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Full and Partial Agonists of Thromboxane Prostanoid Receptor Unveil Fine Tuning of Receptor Superactive Conformation and G Protein Activation
- Source :
- PLoS ONE, Vol 8, Iss 3, p e60475 (2013), PLoS ONE
- Publication Year :
- 2013
-
Abstract
- The intrahelical salt bridge between \(E/D^{3.49}\) and \(R^{3.50}\) within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of \(E^{3.49/6.30}\) in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.
- Subjects :
- Drugs and Devices
Protein Conformation
G protein
Biophysics
lcsh:Medicine
Biology
Cardiovascular
Biochemistry
Cardiovascular Pharmacology
Receptors, Thromboxane A2, Prostaglandin H2
Lipid Mediators
Protein structure
GTP-binding protein regulators
GTP-Binding Proteins
Molecular Cell Biology
Signaling in Cellular Processes
Humans
Point Mutation
Membrane Receptor Signaling
5-HT5A receptor
ddc:610
lcsh:Science
Receptor
G protein-coupled receptor
G protein-coupled receptor kinase
Multidisciplinary
Physics
lcsh:R
Neurochemistry
Lipids
Cell biology
G-Protein Signaling
HEK293 Cells
Amino Acid Substitution
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid
Eicosanoids
Medicine
lcsh:Q
Neurochemicals
Signal transduction
Research Article
Signal Transduction
Protein Binding
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- PLoS ONE, Vol 8, Iss 3, p e60475 (2013), PLoS ONE
- Accession number :
- edsair.doi.dedup.....ce7564f05d96c569e87731b1fa6ea531