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 (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 µm 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.

Adenine nucleotides decrease the apparentKmof endogenous natriuretic peptide receptors for GTP

“…The ATP-independent activation of these receptors is in agreement with most reports. Please note, however, that we are neither currently suggesting nor have we previously suggested that ATP does not increase the guanylyl cyclase activities of these receptors, as has been implied (7,20). With the exception of the very early time points, we, as all other investigators, observe more activity in the presence of ATP (3).…”

“…A recent paper demonstrated that the cross-linking of 8-azido-3Ј-biotinyl-ATP to a cyclase deleted NPR-A construct (20). This labeling was significantly increased following treatment with ANP.…”

“…Two separate groups have recently challenged our findings because they were carried out in highly overexpressing synthetic cells (7,20). Therefore, we investigated whether the same results would be obtained in membranes from cells that express these receptors endogenously.…”

“…However, whether or not these results reflect physiological activation has been called into question (7,20). To address this issue, we tested guanylyl cyclase activity in membranes from BAEC or NIH3T3 fibroblast membranes that endogenously express NPR-A or NPR-B, respectively.…”

Role of Extracellular Domain Dimerization in Agonist-Induced Activation of Natriuretic Peptide Receptor A

“…The cytoplasmic domain includes a kinase homology domain (KHD), which allosterically regulates both peptide binding to the ECD and activation of the effector guanylyl cyclase (GC) (Larose et al, 1991;Duda et al, 2005). The KHD directly binds ATP after activation of the ECD by ANP (Joubert et al, 2005). It is also normally phosphorylated, and its dephosphorylation coincides with desensitization of NPRA to ANP activation (Joubert et al, 2001;Potter et al, 2006).…”