Construct design, isolation and purification of the monomeric form of human GPCR GPR17 for structural and functional studies

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

G protein-coupled receptors (GPCRs) are a family of heptahelical transmembrane proteins consisting of more than 800 representatives in the human genome that regulate most processes in the human body and are targets for up to a third of all modern drugs. Many GPCRs, despite their importance for pharmacology, are still considered orphan, i.e., their endogenous ligand is unknown. Orphan receptor GPR17, belonging to class A GPCR, is expressed mainly in the central nervous system, plays an important role in the formation of the myelin sheath of neurons and is a potential target for the development of new drugs against multiple sclerosis, Alzheimer's disease and ischemia. The aim of this work was to prepare GPR17 for structure-functional studies, starting with the heterologous expression and ending with obtaining a stable protein sample. Screening of various genetically engineered constructs was performed, a number of point mutations were analyzed, and a significant number of potential ligands of this receptor were tested. As a result of the work, the conditions for expression, isolation, and purification of GPR17 were optimized, which together made it possible to obtain a fairly stable and monomeric protein preparation suitable for further structural studies.

Full Text

Restricted Access

About the authors

N. A. Safronova

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Author for correspondence.
Email: mishinalexej@gmail.com
Russian Federation, Dolgoprudny

A. P. Luginina

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Russian Federation, Dolgoprudny

A. A. Sadova

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Russian Federation, Dolgoprudny

M. B. Shevtsov

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Russian Federation, Dolgoprudny

O. V. Moiseeva

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Russian Federation, Dolgoprudny

V. I. Borshchevskiy

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Russian Federation, Dolgoprudny

A. V. Mishin

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Russian Federation, Dolgoprudny

References

  1. Kuneš J., Hojná S., Mráziková L., Montezano A., Touyz R., Maletínská L. // Physiol Res. 2023. V. 72. P. S73–S90. https://doi.org/10.33549/physiolres.935109
  2. Marucci G., Dal Ben D., Lambertucci C., Martí Navia A., Spinaci A., Volpini R., Buccioni M. // Exp. Opin. Ther. Pat. 2019. V. 29. P. 85–95. https://doi.org/10.1080/13543776.2019.1568990
  3. Dziedzic A., Miller E., Saluk-Bijak J., Bijak M. // Int. J. Mol. Sci. 2020. V. 21. P. 1852. https://doi.org/10.3390/ijms21051852
  4. Ou Z., Sun Y., Lin L., You N., Liu X., Li H., Ma Y., Cao L., Han Y., Liu M., Deng Y., Yao L., Lu Q.R., Chen Y. // J. Neurosci. 2016. V. 36. P. 10560–10573. https://doi.org/10.1523/JNEUROSCI.0898-16.2016
  5. Yan S., Conley J.M., Reilly A.M., Stull N.D., Abhyankar S.D., Ericsson A.C., Kono T., Molosh A.I., Kubal C.A., Evans-Molina C., Ren H. // Cell Rep. 2022. V. 38. P. 110179. https://doi.org/10.1016/j.celrep.2021.110179
  6. Ren H., Cook J. R., Kon N., Accili D. // Diabetes. 2015. V. 64. P. 3670–3679. https://doi.org/10.2337/db15-0390
  7. Sriram K., Insel P.A. // Mol. Pharmacol. 2018. V. 93. P. 251–258. https://doi.org/10.1124/mol.117.111062
  8. Khorn P.A., Luginina A.P., Pospelov V.A., Dashevsky D.E., Khnykin A.N., Moiseeva O.V., Safronova N.A., Belousov A.S., Mishin A.V., Borshchevsky V.I. // Biochemistry (Moscow). 2024. V. 89. P. 747–764. https://doi.org/10.1134/S0006297924040138
  9. Ye F., Wong T., Chen G., Zhang Z., Zhang B., Gan S., Gao W., Li J., Wu Z., Pan X., Du Y. // MedComm (Beijing). 2022. V. 3. P. e159. https://doi.org/10.1002/mco2.159
  10. Van Montfort R.L.M., Workman P. // Essays Biochem. 2017. V. 61. P. 431–437. https://doi.org/10.1042/EBC20170052
  11. Chun E., Thompson A.A., Liu W., Roth C.B., Griffith M.T., Katritch V., Kunken J., Xu F., Cherezov V., Hanson M.A., Stevens R.C. // Structure. 2012. V. 20. P. 967–976. https://doi.org/10.1016/j.str.2012.04.010
  12. Гусач А.Ю. // Структурные исследования человеческого цистеинил-лейкотриенового рецептора второго типа для создания новых лекарственных препаратов. Дис. канд. физ.-мат. наук, МФТИ, Москва, 2020.
  13. Ballesteros J.A., Weinstein H. // Methods Neurosci. 1995. V. 25. P. 366–428. https://doi.org/10.1016/S1043-9471(05)80049-7
  14. Bläsius R., Weber R.G., Lichter P., Ogilvie A. // J. Neurochem. 1998. V. 70. P. 1357–1365. https://doi.org/10.1046/j.1471-4159.1998.70041357.x
  15. Cherezov V., Abola E., Stevens R.C. // Methods Mol. Biol. 2010. P. 141–168. https://doi.org/10.1007/978-1-60761-762-4_8
  16. Popov P., Peng Y., Shen L., Stevens R.C., Cherezov V., Liu Z.J., Katritch V. // Elife. 2018. V. 7. P. e34729. https://doi.org/10.7554/eLife.34729
  17. Alexandrov A.I., Mileni M., Chien E.Y.T., Hanson M.A., Stevens R.C. // Structure. 2008. V. 16. P. 351–359. https://doi.org/10.1016/j.str.2008.02.004
  18. Luginina A., Gusach A., Marin E., Mishin A., Brouillette R., Popov P., Shiriaeva A., Besserer-Offroy É., Longpré J.M., Lyapina E., Ishchenko A., Patel N., Polovinkin V., Safronova N., Bogorodskiy A., Edelweiss E., Hu H., Weierstall U., Liu W., Batyuk A., Gordeliy V., Han G. W., Sarret P., Katritch V., Borshchevskiy V., Cherezov V. // Sci. Adv. 2019. V. 5. P. eaax2518. https://doi.org/10.1126/sciadv.aax2518
  19. Ciana P., Fumagalli M., Trincavelli M.L., Verderio C., Rosa P., Lecca D., Ferrario S., Parravicini C., Capra V., Gelosa P., Guerrini U., Belcredito S., Cimino M., Sironi L., Tremoli E., Rovati G.E., Martini C., Abbracchio M.P. // EMBO J. 2006. V. 25. P. 4615–4627. https://doi.org/10.1038/sj.emboj.7601341
  20. Maekawa A., Balestrieri B., Austen K.F., Kanaoka Y. // Proc. Natl. Acad. Sci. USA. 2009. V. 106. P. 11685– 11690. https://doi.org/10.1073/pnas.0905364106
  21. Benned-Jensen T., Rosenkilde M.M. // Br. J. Pharmacol. 2010. V. 159. P. 1092–1105. https://doi.org/10.1111/j.1476-5381.2009.00633.x
  22. Qi A.D., Harden T.K., Nicholas R.A. // J. Pharmacol. Exp. Ther. 2013. V. 347. P. 38–46. https://doi.org/10.1124/jpet.113.207647
  23. Hennen S., Wang H., Peters L., Merten N., Simon K., Spinrath A., Blättermann S., Akkari R., Schrage R., Schröder R., Schulz D., Vermeiren C., Zimmermann K., Kehraus S., Drewke C., Pfeifer A., König G.M., Mohr K., Gillard M., Müller C.E., Lu Q.R., Gomeza J., Kostenis E. // Sci. Signal. 2013. V. 6. P. ra93. https://doi.org/10.1126/scisignal.2004350
  24. Merten N., Fischer J., Simon K., Zhang L., Schröder R., Peters L., Letombe A., Hennen S., Schrage R., Bödefeld T., Vermeiren C., Gillard M., Mohr K., Lu Q.R., Brüstle O., Gomeza J., Kostenis E. // Cell Chem. Biol. 2018. V. 25. P. 775–786.e5. https://doi.org/10.1016/j.chembiol.2018.03.012
  25. Simon K., Merten N., Schröder R., Hennen S., Preis P., Schmitt N., Peters L., Schrage R., Vermeiren C., Gillard M., Mohr K., Gomeza J., Kostenis E. // Mol. Pharmacol. 2017. V. 91. P. 518–532. https://doi.org/10.1124/mol.116.107904
  26. Harrington A.W., Liu C., Phillips N., Nepomuceno D., Kuei C., Chang J., Chen W., Sutton S.W., O’Malley D., Pham L., Yao X., Sun S., Bonaventure P. // Br. J. Pharmacol. 2023. V. 180. P. 401–421. https://doi.org/10.1111/bph.15969

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Additional materials
Download (636KB)
Indexing metadata
3. Fig. 1. Results of gel filtration for the R252-BRIL-V253 construct with various point mutations and their combinations. SE – surface expression.

Download (145KB)
Indexing metadata
4. Fig. 2. Plot showing the thermal shift resulting from point mutations introduced into GPR17. Average values ​​obtained from three independent experiments are shown.

Download (84KB)
Indexing metadata
5. Fig. 3. Schematic representation of the best design. The introduced point mutations are shown in red, the partner protein BRIL is shown in purple, the HA fragment is shown in pink, the FLAG sequence is shown in yellow, 9 histidine residues are shown in blue, the TEV protease site is shown in orange, and atavistic linkers are shown in gray.

Download (496KB)
Indexing metadata
6. Fig. 4. Normalized melting curve (a) and analytical gel filtration HPLC results (b) for the best construct R252-BRIL-V253 with mutations A1313.24L + Y1483.41W + F1583.51Y + F2315.49L + I2345.52L + A3297.57D. Solid line – sample with concentration of 0.1 mg/ml, dotted line – 30 mg/ml.

Download (114KB)
Indexing metadata
7. Fig. 5. Effect of buffer salt composition on GPR17 thermal stability. Melting curves for several receptor constructs are shown. For each construct, the experiment was carried out in three different buffers: blue lines correspond to the buffer with NaCl in the composition, green lines – with KCl, and orange lines – with MgCl2. The salt concentration in this experiment was 500 mM.

Download (158KB)
Indexing metadata

Copyright (c) 2025 Russian Academy of Sciences