title: Full and partial agonists of thromboxane prostanoid receptor unveil fine tuning of receptor superactive conformation and G protein activation.
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.
authors.affiliation: Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy. Valerie.Capra@unimi.it
authors.forename: Valérie
authors.initials: V
authors.lastname: Capra
authors.forename: Marta
authors.initials: M
authors.lastname: Busnelli
authors.forename: Alessandro
authors.initials: A
authors.lastname: Perenna
authors.forename: Manuela
authors.initials: M
authors.lastname: Ambrosio
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Accomazzo
authors.forename: Celine
authors.initials: C
authors.lastname: Galés
authors.forename: Bice
authors.initials: B
authors.lastname: Chini
authors.forename: G Enrico
authors.initials: GE
authors.lastname: Rovati
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D019943:Amino Acid Substitution; D019204:GTP-Binding Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D017354:Point Mutation; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000819:agonists / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D044244:Receptors, Thromboxane A2, Prostaglandin H2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D019204:GTP-Binding Proteins
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