Mutational analysis of G-protein coupled receptor – FFA2 Gayathri Swaminath a,1 , Peter Jaeckel b,1 , Qi Guo a , Mario Cardozo a , Jennifer Weiszmann a , Richard Lindberg a , Yingcai Wang a , Ralf Schwandner b , Yang Li a,⇑ a Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA b Amgen Research-GMBH, Regensburg, Germany article info Article history: Received 30 December 2010 Available online 7 January 2011 Keywords: FFAR2 FFA2 GPR43 Allosteric agonist Short-chain fatty acids AMG7703 abstract FFA2 (GPR43) is a receptor for short-chain fatty acids (SCFAs), acetate, and propionate. FFA2 is predom- inantly expressed in islets, a subset of immune cells, adipocytes, and the gastrointestinal tract which sug- gest a possible role in inflammatory and metabolic conditions. We have previously described the identification and characterization of novel phenylacetamides as allosteric agonists of FFA2. In the cur- rent study, we have investigated the molecular determinants contributing to receptor activation with the endogenous and synthetic ligands as well as allosteric interactions between these two sites. The mutational analysis revealed previously unidentified sites that may allosterically regulate orthosteric ligand’s function as well as residues potentially important for the interactions between orthosteric and allosteric binding sites. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Free fatty acid receptor 2 (FFA2), also known as GPR43, is a member of the G-protein-coupled receptor (GPCR) superfamily [1]. GPCRs are known to bind to structurally diverse ligands and many are molecular targets for a wide range of therapeutic mole- cules [2,3]. FFA2 forms a subfamily with two other closely related receptors, FFA1 (GPR40) and FFA3 (GPR41), which are clustered on chromosome 19q13.1 in humans and share 30–40% sequence identity [1]. All three receptors can be activated by free fatty acids (FFA) but show distinct structure activity relationships for different chain lengths [4,5], with short-chain fatty acids (SCFAs, six or fewer carbon molecules) activating FFA2 and FFA3, and medium- and long-chain fatty acids activating FFA1 [6]. FFA2 can couple to both G ai and G aq [7,8] and is activated by C2 (acetate) and C3 (propio- nate) SCFAs [7,9]. Given the important physiological effects of FFAs, there is sub- stantial interest in exploring this subfamily of receptors as novel targets for various diseases [5]. FFA2 expression has been reported to be enriched in islets, a subset of immune cells, adipocytes, and the gastrointestinal tract, suggesting its potential role in various inflammation and metabolic conditions [7–10]. Clearly, the identi- fication of pharmacological tools and a better understanding of the receptor function will facilitate the development of potential ther- apeutic molecules targeting this receptor. FFA1 has attracted considerable attention given its well-estab- lished effects on pancreatic b-cell function and its potential utility in diabetes treatment [11,12]. Molecular modeling and mutagene- sis studies have identified several charged residues located in transmembrane domains (TM) 5, 6, and 7 that are important for anchoring carboxylate groups in long-chain fatty acids [13,14]. These polar residues are also conserved in FFA2 and FFA3, and sim- ilar to FFA1, they have been shown to be important for FFA2 and FFA3 endogenous ligand binding and function [15,16]. We have recently described the identification and characteriza- tion of a series of phenylacetamide derivatives as FFA2 allosteric agonists [17,18]. We herein report the results of mutagenesis stud- ies on the receptor, and the identification of previous unknown res- idues that may affect receptor activation as well as residues important for allosteric interactions on FFA2. 2. Material and methods 2.1. Materials Reagents were purchased from various sources as follows: so- dium acetate from Sigma (St. Louis, MO), propionate from Fluka Chemie (Taufkirchen, Germany), coelenterazine from P.J.K. GmbH (Kleinblittersdorf, Germany), polyethylenimine from Polysciences (Warrington, WI). Hank’s buffered salt solution (HBSS), Free-style medium and Pluronic F68 from Invitrogen (Carlsbad, CA), AMG7703 (previously known as Phenylacetamide 1) [17] was syn- thesized at Amgen, Inc. (South San Francisco, CA). The detailed syn- thetic routes, solubility properties of AMG7703, and SAR studies have been described elsewhere [18]. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.12.139 ⇑ Corresponding author. E-mail address: yangl@amgen.com (Y. Li). 1 Theses authors contributed equally to this work. Biochemical and Biophysical Research Communications 405 (2011) 122–127 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc