title: Investigation of the Binding Interaction of Fatty Acids with Human G Protein-Coupled Receptor 40 Using a Site-Specific Fluorescence Probe by Flow Cytometry.
abstract: Human G protein-coupled receptor 40 (hGPR40), with medium- and long-chain free fatty acids (FFAs) as its natural ligands, plays an important role in the enhancement of glucose-dependent insulin secretion. To date, information about the direct binding of FFAs to hGPR40 is very limited, and how carbon-chain length affects the activities of FFAs on hGPR40 is not yet understood. In this study, a fluorescein-fasiglifam analogue (F-TAK-875A) conjugate was designed and synthesized as a site-specific fluorescence probe to study the interaction of FFAs with hGPR40. hGPR40 was expressed in human embryonic kidney 293 cells and labeled with F-TAK-875A. By using flow cytometry, competitive binding of FFA and F-TAK-875A to hGPR40-expressed cells was measured. Binding affinities of 18 saturated FFAs, with carbon-chain lengths ranging from C6 to C23, were analyzed. The results showed that the binding potencies of FFAs to hGPR40 were dependent on carbon length. There was a positive correlation between length and binding potency for seven FFAs (C9-C15), with myristic acid (C15) showing the highest potency, 0.2% relative to TAK-875. For FFAs with a length of fewer than C9 or more than C15, they had very weak or no binding. Molecular docking results showed that the binding pocket of TAK-875 in hGPR40 could enclose FFAs with lengths of C15 or fewer. However, for FFAs with lengths longer than C15, part of the alkyl chain extended out of the binding pocket. This study provided insights into the structural dependence of FFAs binding to and activation of hGPR40.
authors.affiliation: State Key Laboratory of Environmental Chemistry and Eco-toxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences , 18 Shuangqing Road, P.O. Box 2871, Beijing 100085, China.
authors.forename: Xiao-Min
authors.initials: XM
authors.lastname: Ren
authors.affiliation: State Key Laboratory of Environmental Chemistry and Eco-toxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences , 18 Shuangqing Road, P.O. Box 2871, Beijing 100085, China.
authors.forename: Lin-Ying
authors.initials: LY
authors.lastname: Cao
authors.affiliation: State Key Laboratory of Environmental Chemistry and Eco-toxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences , 18 Shuangqing Road, P.O. Box 2871, Beijing 100085, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: College of Life and Environmental Science, Minzu University of China , Beijing 100081, China.
authors.forename: Wei-Ping
authors.initials: WP
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Environmental Chemistry and Eco-toxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences , 18 Shuangqing Road, P.O. Box 2871, Beijing 100085, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Environmental Chemistry and Eco-toxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences , 18 Shuangqing Road, P.O. Box 2871, Beijing 100085, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Wan
authors.affiliation: State Key Laboratory of Environmental Chemistry and Eco-toxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences , 18 Shuangqing Road, P.O. Box 2871, Beijing 100085, China.|Institute of Environment and Health, Jianghan University , Wuhan 430056, China.
authors.forename: Liang-Hong
authors.initials: LH
authors.lastname: Guo
mesh_terms: D001572:Benzofurans / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D015195:Drug Design; D005230:Fatty Acids, Nonesterified / Q000737:chemistry / Q000378:metabolism*; D005434:Flow Cytometry; D019793:Fluorescein / Q000737:chemistry / Q000378:metabolism; D005452:Fluoresceins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008744:Methylamines / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008970:Molecular Weight; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011422:Propionates / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013450:Sulfones / Q000737:chemistry / Q000378:metabolism
chemical_list: D001572:Benzofurans; C000609886:F-TAK-875A; C473034:FFAR1 protein, human; D005230:Fatty Acids, Nonesterified; D005452:Fluoresceins; D005456:Fluorescent Dyes; C515630:GW9508; D008024:Ligands; D008744:Methylamines; D009842:Oligopeptides; D011422:Propionates; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D013450:Sulfones; C557331:TAK-875; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C085706:FLAG peptide; D019793:Fluorescein