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

Laura K. Antos, Lincoln R Potter

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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 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 Km for GTP.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Endocrinology and Metabolism
Volume293
Issue number6
DOIs
StatePublished - Dec 1 2007

Fingerprint

Natriuretic Peptides
Peptide Receptors
Adenine Nucleotides
Guanosine Triphosphate
Adenosine Triphosphate
Adenylyl Imidodiphosphate
Phosphorylation
Mutation
Chemical activation
Glutamates
Guanylate Cyclase
Glutamate Receptors
Atrial Natriuretic Factor
Alanine
Binding Sites
Membranes
Kinetics
Substrates

Keywords

  • Adenosine triphosphate
  • Bone growth
  • Cyclic guanosine monophosphate
  • Guanosine triphosphate
  • Guanylyl cyclase
  • Heart failure
  • Hypertension
  • Michaelis-Menten constant

Cite this

@article{056af2c1d59745848ef85d9c58926f62,
title = "Adenine nucleotides decrease the apparent Km of endogenous natriuretic peptide receptors for GTP",
abstract = "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 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 Km for GTP.",
keywords = "Adenosine triphosphate, Bone growth, Cyclic guanosine monophosphate, Guanosine triphosphate, Guanylyl cyclase, Heart failure, Hypertension, Michaelis-Menten constant",
author = "Antos, {Laura K.} and Potter, {Lincoln R}",
year = "2007",
month = "12",
day = "1",
doi = "10.1152/ajpendo.00321.2007",
language = "English (US)",
volume = "293",
journal = "American Journal of Physiology - Cell Physiology",
issn = "0363-6143",
publisher = "American Physiological Society",
number = "6",

}

TY - JOUR

T1 - Adenine nucleotides decrease the apparent Km of endogenous natriuretic peptide receptors for GTP

AU - Antos, Laura K.

AU - Potter, Lincoln R

PY - 2007/12/1

Y1 - 2007/12/1

N2 - 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 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 Km for GTP.

AB - 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 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 Km for GTP.

KW - Adenosine triphosphate

KW - Bone growth

KW - Cyclic guanosine monophosphate

KW - Guanosine triphosphate

KW - Guanylyl cyclase

KW - Heart failure

KW - Hypertension

KW - Michaelis-Menten constant

UR - http://www.scopus.com/inward/record.url?scp=37149013706&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=37149013706&partnerID=8YFLogxK

U2 - 10.1152/ajpendo.00321.2007

DO - 10.1152/ajpendo.00321.2007

M3 - Article

C2 - 17848634

AN - SCOPUS:37149013706

VL - 293

JO - American Journal of Physiology - Cell Physiology

JF - American Journal of Physiology - Cell Physiology

SN - 0363-6143

IS - 6

ER -