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Unlabeled lysophosphatidic acid receptor binding in free solution as determined by a compensated interferometric reader
- Source :
- Journal of Lipid Research, Vol 61, Iss 8, Pp 1244-1251 (2020), Journal of Lipid Research
- Publication Year :
- 2020
- Publisher :
- Elsevier BV, 2020.
-
Abstract
- 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 KD values for chemically distinct LPA species binding to human LPA1 and required only a few nanograms of protein: 1-oleoyl (18:1; KD = 2.08 ± 1.32 nM), 1-linoleoyl (18:2; KD = 2.83 ± 1.64 nM), 1-arachidonoyl (20:4; KD = 2.59 ± 0.481 nM), and 1-palmitoyl (16:0; KD = 1.69 ± 0.1 nM) LPA. These KD 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.
- Subjects :
- 0301 basic medicine
Light
Lysophospholipids
QD415-436
030204 cardiovascular system & hematology
Ligands
Biochemistry
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
Endocrinology
Lysophosphatidic acid
Methods
receptor binding assay
G protein-coupled receptor
Receptors, Lysophosphatidic Acid
Receptor
lysophospholipids
Antagonist
free-solution assay-compensated interferometric reader
Cell Biology
Lipid signaling
interferometry
030104 developmental biology
Membrane
chemistry
molecular interaction
Lipophilicity
Biophysics
lipid signaling
Biological Assay
lipids (amino acids, peptides, and proteins)
Protein Binding
Subjects
Details
- ISSN :
- 00222275
- Volume :
- 61
- Database :
- OpenAIRE
- Journal :
- Journal of Lipid Research
- Accession number :
- edsair.doi.dedup.....f19c521184c5e01d54c42c8468bc5dc1