstrate that this receptor (NPR‐A‐ΔGC) can be directly labeled by 8‐azido‐3′‐biotinyl‐ATP and that labeling is highly increased following ANP treatment. The mutant receptor ΔKC, which does not contain the KHD, is not labeled. Photoaffinity labeling of the NPR‐A‐ΔGC is reduced by 50% in the presence of 550 µ m 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.