Unlabeled lysophosphatidic acid receptor binding in free solution as determined by a compensated interferometric...

Native interactions between lysophospholipids (LPs) and their cognate LP receptors are difficult to measure because of lipophilicity and/or the adhesive properties of lipids, which contribute to high levels of nonspecific binding in cell membrane preparations. Here, we report development of a free-solution assay (FSA) where label-free LPs bind to their cognate G protein-coupled receptors (GPCRs), combined with a recently reported compensated interferometric reader (CIR) to quantify native binding interactions between receptors and ligands. As a test case, the binding parameters between lysophosphatidic acid (LPA) receptor 1 (LPA₁; one of six cognate LPA GPCRs) and LPA were determined. FSA-CIR detected specific binding through the simultaneous real-time comparison of bound versus unbound species by measuring the change in the solution dipole moment produced by binding-induced conformational and/or hydration changes. FSA-CIR identified K values for chemically distinct LPA species binding to human LPA₁ and required only a few nanograms of protein: 1-oleoyl (18:1; K = 2.08 ± 1.32 nM), 1-linoleoyl (18:2; K = 2.83 ± 1.64 nM), 1-arachidonoyl (20:4; K = 2.59 ± 0.481 nM), and 1-palmitoyl (16:0; K = 1.69 ± 0.1 nM) LPA. These K values compared favorably to those obtained using the previous generation back-scattering interferometry system, a chip-based technique with low-throughput and temperature sensitivity. In conclusion, FSA-CIR offers a new increased-throughput approach to assess quantitatively label-free lipid ligand-receptor binding, including nonactivating antagonist binding, under near-native conditions.