G protein-coupled receptor (GPCR); G protein, high-throughput screening (HTS); drug screening; pharmacology; functional selectivity; biased signaling; complementation assay; dopamine; Nanoluciferase
Abstract :
[en] G protein-coupled receptors (GPCRs) are
currently the target of more than 30% of the
marketed medicines. However, there is an important
medical need for ligands with improved
pharmacological activities on validated drug targets.
Moreover, most of these ligands remain poorly
characterized, notably because of a lack of
pharmacological tools. Thus, there is an important
demand for innovative assays that can detect and
drive the design of compounds with novel or
improved pharmacological properties. In particular,
a functional and screening-compatible GPCR-G
protein interaction assay is still unavailable. Here,
we report on a nanoluciferase-based
complementation technique to detect ligands that
promote a GPCR-G protein interaction. We
demonstrate that our system can be used to profile
compounds with regard to the G proteins they
activate through a given GPCR. Furthermore, we
established a proof of applicability of screening for
distinct G proteins on dopamine receptor D2 whose
differential coupling to Gαi/o family members has
been extensively studied. In a D2-Gαi1 versus D2-
Gαo screening, we retrieved five agonists that are
currently being used in antiparkinsonian
medications. We determined that in this assay,
piribedil and pergolide are full agonists for the
recruitment of Gαi1 but are partial agonists for Gαo,
that the agonist activity of ropinirole is biased in
favor of Gαi1 recruitment, and that the agonist
activity of apomorphine is biased for Gαo. We
proposed that this newly developed assay could be
used to develop molecules that selectively modulate
a particular G protein pathway.
Research Center/Unit :
Giga-Signal Transduction - ULiège Centre Interfacultaire de Recherche du Médicament - CIRM
Disciplines :
Life sciences: Multidisciplinary, general & others Biotechnology Pharmacy, pharmacology & toxicology Biochemistry, biophysics & molecular biology
Author, co-author :
Laschet, Céline ; Université de Liège - ULiège > Département de pharmacie > Chimie pharmaceutique
Dupuis, Nadine
Hanson, Julien ; Université de Liège - ULiège > Département de pharmacie > Chimie pharmaceutique
Language :
English
Title :
A dynamic and screening-compatible nanoluciferase-based complementation assay enables profiling of individual GPCR-G protein interactions
Publication date :
15 March 2019
Journal title :
Journal of Biological Chemistry
ISSN :
0021-9258
eISSN :
1083-351X
Publisher :
American Society for Biochemistry and Molecular Biology, United States - Maryland
F.R.S.-FNRS - Fonds de la Recherche Scientifique [BE] FWB - Fédération Wallonie-Bruxelles [BE] Fonds Léon Fredericq [BE] FRIA - Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture [BE]
Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schiöth, H. B., and Gloriam, D. E. (2017) Trends in GPCR drug discovery: new agents, targets and indications. Nat. Rev. Drug Discov. 16, 829 – 842 CrossRef Medline
Sriram, K., and Insel, P. A. (2018) G protein– coupled receptors as targets for approved drugs: how many targets and how many drugs? Mol. Pharmacol. 93, 251–258 CrossRef Medline
Davenport, A. P., Alexander, S. P., Sharman, J. L., Pawson, A. J., Benson, H. E., Monaghan, A. E., Liew, W. C., Mpamhanga, C. P., Bonner, T. I., Neubig, R. R., Pin, J.-P., Spedding, M., and Harmar, A. J. (2013) International union of basic and clinical pharmacology. LXXXVIII. G protein– coupled receptor list: recommendations for new pairings with cognate ligands. Pharmacol. Rev. 65, 967–986 CrossRef Medline
Ngo, T., Kufareva, I., Coleman, J. L. J., Graham, R. M., Abagyan, R., and Smith, N. J. (2016) Identifying ligands at orphan GPCRs: current status using structure-based approaches. Br. J. Pharmacol. 173, 2934 –2951 CrossRef Medline
Laschet, C., Dupuis, N., and Hanson, J. (2018) The G protein– coupled receptors deorphanization landscape. Biochem. Pharmacol. 153, 62–74 CrossRef Medline
Roth, B. L., and Kroeze, W. K. (2015) Integrated approaches for genome-wide interrogation of the druggable non-olfactory G protein– coupled receptor superfamily. J. Biol. Chem. 290, 19471–19477 CrossRef Medline
Kenakin. (2012) Biased signalling and allosteric machines: new vistas and challenges for drug discovery. Br. J. Pharmacol. 165, 1659 –1669 CrossRef Medline
Oldham, W. M., and Hamm, H. E. (2008) Heterotrimeric G protein activation by G-protein-coupled receptors. Nat. Rev. Mol. Cell Biol. 9, 60 –71 CrossRef Medline
Wettschureck, N., and Offermanns, S. (2005) Mammalian G proteins and their cell type specific functions. Physiol. Rev. 85, 1159 –1204 CrossRef Medline
Rajagopal, S., and Shenoy, S. K. (2018) GPCR desensitization: acute and prolonged phases. Cell. Signal. 41, 9 –16 CrossRef Medline
Milligan, G., and Kostenis, E. (2006) Heterotrimeric G-proteins: a short history. Br. J. Pharmacol. 147, S46 –S55 Medline
Williams, C. (2004) cAMP detection methods in HTS: selecting the best from the rest. Nat. Rev. Drug Discov. 3, 125–135 CrossRef Medline
Gilissen, J., Geubelle, P., Dupuis, N., Laschet, C., Pirotte, B., and Hanson, J. (2015) Forskolin-free cAMP assay for Gi-coupled receptors. Biochem. Pharmacol. 98, 381–391 CrossRef Medline
Ma, Q., Ye, L., Liu, H., Shi, Y., and Zhou, N. (2017) An overview of Ca2 mobilization assays in GPCR drug discovery. Expert Opin. Drug Discov. 12, 511–523 CrossRef Medline
Inoue, A., Ishiguro, J., Kitamura, H., Arima, N., Okutani, M., Shuto, A., Higashiyama, S., Ohwada, T., Arai, H., Makide, K., and Aoki, J. (2012) TGF shedding assay: an accurate and versatile method for detecting GPCR activation. Nat Methods 9, 1021–1029 CrossRef Medline
Milligan, G. (2003) Principles: extending the utility of [35S]GTPS binding assays. Trends Pharmacol. Sci. 24, 87–90 CrossRef Medline
Schröder, R., Janssen, N., Schmidt, J., Kebig, A., Merten, N., Hennen, S., Müller, A., Blättermann, S., Mohr-Andrä, M., Zahn, S., Wenzel, J., Smith, N. J., Gomeza, J., Drewke, C., Milligan, G., et al. (2010) Deconvolution of complex G protein– coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat. Biotechnol. 28, 943–949 CrossRef Medline
Kostenis, E., Waelbroeck, M., and Milligan, G. (2005) Techniques: promiscuous G proteins in basic research and drug discovery. Trends Pharmacol. Sci. 26, 595– 602 CrossRef Medline
Lohse, M. J., Nuber, S., and Hoffmann, C. (2012) Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling. Pharmacol. Rev. 64, 299 –336 CrossRef Medline
Boute, N., Jockers, R., and Issad, T. (2002) The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol. Sci. 23, 351–354 CrossRef Medline
Machleidt, T., Woodroofe, C. C., Schwinn, M. K., Méndez, J., Robers, M. B., Zimmerman, K., Otto, P., Daniels, D. L., Kirkland, T. A., and Wood, K. V. (2015) NanoBRET—a novel BRET platform for the analysis of protein–protein interactions. ACS Chem. Biol. 10, 1797–1804 CrossRef Medline
Hoffmann, C., Gaietta, G., Bünemann, M., Adams, S. R., Oberdorff-Maass, S., Behr, B., Vilardaga, J.-P., Tsien, R. Y., Ellisman, M. H., and Lohse, M. J. (2005) A FlAsH-based FRET approach to determine G protein– coupled receptor activation in living cells. Nat. Methods 2, 171–176 CrossRef Medline
Hall, M. P., Unch, J., Binkowski, B. F., Valley, M. P., Butler, B. L., Wood, M. G., Otto, P., Zimmerman, K., Vidugiris, G., Machleidt, T., Robers, M. B., Benink, H. A., Eggers, C. T., Slater, M. R., Meisenheimer, P. L., et al. (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848 –1857 CrossRef Medline
Dixon, A. S., Schwinn, M. K., Hall, M. P., Zimmerman, K., Otto, P., Lub-ben, T. H., Butler, B. L., Binkowski, B. F., Machleidt, T., Kirkland, T. A., Wood, M. G., Eggers, C. T., Encell, L. P., and Wood, K. V. (2016) NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem. Biol. 11, 400 – 408 CrossRef Medline
Beaulieu, J.-M., Espinoza, S., and Gainetdinov, R. R. (2015) Dopamine receptors–IUPHAR review 13. Br. J. Pharmacol. 172, 1–23 CrossRef Medline
Panula, P., Chazot, P. L., Cowart, M., Gutzmer, R., Leurs, R., Liu, W. L., Stark, H., Thurmond, R. L., and Haas, H. L. (2015) International union of basic and clinical pharmacology. XCVIII. Histamine receptors. Pharmacol. Rev. 67, 601– 655 CrossRef Medline
Gilissen, J., Jouret, F., Pirotte, B., and Hanson, J. (2016) Insight into SUCNR1 (GPR91) structure and function. Pharmacol. Ther. 159, 56 – 65 CrossRef Medline
Galés, C., Rebois, R. V., Hogue, M., Trieu, P., Breit, A., Hébert, T. E., and Bouvier, M. (2005) Real-time monitoring of receptor and G-protein interactions in living cells. Nat Methods 2, 177–184 CrossRef Medline
Hein, P., Frank, M., Hoffmann, C., Lohse, M. J., and Bünemann, M. (2005) Dynamics of receptor/G protein coupling in living cells. EMBO J. 24, 4106 – 4114 CrossRef Medline
Beaulieu, J. M., and Gainetdinov, R. R. (2011) The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol. Rev. 63, 182–217 CrossRef Medline
Lane, J. R., Powney, B., Wise, A., Rees, S., and Milligan, G. (2007) Protean agonism at the dopamine D2 receptor: (S)-3-(3-hydroxyphenyl)-N-pro-pylpiperidine is an agonist for activation of Go1 but an antagonist/inverse agonist for Gi1,Gi2, and Gi3. Mol. Pharmacol. 71, 1349 –1359 CrossRef Medline
Gazi, L., Nickolls, S. A., and Strange, P. G. (2003) Functional coupling of the human dopamine D2 receptor with Gi1, Gi2, Gi3, and Go G proteins: evidence for agonist regulation of G protein selectivity. Br. J. Pharmacol. 138, 775–786 CrossRef Medline
Masuho, I., Ostrovskaya, O., Kramer, G. M., Jones, C. D., Xie, K., and Martemyanov, K. A. (2015) Distinct profiles of functional discrimination among G proteins determine the actions of G protein– coupled receptors. Sci. Signal. 8, ra123–ra123 CrossRef Medline
Freedman, S. B., Patel, S., Marwood, R., Emms, F., Seabrook, G. R., Knowles, M. R., and McAllister, G. (1994) Expression and pharmacological characterization of the human D3 dopamine receptor. J. Pharmacol. Exp. Ther. 268, 417– 426 Medline
Zhang, J., Chung, T. D., and Oldenburg, K. R. (1999) A simple statistical parameter for use in evaluation and validation of high-throughput screening assays. J. Biomol. Screen. 4, 67–73 CrossRef Medline
Kenakin, T., Watson, C., Muniz-Medina, V., Christopoulos, A., and Novick, S. (2012) A simple method for quantifying functional selectivity and agonist bias. ACS Chem. Neurosci. 3, 193–203 CrossRef Medline
Michnick, S. W., Ear, P. H., Manderson, E. N., Remy, I., and Stefan, E. (2007) Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nat. Rev. Drug Discov. 6, 569 –582 CrossRef Medline
Magliery, T. J., Wilson, C. G., Pan, W., Mishler, D., Ghosh, I., Hamilton, A. D., and Regan, L. (2005) Detecting protein-protein interactions with a green fluorescent protein fragment Reassembly trap: scope and mechanism. J. Am. Chem. Soc. 127, 146 –157 CrossRef Medline
Dupuis, N., Laschet, C., Franssen, D., Szpakowska, M., Gilissen, J., Geubelle, P., Soni, A., Parent, A.-S., Pirotte, B., Chevigné, A., Twizere, J.-C., and Hanson, J. (2017) Activation of the orphan G protein– coupled receptor GPR27 by surrogate ligands promotes -Arrestin 2 recruitment. Mol. Pharmacol. 91, 595– 608 CrossRef Medline
Hattori, M., Tanaka, M., Takakura, H., Aoki, K., Miura, K., Anzai, T., and Ozawa, T. (2013) Analysis of temporal patterns of GPCR-arrestin interactions using split luciferase-fragment complementation. Mol. BioSyst. 9, 957–964 CrossRef Medline
Szpakowska, M., Nevins, A. M., Meyrath, M., Rhainds, D., D’huys, T., Guité-Vinet, F., Dupuis, N., Gauthier, P.-A., Counson, M., Kleist, A., St-Onge, G., Hanson, J., Schols, D., Volkman, B. F., Heveker, N., and Chevigné, A. (2018) Different contributions of chemokine N-terminal features attest to a different ligand binding mode and a bias towards activation of ACKR3/CXCR7 compared with CXCR4 and CXCR3. Br. J. Pharmacol. 175, 1419 –1438 CrossRef Medline
England, C. G., Ehlerding, E. B., and Cai, W. (2016) NanoLuc: a small luciferase is brightening up the field of bioluminescence. Bioconjug. Chem. 27, 1175–1187 CrossRef Medline
Liu, R., Wong, W., and IJzerman, A. P. (2016) Human G protein– coupled receptor studies in Saccharomyces cerevisiae. Biochem. Pharmacol. 114, 103–115 CrossRef Medline
Wan, Q., Okashah, N., Inoue, A., Nehmé, R., Carpenter, B., Tate, C. G., and Lambert, N. A. (2018) Mini G protein probes for active G protein– coupled receptors (GPCRs) in live cells. J. Biol. Chem. 293, 7466 –7473 CrossRef Medline
Galés, C., Van Durm, J. J., Schaak, S., Pontier, S., Percherancier, Y., Audet, M., Paris, H., and Bouvier, M. (2006) Probing the activation-promoted structural rearrangements in preassembled receptor–G protein complexes. Nat. Struct. Mol. Biol. 13, 778 –786 CrossRef Medline
Saulière, A., Bellot, M., Paris, H., Denis, C., Finana, F., Hansen, J. T., Altié, M.-F., Seguelas, M.-H., Pathak, A., Hansen, J. L., Sénard, J.-M., and Galés, C. (2012) Deciphering biased-agonism complexity reveals a new active AT1 receptor entity. Nat. Chem. Biol. 8, 622– 630 CrossRef Medline
Obeso, J. A., Rodriguez-Oroz, M. C., Goetz, C. G., Marin, C., Kordower, J. H., Rodriguez, M., Hirsch, E. C., Farrer, M., Schapira, A. H., and Halliday, G. (2010) Missing pieces in the Parkinson’s disease puzzle. Nat. Med. 16, 653– 661 CrossRef Medline
Oertel, W., and Schulz, J. B. (2016) Current and experimental treatments of Parkinson disease: a guide for neuroscientists. J Neurochem. 139, Suppl. 1, 325–337 CrossRef Medline
De Keyser, J., De Backer, J. P., Wilczak, N., and Herroelen, L. (1995) Dopamine agonists used in the treatment of Parkinson’s disease and their selectivity for the D1, D2, and D3 dopamine receptors in human striatum. Prog. Neuropsychopharmacol. Biol. Psychiatry 19, 1147–1154 CrossRef Medline
Cordeaux, Y., Nickolls, S. A., Flood, L. A., Graber, S. G., and Strange, P. G. (2001) Agonist regulation of D(2) dopamine receptor/G protein interaction. Evidence for agonist selection of G protein subtype. J. Biol. Chem. 276, 28667–28675 CrossRef Medline
Winpenny, D., Clark, M., and Cawkill, D. (2016) Biased ligand quantification in drug discovery: from theory to high-throughput screening to identify new biased opioid receptor agonists. Br. J. Pharmacol. 173, 1393–1403 CrossRef Medline
Yano, H., Cai, N. S., Javitch, J. A., and Ferré, S. (2018) Luciferase complementation based-detection of G-protein-coupled receptor activity. Bio-Techniques 65, 9 –14 CrossRef Medline
Harding, S. D., Sharman, J. L., Faccenda, E., Southan, C., Pawson, A. J., Ireland, S., Gray, A. J. G., Bruce, L., Alexander, S. P. H., Anderton, S., Bryant, C., Davenport, A. P., Doerig, C., Fabbro, D., Levi-Schaffer, F., et al. (2018) The IUPHAR/BPS guide to pharmacology in 2018: updates and expansion to encompass the new guide to immunopharmacology. Nucleic Acids Res. 46, D1091–D1106 CrossRef Medline
Guan, X. M., Kobilka, T. S., and Kobilka, B. (1992) Enhancement of membrane insertion and function in a type IIIb membrane protein following introduction of a cleavable signal peptide. J. Biol. Chem. 267, 21995–21998 Medline
