QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.108.052225v1 75/1/85    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hara, T. Articles by Tsujimoto, G. Search for Related Content PubMed PubMed Citation Articles by Hara, T. Articles by Tsujimoto, G.

Flow Cytometry-Based Binding Assay for GPR40 (FFAR1; Free Fatty Acid Receptor 1)

Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan (T.H., A.H., Q.S., T.K., C.I., K.S., G.T.); and Department of Pharmacology, Saitama Medical University, Saitama, Japan (T.A.)

GPR40 is a G protein-coupled receptor (GPCR) whose endogenous ligands have recently been identified as medium- and long-chain free fatty acids (FFAs), and it is thought to play an important role in insulin release. Despite recent research efforts, much still remains unclear in our understanding of its pharmacology, mainly because the receptor-ligand interaction has not been analyzed directly. To study the pharmacology of GPR40 in a more direct fashion, we developed a flow cytometry-based binding assay. FLAG-tagged GPR40 protein was expressed in Sf9 cells, solubilized, immobilized on immunomagnetic beads, and labeled with the fluorescent probe C1-BODIPY-C12. Flow cytometry analysis showed that C1-BODIPY-C12 specifically labels a single class of binding site in a saturable and reversible manner with an apparent dissociation constant of 3 µM. The FFAs that activate GPR40 competed with C1-BODIPY-C12 binding; thus, medium- to long-chain FFAs could compete, whereas short-chain FFAs and methyl linoleate had no inhibitory effect. Furthermore, ligands that are known to activate GPR40 competed for binding in a concentration-dependent manner. All the ligands that inhibited the binding promoted phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in human embryonic kidney (HEK) 293 cells that expressed GPR40 and [Ca²⁺]_i responses in mouse insulinoma (MIN6) cells that natively express GPR40; however, pioglitazone, a thiazolidinedione that failed to compete for the binding, did not activate ERK or [Ca²⁺]_i response. This study showed that a flow cytometry-based binding assay can successfully identify direct interactions between GPR40 and its ligands. This approach would be of value in studying the pharmacology of GPCRs.

Received for publication September 23, 2008.

Accepted for publication October 16, 2008.

Address correspondence to: Dr. Akira Hirasawa, Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan. E-mail: akira_h{at}pharm.kyoto-u.ac.jp

This article has been cited by other articles:

				K. Yanagida, K. Masago, H. Nakanishi, Y. Kihara, F. Hamano, Y. Tajima, R. Taguchi, T. Shimizu, and S. Ishii Identification and Characterization of a Novel Lysophosphatidic Acid Receptor, p2y5/LPA6 J. Biol. Chem., June 26, 2009; 284(26): 17731 - 17741. [Abstract] [Full Text] [PDF]