Ligand-specific conformation determines agonist activation and antagonist blockade in purified human thromboxane A2 receptor.

ABSTRACT

The binding of an agonist to a G protein-coupled receptor (GPCR) causes its coupling to different G proteins, which mediate signaling. However, the binding of an antagonist to the same site of the GPCR could not induce coupling. To understand the molecular mechanism involved, the structural flexibility of the purified human thromboxane A2 receptor (TP) was characterized by spectroscopic approaches, while bound to an agonist or antagonist. Circular dichroism not only revealed that the purified TP adopted more than 50% helical conformation in solution but also showed that the antagonist, SQ29,548, could induce more of a beta-sheet structure in the TP than that of the agonist, U46619. Also, fluorescence studies showed that the antagonist induced the intrinsic Trp fluorescence signal change more than the agonist. Furthermore, three of the nine tryptophan residues involved in the different ligand-based structural changes were demonstrated by NMR spectroscopy. Low pH-induced changes in the receptor conformation and molecular interaction field dramatically increased the agonist binding but did not significantly affect the antagonist binding. Different conformational changes were also observed in the TP reconstituted into phosphatidylcholine/phosphatidylserine/phosphatydylethanolamine-formed liposomes. These studies are the first to show a possible mechanism of the ligand-specific conformation-dependent agonist activation and antagonist blockage in the GPCR.