Ligand-induced activation of ERK1/2 signaling by constitutively active Gs-coupled 5-HT receptors


5-HT4R, 5-HT6R, and 5-HT7AR are three constitutively active Gs-coupled 5-HT receptors that have key roles in brain development, learning, memory, cognition, and other physiological processes in the central nervous system. In addition to Gs signaling cascade mediated by these three 5-HT receptors, the ERK1/2 signaling which is dependent on cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) activation downstream of Gs signaling has also been widely studied. In this study, we investigated these two signaling pathways originating from the three Gs-coupled 5-HT receptors in AD293 cells. We found that the phosphorylation and activation of ERK1/2 are ligand-induced, in contrast to the constitutively active Gs signaling. This indicates that Gs signaling alone is not sufficient for ERK1/2 activation in these three 5-HT receptors. In addition to Gs, we found that β-arrestin and Fyn are essential for the activation of ERK1/2. Together, these results put forth a novel mechanism for ERK1/2 activation involving the cooperative action of Gs, β-arrestin, and Fyn.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7


  1. 1.

    Ranjan R, Dwivedi H, Baidya M, Kumar M, Shukla AK. Novel structural insights into GPCR-beta-Arrestin interaction and signaling. Trends Cell Biol. 2017;27:851–62.

  2. 2.

    Peterson YK, Luttrell LM. The diverse roles of arrestin scaffolds in G protein-coupled receptor signaling. Pharmacol Rev. 2017;69:256–97.

  3. 3.

    Dikic I, Blaukat A. Protein tyrosine kinase-mediated pathways in G protein-coupled receptor signaling. Cell Biochem Biophys. 1999;30:369–87.

  4. 4.

    Shukla AK. Biasing GPCR signaling from inside. Sci Signal. 2014;7:pe3.

  5. 5.

    Leroy D, Missotten M, Waltzinger C, Martin T, Scheer A. G protein-coupled receptor-mediated ERK1/2 phosphorylation: towards a generic sensor of GPCR activation. J Recept Signal Transduct Res. 2007;27:83–97.

  6. 6.

    Ahn S, Shenoy SK, Wei H, Lefkowitz RJ. Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor. J Biol Chem. 2004;279:35518–25.

  7. 7.

    Wei H, Ahn S, Shenoy SK, Karnik SS, Hunyady L, Luttrell LM, et al. Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2. Proc Natl Acad Sci USA. 2003;100:10782–7.

  8. 8.

    Yoon S, Seger R. The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors. 2006;24:21–44.

  9. 9.

    Carmona-Rosas G, Alcantara-Hernandez R, Hernandez-Espinosa DA. Dissecting the signaling features of the multi-protein complex GPCR/beta-arrestin/ERK1/2. Eur J Cell Biol. 2018;97:349–58.

  10. 10.

    Saini DK, Kalyanaraman V, Chisari M, Gautam N. A family of G protein betagamma subunits translocate reversibly from the plasma membrane to endomembranes on receptor activation. J Biol Chem. 2007;282:24099–108.

  11. 11.

    Jordan JD, Carey KD, Stork PJ, Iyengar R. Modulation of rap activity by direct interaction of Galpha(o) with Rap1 GTPase-activating protein. J Biol Chem. 1999;274:21507–10.

  12. 12.

    Mochizuki N, Ohba Y, Kiyokawa E, Kurata T, Murakami T, Ozaki T, et al. Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G alpha(i). Nature. 1999;400:891–4.

  13. 13.

    Inglese J, Koch WJ, Touhara K, Lefkowitz RJ. G beta gamma interactions with PH domains and Ras-MAPK signaling pathways. Trends Biochem Sci. 1995;20:151–6.

  14. 14.

    Garbison KE, Heinz BA, Lajiness ME, Weidner JR, Sittampalam GS. Phospho-ERK assays. In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, et al., editors. Assay guidance manual. Bethesda, MD; 2004.

  15. 15.

    Franco R, Martinez-Pinilla E, Navarro G, Zamarbide M. Potential of GPCRs to modulate MAPK and mTOR pathways in Alzheimer’s disease. Prog Neurobiol. 2017;149-150:21–38.

  16. 16.

    Ahn S, Wei H, Garrison TR, Lefkowitz RJ. Reciprocal regulation of angiotensin receptor-activated extracellular signal-regulated kinases by beta-arrestins 1 and 2. J Biol Chem. 2004;279:7807–11.

  17. 17.

    Shenoy SK, Drake MT, Nelson CD, Houtz DA, Xiao K, Madabushi S, et al. beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor. J Biol Chem. 2006;281:1261–73.

  18. 18.

    Gaborik Z, Jagadeesh G, Zhang M, Spat A, Catt KJ, Hunyady L. The role of a conserved region of the second intracellular loop in AT1 angiotensin receptor activation and signaling. Endocrinology. 2003;144:2220–8.

  19. 19.

    Grundmann M, Merten N, Malfacini D, Inoue A, Preis P, Simon K, et al. Lack of beta-arrestin signaling in the absence of active G proteins. Nat Commun. 2018;9:341.

  20. 20.

    Singh AP, Gupta AK, Pardeshi R, Shukla AK. Aplasia cutis congenita in a newborn: a rare case. J Indian Assoc Pediatr Surg. 2018;23:175–7.

  21. 21.

    Cattaneo F, Guerra G, Parisi M, De Marinis M, Tafuri D, Cinelli M, et al. Cell-surface receptors transactivation mediated by g protein-coupled receptors. Int J Mol Sci. 2014;15:19700–28.

  22. 22.

    Che J, Chan ES, Cronstein BN. Adenosine A2A receptor occupancy stimulates collagen expression by hepatic stellate cells via pathways involving protein kinase A, Src, and extracellular signal-regulated kinases 1/2 signaling cascade or p38 mitogen-activated protein kinase signaling pathway. Mol Pharmacol. 2007;72:1626–36.

  23. 23.

    Magalhaes AC, Dunn H, Ferguson SS. Regulation of GPCR activity, trafficking and localization by GPCR-interacting proteins. Br J Pharmacol. 2012;165:1717–36.

  24. 24.

    Claeysen S, Sebben M, Becamel C, Bockaert J, Dumuis A. Novel brain-specific 5-HT4 receptor splice variants show marked constitutive activity: role of the C-terminal intracellular domain. Mol Pharmacol. 1999;55:910–20.

  25. 25.

    Krobert KA, Levy FO. The human 5-HT7 serotonin receptor splice variants: constitutive activity and inverse agonist effects. Br J Pharmacol. 2002;135:1563–71.

  26. 26.

    Purohit A, Herrick-Davis K, Teitler M. Creation, expression, and characterization of a constitutively active mutant of the human serotonin 5-HT6 receptor. Synapse. 2003;47:218–24.

  27. 27.

    Teitler M, Herrick-Davis K, Purohit A. Constitutive activity of G-protein coupled receptors: emphasis on serotonin receptors. Curr Top Med Chem. 2002;2:529–38.

  28. 28.

    Shukla AK, Xiao K, Lefkowitz RJ. Emerging paradigms of beta-arrestin-dependent seven transmembrane receptor signaling. Trends Biochem Sci. 2011;36:457–69.

  29. 29.

    Norum JH, Hart K, Levy FO. Ras-dependent ERK activation by the human G(s)-coupled serotonin receptors 5-HT4(b) and5-HT7(a). J Biol Chem. 2003;278:3098–104.

  30. 30.

    Yun HM, Kim S, Kim HJ, Kostenis E, Kim JI, Seong JY, et al. The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. J Biol Chem. 2007;282:5496–505.

  31. 31.

    Barthet G, Framery B, Gaven F, Pellissier L, Reiter E, Claeysen S, et al. 5-hydroxytryptamine 4 receptor activation of the extracellular signal-regulated kinase pathway depends on Src activation but not on G protein or beta-arrestin signaling. Mol Biol Cell. 2007;18:1979–91.

  32. 32.

    Hill CS, Marais R, John S, Wynne J, Dalton S, Treisman R. Functional analysis of a growth factor-responsive transcription factor complex. Cell. 1993;73:395–406.

  33. 33.

    Cheng Z, Garvin D, Paguio A, Stecha P, Wood K, Fan F. Luciferase reporter assay system for deciphering GPCR pathways. Curr Chem Genom. 2010;4:84–91.

  34. 34.

    Chen Y, Xu Z, Wu D, Li J, Song C, Lu W, et al. Luciferase reporter gene assay on human 5-HT receptor: which response element should be chosen? Sci Rep. 2015;5:8060.

  35. 35.

    Stratowa C, Himmler A, Czernilofsky AP. Use of a luciferase reporter system for characterizing G-protein-linked receptors. Curr Opin Biotechnol. 1995;6:574–81.

  36. 36.

    Conway S, Canning SJ, Howell HE, Mowat ES, Barrett P, Drew JE, et al. Characterisation of human melatonin mt(1) and MT(2) receptors by CRE-luciferase reporter assay. Eur J Pharmacol. 2000;390:15–24.

  37. 37.

    Nordemann U, Wifling D, Schnell D, Bernhardt G, Stark H, Seifert R, et al. Luciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts. PLoS ONE. 2013;8:e73961.

  38. 38.

    Zhao LH, Yin Y, Yang D, Liu B, Hou L, Wang X, et al. Differential requirement of the extracellular domain in activation of class B G protein-coupled receptors. J Biol Chem. 2016;291:15119–30.

  39. 39.

    Yin Y, de Waal PW, He Y, Zhao LH, Yang D, Cai X, et al. Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors. J Biol Chem. 2017;292:9865–81.

  40. 40.

    Baghaei KA. Deorphanization of human olfactory receptors by luciferase and Ca-imaging methods. Methods Mol Biol. 2013;1003:229–38.

  41. 41.

    Wu L, Zhang W, Qiu X, Wang C, Liu Y, Wang Z, et al. Identification of alkaloids from Corydalis yanhusuo W. T. Wang as dopamine D(1) receptor antagonists by using CRE-luciferase reporter gene assay. Molecules 2018;23:2585.

  42. 42.

    Shukla AK, Goto M, Xu X, Nawaoka K, Suwardy J, Ohkubo T, et al. Voltage-controlled magnetic anisotropy in Fe1-xCox/Pd/MgO system. Sci Rep. 2018;8:10362.

  43. 43.

    Stoddard EG, Volk RF, Carson JP, Ljungberg CM, Murphree TA, Smith JN, et al. Multifunctional activity-based protein profiling of the developing lung. J Proteome Res. 2018;17:2623–34.

  44. 44.

    Stork PJ, Schmitt JM. Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol. 2002;12:258–66.

  45. 45.

    Koch WJ, Hawes BE, Inglese J, Luttrell LM, Lefkowitz RJ. Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuates G beta gamma-mediated signaling. J Biol Chem. 1994;269:6193–7.

  46. 46.

    Garnovskaya MN, van Biesen T, Hawe B, Casanas Ramos S, Lefkowitz RJ, Raymond JR. Ras-dependent activation of fibroblast mitogen-activated protein kinase by 5-HT1A receptor via a G protein beta gamma-subunit-initiated pathway. Biochemistry. 1996;35:13716–22.

  47. 47.

    George J, Singh R, Mahmood Z, Shukla Y. Toxicoproteomics: new paradigms in toxicology research. Toxicol Mech Methods. 2010;20:415–23.

  48. 48.

    Kang Y, Zhou XE, Gao X, He Y, Liu W, Ishchenko A, et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature. 2015;523:561–7.

  49. 49.

    Bohn LM, Schmid CL. Serotonin receptor signaling and regulation via beta-arrestins. Crit Rev Biochem Mol Biol. 2010;45:555–66.

Download references


This work was supported in part by the National Natural Science Foundation (31770796 to Yi Jiang), the National Science and Technology Major Project (2018ZX09711002-002-002 to Yi Jiang), the Ministry of Science and Technology of China (XDB08020303 to H. Eric Xu), the K.C. Wong Education Foundation (to Yi JIANG), the Youth Innovation Promotion Association of CAS (to Yi Jiang), and the Outstanding Young Scientist Foundation (CAS, to Yi Jiang).

Author contributions

PL conducted the experiments and wrote the first draft of the paper; Y-lY, TW, LH, X-xW, MW, and G-gZ performed the experiments; PL, YS, and YJ analyzed the results; HEX and YJ supervised the project and wrote the manuscript, with contributions from all the authors.

Author information

Correspondence to H. Eric Xu or Yi Jiang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Suplementary Figure legends

Suplementary Tables

Suplementary Figure 1

Suplementary Figure 2

Suplementary Figure 3

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark


  • 5-HT4R
  • 5-HT6R
  • 5-HT7AR
  • 5-HT
  • ERK1/2
  • Gs
  • β-arrestin
  • Fyn
  • Src