Adenine nucleotides decrease the apparentK_mof endogenous natriuretic peptide receptors for GTP

Abstract

Antos LK, Potter LR. Adenine nucleotides decrease the apparent K m of endogenous natriuretic peptide receptors for GTP. Am J Physiol Endocrinol Metab 293: E1756-E1763, 2007. First published September 11, 2007 doi:10.1152/ajpendo.00321.2007.-Natriuretic peptide receptors A (NPR-A) and B (NPR-B) mediate most effects of natriuretic peptides by synthesizing cGMP. ATP increases the activity of these receptors by an unknown mechanism. We recently reported that a nonhydrolyzable form of ATP, adenylyl imidodiphosphate (AMPPNP), stabilizes but is not required for the activation of NPR-A and NPR-B in membranes from highly overexpressing cells. Here, we repeated these studies on receptors expressed in endogenous settings. Kinetic analysis indicated that both AMPPNP and ATP dramatically decrease the apparent Km of both receptors for GTP but had little effect on the Vmax. The EC50 for AMPPNP decreased as substrate concentration increased whereas the magnitude of the effect was greater at lower GTP concentrations. ATP increased the activity of a mutant receptor containing glutamates substituted for all known phosphorylation sites similarly to the wildtype receptor, consistent with a phosphorylation independent mechanism. Finally, the putative ATP binding sites were investigated. Mutation of the ATP modulatory domain region had no effect, but mutation of K535A dramatically diminished ANP-dependent cyclase activity in a manner that was unresponsive to ATP. Mutation of the highly conserved 630-KSS to AAA (all alanines) resulted in an expressed receptor that had no detectable guanylyl cyclase activity. We conclude that ATP is not required for the initial activation of NPRs but does increase activity over time by reducing the apparent

ATP Potentiates Competitive Inhibition of Guanylyl Cyclase A and B by the Staurosporine Analog, Gö6976

“…The ability of ATP to increase the potency of Gö6976 is also consistent with reduced IC 50 values in whole cell experiments where intracellular ATP concentrations are higher and GTP concentrations are lower than those used in our assays (15). The reciprocal regulation between the ATP and GTP sites in GC-A and GC-B is consistent with previous data showing that ATP decreases the K m for GTP and that GTP decreases the EC 50 for ATP (14).…”

“…ATP is also an allosteric activator that reduces the Michaelis-Menten constant without affecting maximal velocities (14). In a reciprocal manner, GTP decreases the EC 50 for ATP.…”

Natriuretic Peptides: Their Structures, Receptors, Physiologic Functions and Therapeutic Applications

“…Phosphorylation is essential for activation of NPR-A and dephosphorylation is a mechanism of desensitization in response to prolonged ANP exposure or protein kinase C activation (Potter and Garbers 1992, 1994). Although ATP increases ANP-dependent guanylyl cyclase activity, the mechanism for this effect is debatable (Antos et al 2005; Antos and Potter 2007; Burczynska et al 2007; Joubert et al 2005). Our recent data indicate that ATP reduces the K m for NPR-A (Antos and Potter 2007).…”

A Functional Kinase Homology Domain Is Essential for the Activity of Photoreceptor Guanylate Cyclase 1

“…Surprisingly, ATP, besides being used for this phosphorylation, also directly binds to this domain, enhancing the peptide ligand-dependent cyclase activity (16,17). Whether ATP is absolutely required for such ligand-induced activation or just potentiates it is still controversial (17)(18)(19).…”