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The G 12 Family of G Proteins as a Reporter of Thromboxane A 2 Receptor Activity

2006; American Society for Pharmacology and Experimental Therapeutics; Volume: 69; Issue: 4 Linguagem: Inglês

10.1124/mol.105.019703

1521-0111

Li Zhang, Cherisse DiLizio, David Kim, Emer M. Smyth, David R. Manning,

Protein Kinase Regulation and GTPase Signaling

Despite advances in the understanding of pathways regulated by the G 12 family of heterotrimeric G proteins, much regarding the engagement of this family by receptors remains unclear. We explore here, using the thromboxane A 2 receptor TPα, the ability of G 12 and G 13 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-PGF 2α ), among other ligands examined, to activate G 12 and G 13 through TPα explicitly. We were also interested in the functionality of TPα-Gα fusion proteins germane to G 12 and G 13 . Using fusion proteins in Spodoptera frugiperda (Sf9) cells and independently expressed proteins in human embryonic kidney 293 cells, and using guanosine 5′- O -(3-[ 35 S]thio)triphosphate binding to evaluate Gα activation directly, we found for Gα that no ligand tested, including 8-iso-prostaglandin F (8-iso-PGF 2α and a purported antagonist (pinane thromboxane A 2 ), was silent. The activity of agonists was especially pronounced when evaluated for TPα-Gα 13 and in the context of receptor reserve. Agonist activity for 8-iso-PGF 2 was diminished and that for pinane thromboxane A nonexistent when Gα 12 was the reporter. These data establish that G 12 and G 13 can report differentially potency and efficacy and underscore the relevance of receptor and G protein context.