Utilization of the Tango TM β-Arrestin Recruitment Technology for Cell-Based EDG Receptor Assay Development and Interrogation

Abstract

Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. these LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. these receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. this complicates examination and comparison of these receptors across the entire family. the tango™ technology uses the conserved β-arrestin-dependent receptor deactivation process to allow interrogation of the EDG and related receptors with a single functional assay. this method also isolates the target receptor signal, allowing the use of tissue culture cell lines regardless of their endogenous receptor expression. the authors describe the use of this technique to build cell-based receptor-specific assays for all 8 members of the EDG receptor family as well as the related LPA receptors GPR23, GPR92, and GPR87. In addition, they demonstrate the value of this technology for identification and investigation of functionally selective receptor compounds as demonstrated by the immunosuppressive compound FtY720-P and its action at the EDG₁ and EDG₃ receptors. (Journal of Biomolecular Screening 2009:1134-1141)

Keywords

GPCR EDG LPA S1P tango™β-arrestin β-lactamase

References

Chun J. , Goetzl E. , Hla t , Igarashi Y. , Lynch K. , Moolenaar W. , et al: International union of pharmacology. XXXIV. Lysophospholipid receptor nomenclature. Pharmacol Rev 2002;54:265-269.

Lee C. , Rivera R. , Dubin A. , Chun J. : LPA4/GPR23 is a lysophosphatidic acid receptor utilizing Gs-, Gq/Gi-mediated calcium signaling and G12/13-mediated Rho activation. J Biol Chem 2007;282:4310-4317.

Lee C. , Rivera R. , Gardell S. , Dubin A. , Chun J. : GPR92 as a new G12/13-and Gq-coupled lysophosphatidic acid receptor that increases cAMP, LPA5. J Biol Chem 2006;281:23589-23597.

tabata K. , Baba K. , Shiraishi A. , Ito M. , Fujita N. : the orphan GPCR GPR87 was deorphanized and shown to be a lysophosphatidic acid receptor. Biochem Biophys Res Commun 2007;363:861-866.

Murakami M. , Shiraishi A. , tabata K. , Fujita N. : Identification of the orphan GPCR, P2Y10 receptor as the sphingosing 1-phosphate and lysophosphatidic acid receptor. Biochem Biophys Res Commun 2008;371:702-712.

Gardell S. , Dubin A. , Chun J. : Emerging roles for lysophospholipid signaling. Trends Mol Med 2006;12:65-75.

Holdsworth G. , Slocombe P. , Hutchinson G. , Milligan G. : Analysis of endogenous S1P and LPA receptor expression in CHO-K1 cells. Gene 2005;350:59-63.

Wang F. , Van Brocklyn J. , Hobson J. , Movafagh S. , Zukowska-Grojec Z. , Milstien S. , et al: Sphingosine 1-phosphate stimulates cell migration through a Gi-coupled cell surface receptor. J Biol Chem 1999;274:35343-35350.

Hanson G. , Hanson B. : Fluorescent probes for cellular assays. Comb Chem High Throughput Screen 2008;11:505-513.

10.

Barnea G. , Strapps W. , Herrada G. , Berman Y. , Ong J. , Kloss B. , et al: the genetic design of signaling cascades to record receptor activation. Proc Natl Acad Sci USA 2008;105:64-69.

11.

Hanson BJ , Wetter J. , Bercher MR , Kopp L. , Vedvik KL , Zielinski T , et al: A homogeneous fluorescent live-cell assay for measuring 7-transmembrane receptor activity and agonist functional selectivity through beta-arrestin recruitment. J Biomol Screen 2009 Jun 16. [Epub ahead of print]

12.

Doucette C. , Vedvik K. , Koepnick E. , Bergsma A. , thomson B. , Turek-Etienne TC : Kappa opioid receptor screen with the tango beta-arrestin recruitment technology and characterization of hits with second-messenger assays. J Biomol Screen 2009;14:381-394.

13.

Brinkmann V. : FtY720: mechanism of action and potential benefit in organ transplant. Yonsei Med J 2004;45:991-997.

14.

Germana Sanna M. , Liao J. , Jo E. , Alfonso C. , Ahn M. , Peterson MS , et al: Sphingosine 1-Phosphate receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate . J Biol Chem 2004;279: 13839-13848.

15.

Keller CD , Gil PR , tölle M. , van der Giet M. , Chun J. , Radeke HH , et al: Immunomodulator FtY720 induces myofibroblast differentiation via the lysophospholipid receptor S1P3 and Smad3 signaling. Am J Pathol 2007; 170:281-292.

16.

Sensken S. , Stäubert C. , Keul P. , Levkau B. , Schöneberg T , Gräler M. : Selective activation of G alpha i mediated signalling of S1P3 by FtY720phosphate . Cell Signal 2008;20:1125-1133.

17.

Im D. , Heise C. , Harding M. , George S. , O’Dowd B. , theodorescu D. , et al: Molecular cloning and characterization of a lysophosphatidic acid receptor, Edg-7, expressed in prostate. Mol Pharmacol 2000; 57:753-759.

18.

Heise C. , Santos W. , Schreihofer A. , Heasley B. , Mukhin Y. , Macdonald T. , et al: Activity of 2-substituted lysophosphatidic acid (LPA) analogs at LPA receptors: discovery of a LPA1/LPA3 receptor antagonist. Mol Pharmacol 2001;60:1173-1180.

19.

Ancellin N. , Hla T : Differential pharmacological properties and signal transduction of the sphingosine 1-phosphate receptors EDG-1, EDG-3, and EDG-5. J Biol Chem 1999;274:18997-19002.

20.

Van Brocklyn J. , Gräler M. , Bernhardt G. , Hobson J. , Lipp M. , Spiegel S. : Sphingosine-1-phosphate is a ligand for the G protein-coupled receptor EDG-6. Immunobiology 2000;95:2624-2629.

21.

Im D. , Heise C. , Ancellin N. , O’Dowd B. , Shei G. , Heavens R. , et al: Characterization of a novel sphingosine 1-phosphat receptor, EDG-8. J Biol Chem 2000;275:14281-14286.

22.

Heasley B. , Jarosz R. , Lynch K. , Macdonald T : Initial structure-activity relationships of lysophosphatidic acid receptor antagonists: discovery of a high-affinity LPA1/LPA3 receptor antagonist. Bioorg Med Chem 2004; 14:2735-2740.

23.

Mandala S. , Hajdu R. , Bergstrom J. , Quackenbush E. , Xie J. , Milligan J. , et al: Alteration of lymphocyte trafficking by sphingosine 1-phosphate receptor agonists. Science 2008;296:346-349.

24.

Gräler MH , Goetzl EJ : the immunosuppressant FtY720 down-regulates sphingosine 1-phosphate G protein-coupled receptors. Retrieved from http://www.fasebj.org/cgi/reprint/03-0910fjev1