Structural and functional characterization of the first intracellular loop of human thromboxane A2 receptor.

The conformation of a constrained peptide mimicking the putative first intracellular domain (iLP1) of thromboxane A(2) receptor (TP) was determined by (1)H 2D NMR spectroscopy. Through completed assignments of TOCSY, DQF-COSY, and NOESY spectra, a NMR structure of the peptide showed a beta-turn in residues 56-59 and a short helical structure in the residues 63-66. It suggests that residues 63-66 may be part of the second transmembrane domain (TM), and that Arg60, in an exposed position on the outer surface of the loop, may be involved in signaling through charge contact with Gq protein. The sequence alignment of Lys residue in the same position of other prostanoid receptors mediates different G protein couplings, suggesting that the chemical properties of Arg and Lys may also affect the receptor signaling activity. These hypotheses were supported by mutagenesis studies, in which the mutant of Arg60Leu completely lost activity in increasing intracellular calcium level through Gq coupling, and the mutant of Arg60Lys retained only about 35% signaling activity. The difference between the side chain functions of Lys and Arg in effecting the signaling was discussed.