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This Article Full Text Full Text (PDF) All Versions of this Article: 293/6/E1756    most recent 00321.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Antos, L. K. Articles by Potter, L. R. Search for Related Content PubMed PubMed Citation Articles by Antos, L. K. Articles by Potter, L. R.

Adenine nucleotides decrease the apparent K_m of endogenous natriuretic peptide receptors for GTP

¹Department of Biochemistry, Molecular Biology and Biophysics, and ²Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota

Submitted 23 May 2007 ; accepted in final form 5 September 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 K_m of both receptors for GTP but had little effect on the V_max. 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 wild-type 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 K_m for GTP.

guanylyl cyclase; cyclic guanosine monophosphate; guanosine triphosphate; adenosine triphosphate; Michaelis-Menten constant; heart failure; hypertension; bone growth