Structural and Functional Analysis of the C-Terminus of Gαq in Complex with the Human Thromboxane A2 Receptor Provides Evidence of Constitutive Activity

摘要

The human thromboxane A2 (TXA2) receptor (TP) is known to mediate platelet aggregation andnvasoconstriction. The receptor predominantly interacts with the Gq protein, thereby activating phospholi-npase C and increasing the intracellular calcium level. In this study, we synthesized a 15-residue peptidencorresponding to the C-terminal domain of the Gq protein R subunit (GRq-Ct peptide) and characterized itsninteraction with recombinant TP purified from a baculovirus expression system in the presence and absencenof an agonist using fluorescence and NMR spectroscopic studies. With fluorescence binding assays, wendemonstrated that the GRq-Ct peptide was bound to TP, in the absence of the agonist, with a Kd value ofnapproximately 17 μM. Interestingly, upon addition of the agonist, U46619, the GRq-Ct peptide’s bindingnaffinity for this activated TP was reduced, thereby increasing the Kd value to approximately 240 μM. NMRnexperiments demonstrated that the TP-boundGRq-Ct peptide shows a different affinity and conformation, innthe absence and presence of the agonist, U46619. This suggested there is the possibility of ligand-freenconstitutive TP signaling throughGR binding. Thus, anHEK293 cell line that stably expresses human TP andnlacks the ability to produce TXA2 was created by gene transfer and G418 selection. In comparison with thencontrol cells, the stable cell line showed significant GR-mediated ligand-free calcium signaling. The studynindicates a promising new outlook for the examination of prostanoid receptor-G-protein interactions inngreater detail using integrated NMR spectroscopy, the purified receptor, and the stable cell line.