Structural and functional analysis of the C-terminus of Galphaq in complex with the human thromboxane A2 receptor provides evidence of constitutive activity.
The human thromboxane A(2) (TXA(2)) receptor (TP) is known to mediate platelet aggregation and vasoconstriction. The receptor predominantly interacts with the Gq protein, thereby activating phospholipase C and increasing the intracellular calcium level. In this study, we synthesized a 15-residue peptide corresponding to the C-terminal domain of the Gq protein alpha subunit (Galphaq-Ct peptide) and characterized its interaction with recombinant TP purified from a baculovirus expression system in the presence and absence of an agonist using fluorescence and NMR spectroscopic studies. With fluorescence binding assays, we demonstrated that the Galphaq-Ct peptide was bound to TP, in the absence of the agonist, with a K(d) value of approximately 17 muM. Interestingly, upon addition of the agonist, U46619, the Galphaq-Ct peptide's binding affinity for this activated TP was reduced, thereby increasing the K(d) value to approximately 240 muM. NMR experiments demonstrated that the TP-bound Galphaq-Ct peptide shows a different affinity and conformation, in the absence and presence of the agonist, U46619. This suggested there is the possibility of ligand-free constitutive TP signaling through Galpha binding. Thus, an HEK293 cell line that stably expresses human TP and lacks the ability to produce TXA(2) was created by gene transfer and G418 selection. In comparison with the control cells, the stable cell line showed significant Galpha-mediated ligand-free calcium signaling. The study indicates a promising new outlook for the examination of prostanoid receptor-G-protein interactions in greater detail using integrated NMR spectroscopy, the purified receptor, and the stable cell line.
Chillar
Annirudha
A
Center for Experimental Therapeutics and Pharmacoinformatics, Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX 77204, USA.
Wu
Jiaxin
J
Cervantes
Vanessa
V
Ruan
Ke-He
KH
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D020013:Calcium Signaling; D002460:Cell Line; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011982:Receptors, Prostaglandin; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000819:agonists / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D010446:Peptide Fragments; D011982:Receptors, Prostaglandin; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D043802:GTP-Binding Protein alpha Subunits, Gq-G11