Fig. 5.

CRTH2–POPC interaction in holo-1 simulation and mutagenesis data. (A) Interaction of a bilayer POPC molecule, with the residues lining the entry port in holo-1 simulation. The structures of the receptor at t = 0 ns (translucent gray) and t = 1 μs (green) are shown in ribbon representation. The entry port residues (pink), bound 15mPGD₂ (yellow), POPC (gray), and binding site residues R170^ECL2 and K210^5.42 (purple) are shown in sphere representation. (B) Close up of the CRTH2 entry port–POPC interaction, with the bound POPC (gray) and entry port residues (pink) shown in ball-and-stick and stick representation, respectively. The interactions between the POPC and the protein residues are shown as black dotted lines. (C) Evolution of CRTH2–POPC intermolecular salt bridges during the 1-µs holo-1 simulation, with the dotted black line indicating the cutoff distance of 4.0 Å. (D) Saturation-binding assays on different CRTH2 constructs using ³H-PGD₂. All CRTH2 constructs were transiently expressed in human embryonic kidney 293 cells, and cell membranes were prepared and used in the ligand-binding assays. Expression of each construct was confirmed by cell surface staining. Nonspecific binding measured in the presence of access amount of PGD₂ was subtracted. The dissociation equilibrium constants (K_ds) of PGD₂ for the wtCRTH2 and the R284A mutant are listed in the table shown on the right of panel D. For other CRTH2 mutants, no saturable ³H-PGD₂ binding was observed, indicating mostly nonspecific binding. Each data point in the left panel is shown as mean ± SEM and n = 3.