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. 2022 Apr 27;11(9):1473.
doi: 10.3390/cells11091473.

β-Arrestin2 Is Critically Involved in the Differential Regulation of Phosphosignaling Pathways by Thyrotropin-Releasing Hormone and Taltirelin

Zdenka Drastichova  1 , Radka Trubacova  1 , Jiri Novotny  1
Affiliations

β-Arrestin2 Is Critically Involved in the Differential Regulation of Phosphosignaling Pathways by Thyrotropin-Releasing Hormone and Taltirelin

Zdenka Drastichova et al. Cells. .

Abstract

In recent years, thyrotropin-releasing hormone (TRH) and its analogs, including taltirelin (TAL), have demonstrated a range of effects on the central nervous system that represent potential therapeutic agents for the treatment of various neurological disorders, including neurodegenerative diseases. However, the molecular mechanisms of their actions remain poorly understood. In this study, we investigated phosphosignaling dynamics in pituitary GH1 cells affected by TRH and TAL and the putative role of β-arrestin2 in mediating these effects. Our results revealed widespread alterations in many phosphosignaling pathways involving signal transduction via small GTPases, MAP kinases, Ser/Thr- and Tyr-protein kinases, Wnt/β-catenin, and members of the Hippo pathway. The differential TRH- or TAL-induced phosphorylation of numerous proteins suggests that these ligands exhibit some degree of biased agonism at the TRH receptor. The different phosphorylation patterns induced by TRH or TAL in β-arrestin2-deficient cells suggest that the β-arrestin2 scaffold is a key factor determining phosphorylation events after TRH receptor activation. Our results suggest that compounds that modulate kinase and phosphatase activity can be considered as additional adjuvants to enhance the potential therapeutic value of TRH or TAL.

Keywords: GH1 cells; TRH receptor; small GTPase-mediated signaling; taltirelin; thyrotropin-releasing hormone; β-arrestin2.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Alterations in phosphorylation of phosphoproteins involved in Ras GTPase-mediated signal transduction associated with the PI3K/Akt/mTOR pathway. Phosphoproteins with changes in phosphorylation after β-arrestin2 knockdown and TRH or TAL treatments in wild-type or β-arrestin2 knockdown cells are labeled with the gene name and protein ID in the UniProt Database (www.uniprot.org, accessed on 1 August 2021). Phosphosites are labeled with the abbreviations of the amino acid residues (S, serine; T, threonine; Y, tyrosine) and the number that determines the position in the amino acid sequence of the protein. Small up and down arrows represent an increase and a decrease in phosphorylation, respectively. Two or one arrows represent qualitative and quantitative changes, respectively. The five pairwise experimental groups are distinguished by color (β-arrestin2 knockdown to control, red; TRH to control, yellow; β-arrestin2 knockdown + TRH to control, brown; TAL to control, blue; β-arrestin2 knockdown + TAL to control, green). Associations between proteins are shown by arrows that determine stimulatory or inhibitory effects. Red arrows and crosses represent confirmed relationships between phosphorylation on the protein phosphosite and the phosphorylating kinase or downstream protein. Associations and interactions between proteins were ordered according to [44,45,46,47,48,50,52,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72]. Abbreviations: AMPK: 5′-AMP-activated protein kinase; CDK4: cyclin-dependent kinase 4; CK2β: casein kinase 2β; EGFR: epidermal growth factor receptor; ERK1/2: extracellular signal-regulated kinase 1; GPCR: G-protein coupled receptor; Map3k: mitogen-activated protein kinase kinase kinase; Map4k: mitogen-activated protein kinase kinase kinase kinase; MEK1/2: dual specificity mitogen-activated protein kinase kinase 1; mTOR: mechanistic target of rapamycin; PAK4: serine/threonine p21-activated kinase 4; PIP2: phosphatidylinositol 4,5-bisphosphate; PI3K: phosphoinositide 3-kinase; RPS6KC1: ribosomal protein S6 kinase C1; Stk3: serine/threonine protein kinase 3; Rptor: regulatory-associated protein of mTOR; Rragc: Ras-related GTP-binding protein C; Src: proto-oncogene tyrosine-protein kinase Src; Taok3: serine/threonine protein kinase thousand and one amino acid protein 3; TSC: tuberous sclerosis protein; Ulk1: Unc-51-like kinase 1.
Figure 2
Figure 2
Alterations in phosphorylation of phosphoproteins involved in Ras GTPase-mediated signal transduction associated with Grb2/Sos/Ras/Raf/MEK/ERK. The description of the figure is the same as in Figure 1. Associations and interactions between proteins were ordered according to [60,64,65,76,79,80,84,85,86,87,88,89]. Abbreviations: Afdn: afadin; Adcy6: adenylyl cyclase 6; Cnksr1: connector enhancer of kinase suppressor 1; Dab2ip: disabled homolog 2-interacting protein; Dclk1: doublecortin-like kinase 1; Eps8l2: epidermal growth factor receptor kinase substrate 8-like protein 2; Erbb3: receptor tyrosine-protein kinase erbB-3; Gab1: Grb2-associated-binding protein 1; Grb2: growth factor receptor-bound protein2; KSR: kinase suppressor of Ras; PKA: cAMP-dependent protein kinase A; Prkar: cAMP-dependent protein kinase regulatory subunit; Rassf5: Ras association domain-containing protein 5; Nf1: neurofibromin 1; Riok2: serine/threonine protein kinase RIO2; RPS6KA3: ribosomal protein S6 kinase α3; Rreb1: Ras-responsive element-binding protein 1; Sos: son of sevenless homolog 1.
Figure 3
Figure 3
Alterations in phosphorylation of phosphoproteins involved in Rho GTPase-mediated signal transduction. The description of the figure is the same as in Figure 1. Associations and interactions between proteins were ordered according to [44,69,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117]. Abbreviations: Abl2: Abelson tyrosine-protein kinase 2; Akap13: A-kinase anchor protein 13; Arhgef: Rho guanine nucleotide exchange factor; Arhgap: Rho GTPase-activating protein; Camk2β: calcium/calmodulin-dependent protein kinase type II subunit β; Dvl: dishevelled protein; Ezh2: histone-lysine N-methyltranferase; Kif13: kinesin family member 13A; Mcf2l: Mcf2-transforming sequence-like protein; Myo9b: unconventional myosin-IXb; Itsn: intersectin; Pak6, p21-activated kinase 6; PKN1: serine/threonine-protein kinase N1; PLCε: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase ε; PKCδ: protein kinase Cδ; PKCε: protein kinase Cε; PKD: serine/threonine-protein kinase D; Ppp1r14a: protein phosphatase 1 regulatory subunit 14A; Ppp1r9b: neurabin-2; Prex2: phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 protein; Rock: Rho-associated protein kinase; Slk: STE20-like serine/threonine-protein kinase; Snrk: SNF-related serine/threonine-protein kinase; Stk24: serin/threonine-protein kinase 24; Tns: tensin; Trio: Triple functional domain protein; Zak: leucine zipper- and sterile α motif-containing kinase.
Figure 4
Figure 4
Alterations in phosphorylation of phosphoproteins involved in Rac GTPase-mediated signal transduction. The description of the figure is the same as in Figure 1. Associations and interactions between proteins were ordered according to [44,72,94,98,100,102,110,111,114,117,118,119,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145]. Abbreviations: Cdkl5: cyclin-dependent kinase-like 5; Cttn: Src substrate cortactin; Dock: dedicator of cytokinesis protein; Dyrk1b: dual specificity tyrosine-phosphorylation-regulated kinase 1B; Farp2: FERM, ARH/RhoGEF and pleckstrin domain protein 2; Kalrn: kalirin; Marcks, myristoylated alanine-rich C-kinase substrate; Nisch: nischarin; Pak1: p21-activated kinase 1; Pak2: p21-activated kinase 2; PLCβ3: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase β3; Ptk2β: protein-tyrosine kinase 2β; Srgap2: SLIT-ROBO Rho GTPase-activating protein 2; Srpk: SRSF protein kinase; Tiam1: TIAM Rac1-associated GEF 1; Wnk2: serine/threonine-protein kinase with no lysine 2.
Figure 5
Figure 5
Alterations in phosphorylation of phosphoproteins involved in Cdc42 GTPase-mediated signal transduction. The description of the figure is the same as in Figure 1. Associations and interactions between proteins were arranged according to [94,101,107,111,114,117,121,124,128,129,133,137,140,144,145,150,151,152,153,154,155,156,157]. Abbreviations: Cblb: E3 ubiquitin-protein ligase CBL-B; Cdc42bpa: Cdc42-binding protein kinase α; Cdc42bpb: Cdc42-binding protein kinase β; Cdc42ep4: Cdc42 effector protein 4; Git1: ARF GTPase-activating protein GIT1; Plekhg3: pleckstrin homology domain-containing family G member 3; Scrib: protein scribble homolog.
Figure 6
Figure 6
Alterations in phosphorylation of phosphoproteins involved in Arf GTPase-mediated signal transduction. The description of the figure is the same as in Figure 1. Associations and interactions between proteins were ordered according to [145,147,158,159,160,161,162,163,164]. Abbreviations: Agfg1: Arf-GAP domain and FG repeat-containing protein 1; AP3: adaptor complex protein 3; Arap1: Arfgap with RhoGAP domain, ANK repeat and PH domain-containing protein 1; Arfgap: ADP-ribosylation factor GTPase-activating protein; Arfgef: Brefeldin A-inhibited guanine nucleotide-exchange protein; Arfip1, arfaptin 1; Asap: ArfGAP with SH3 domain, ANK repeat and PH domain-containing protein; Bin1: Myc box-dependent-interacting protein 1; Btbd8: AP2-interacting clathrin-endocytosis protein; Gbf1: Golgi brefeldin A-resistant guanine nucleotide exchange factor 1; PLCδ1: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase δ1; Smap2: Small Arfgap.
Figure 7
Figure 7
Alterations in phosphorylation of phosphoproteins involved in Rab GTPase-mediated signal transduction. The description of the figure is the same as in Figure 1. Associations and interactions between proteins were ordered according to [130,165,166,167,169,172,173,176,178,179,181,182,183,187,188,189,190,191,192,193]. Abbreviations I: AP: autophagosomes; EEs: early endosomes; LEs: late endosomes; LYS: lysosomes; REs: recycling endosomes; SVs: secretory vesicles. Abbreviations II: Dennd: DENN domain-containing proteins; Gapvd1: GTPase-activating protein and VPS9 domain-containing protein 1; Madd: MAP kinase-activating death domain protein; Pdzd8: PDZ domain-containing protein 8; Rabep: Rab GTPase-binding effector protein; Rabgap: Rab GTPase-activating protein 1; Rab3ip: Rab-3A-interacting protein; Rab11fip: Rab11 family-interacting proteins; Rbsn: rabenosyn-5; Tbc1d: TBC1 domain family members.
Figure 8
Figure 8
Alterations in phosphorylation of phosphoproteins involved in Ral GTPase-mediated signal transduction. The description of the figure is the same as in Figure 1. Associations and interactions between proteins were ordered according to [71,160,161,194,202,203,204,205]. Abbreviations: Aak1: AP2-associated protein kinase 1; AP2: adaptor protein complex 2; Gak: cyclin-G-associated kinase; Ralbp1: RalA-binding protein 1; RalGAP: Ral GTPase-activating protein; Ralgps2: Ras-specific guanine nucleotide-releasing factor RalGPS2.
Figure 9
Figure 9
Alterations in phosphorylation of phosphoproteins involved in Ran GTPase-mediated signal transduction. The description of the figure is the same as in Figure 1. Associations and interactions between proteins were ordered according to [207,209,210,211]. Abbreviations: Mycbp2: RCR-type E3 ubiquitin transferase; Ranbp: Ran-binding protein; RanGAP: Ran GTPase-activating protein; Vrk3: serine/threonine-protein vaccinia-related kinase 3.
Figure 10
Figure 10
Alterations in phosphorylation of phosphoproteins involved in Rap GTPase-mediated signal transduction. The description of the figure is the same as in Figure 1. Associations and interactions between proteins were ordered according to [212,213,214,215,216,217,218]. Abbreviations: Bcr: breakpoint cluster region protein; Crk: adapter molecule crk; Radil: Ras-associating and dilute domain-containing protein; Rap1gap: Rap1 GTPase-activating protein 1; Rapgef: Rap guanine nucleotide exchange factor; Sipa: signal-induced proliferation-associated proteins; Tnik: TRAF2 and NCK-interacting protein kinase.
Figure 11
Figure 11
Alterations in phosphorylation of phosphoproteins involved in β-catenin-mediated signal transduction. The description of the figure is the same as in Figure 1. Associations and interactions between proteins were ordered according to [117,219,220,221,222,223,224,225,226]. Abbreviations: Dact3: dishevelled-binding antagonist of β-catenin 3; GSK3β: glycogen synthase kinase-3β; Psen1: presenilin 1; Tnks1bp1: 182 kDa tankyrase-1-binding protein 1.
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References

    1. Tashjian A.H., Barowsky N.J., Jensen D.K. Thyrotropin releasing hormone–direct evicence for stimulation of prolactin production by pituitary cells in culture. Biochem. Biophys. Res. Commun. 1971;43:516–523. doi: 10.1016/0006-291X(71)90644-9. - DOI - PubMed
    1. Kanasaki H., Oride A., Mijiddorj T., Kyo S. Role of thyrotropin-releasing hormone in prolactin-producing cell models. Neuropeptides. 2015;54:73–77. doi: 10.1016/j.npep.2015.08.001. - DOI - PubMed
    1. Drastichova Z., Bourova L., Hejnova L., Jedelsky P., Svoboda P., Novotny J. Protein Alterations Induced by Long-Term Agonist Treatment of HEK293 Cells Expressing Thyrotropin-Releasing Hormone Receptor and G(11)alpha Protein. J. Cell. Biochem. 2010;109:255–264. doi: 10.1002/jcb.22409. - DOI - PubMed
    1. Koo K.B., Suh H.J., Ra K.S., Choi J.W. Protective Effect of Cyclo(His-Pro) on Streptozotocin-Induced Cytotoxicity and Apoptosis In Vitro. J. Microbiol. Biotechnol. 2011;21:218–227. doi: 10.4014/jmb.1012.12003. - DOI - PubMed
    1. Luo L., Luo J.Z., Jackson I. Tripeptide amide L-pyroglutamyl-histidyl-L-prolineamide (L-PHP-thyrotropin-releasing hormone, TRH) promotes insulin-producing cell proliferation. Curr. Aging Sci. 2013;6:8–13. doi: 10.2174/1874609811306010002. - DOI - PubMed

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This study was supported by the Charles University Institutional Research Fund (no. SVV-260571/2020) and by the project BIOCEV—Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (no. CZ.1.05/1.1.00/02.0109), funded from the European Regional Development Fund.

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