title: The G12 family of G proteins as a reporter of thromboxane A2 receptor activity.
abstract: Despite advances in the understanding of pathways regulated by the G12 family of heterotrimeric G proteins, much regarding the engagement of this family by receptors remains unclear. We explore here, using the thromboxane A2 receptor TPalpha, the ability of G12 and G13 to report differences in the potency and efficacy of receptor ligands. We were interested especially in the potential of the isoprostane 8-iso-prostaglandin F (8-iso-PGF2alpha), among other ligands examined, to activate G12 and G13 through TPalpha explicitly. We were also interested in the functionality of TPalpha-Galpha fusion proteins germane to G12 and G13. Using fusion proteins in Spodoptera frugiperda (Sf9) cells and independently expressed proteins in human embryonic kidney 293 cells, and using guanosine 5'-O-(3-[35S]thio)triphosphate binding to evaluate Galpha activation directly, we found for Galpha that no ligand tested, including 8-iso-prostaglandin F (8-iso-PGF2alpha and a purported antagonist (pinane thromboxane A2), was silent. The activity of agonists was especially pronounced when evaluated for TPalpha-Galpha13 and in the context of receptor reserve. Agonist activity for 8-iso-PGF2 was diminished and that for pinane thromboxane A nonexistent when Galpha12 was the reporter. These data establish that G12 and G13 can report differentially potency and efficacy and underscore the relevance of receptor and G protein context.
authors.affiliation: Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Cherisse
authors.initials: C
authors.lastname: DiLizio
authors.forename: David
authors.initials: D
authors.lastname: Kim
authors.forename: Emer M
authors.initials: EM
authors.lastname: Smyth
authors.forename: David R
authors.initials: DR
authors.lastname: Manning
mesh_terms: D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002460:Cell Line; D015237:Dinoprost / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005231:Fatty Acids, Unsaturated; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism*; D006801:Humans; D006834:Hydrazines / Q000378:metabolism; D011869:Radioligand Assay; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000502:physiology*; D018411:Spodoptera
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; D006834:Hydrazines; D044244:Receptors, Thromboxane A2, Prostaglandin H2; C075750:8-epi-prostaglandin F2alpha; C045749:SQ 29548; D015237:Dinoprost; D044365:GTP-Binding Protein alpha Subunits, G12-G13