The G12 family of G proteins as a reporter of thromboxane A2 receptor activity.

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 TP alpha, 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-2 alpha (8-iso-PGF(2 alpha)), among other ligands examined, to activate G(12) and G(13) through TP alpha explicitly. We were also interested in the functionality of TP alpha-G alpha 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-[S-35] thio) triphosphate binding to evaluate G alpha activation directly, we found for G alpha(13) that no ligand tested, including 8-iso-prostaglandin F-2 alpha (8-iso-PGF2 alpha) and a purported antagonist (pinane thromboxane A(2)), was silent. The activity of agonists was especially pronounced when evaluated for TP alpha-G alpha(13) and in the context of receptor reserve. Agonist activity for 8-iso-PGF(2 alpha) was diminished and that for pinane thromboxane A(2) nonexistent when G alpha(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.