Download PDF
Thromb Haemost
DOI: 10.1055/a-2442-9031
Cellular Haemostasis and Platelets

Distinct Role of GRK3 in Platelet Activation by Desensitization of G Protein-Coupled Receptors

Preeti K. Chaudhary
1   Laboratory of Veterinary Pathology and Platelet Signaling, College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
,
Sanggu Kim
1   Laboratory of Veterinary Pathology and Platelet Signaling, College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
,
Satya P. Kunapuli
2   Sol Sherry Thrombosis Research Center and Department of Cardiovascular Sciences, Lewis Katz School of Medicine, Temple University, Philadelphia, Pennsylvania, United States
,
Soochong Kim
1   Laboratory of Veterinary Pathology and Platelet Signaling, College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
› Author Affiliations Funding This research was funded by the Chungbuk National University BK21 program (2024), the National Research Foundation (NRF) of Korea (NRF-2022R1A2C1003638), and the Basic Research Laboratory Program (2022R1A4A1025557) through the NRF of Korea funded by the Ministry of Science and ICT.
› Further Information
Also available at
    Permissions and Reprints


Abstract

Background Many platelet agonists mediate their cellular effects through G protein-coupled receptors (GPCRs) to induce platelet activation, and GPCR kinases (GRKs) have been demonstrated to have crucial roles in most GPCR functions in other cell types. Here, we investigated the functional role of GRK3 and the molecular basis for the regulation of GPCR desensitization by GRK3 in platelets.

Methods We used mice lacking GRK3 as well as β-arrestin2, which has been shown to be important in GPCR function in platelets.

Results Platelet aggregation and dense granule secretion induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in both GRK3 −/− and β-arrestin2 −/− platelets compared with wild-type (WT) platelets, whereas non-GPCR agonist collagen-induced platelet aggregation and secretion were not affected. We have previously shown that GRK6 is not involved in the regulation of Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors. Interestingly, in contrast to GRK6, platelet aggregation induced by costimulation of serotonin and epinephrine, which activate 5-HT2A and α2A adrenergic receptors, respectively, was significantly potentiated in GRK3 −/− platelets, suggesting that GRK3 is involved in general GPCR regulation. In addition, platelet aggregation in response to the second challenge of adenosine diphosphate was restored in GRK3 −/− platelets, whereas restimulation of the agonist failed to induce aggregation in WT platelets, confirming that GRK3 contributes to general GPCR desensitization. Furthermore, 2-MeSADP- and AYPGKF-induced AKT and ERK phosphorylation were significantly potentiated in GRK3 −/− platelets. Finally, GRK3 −/− mice showed shorter tail bleeding times compared with WT, indicating that GRK3 −/− mice is more susceptible to hemostasis.

Conclusion GRK3 plays a crucial role in the regulation of platelet activation through general GPCR desensitization in platelets.

Keywords

GRK3 - β-arrestin2 - GPCR - desensitization - platelets

Authors' Contribution

Conceptualization, P.K.C. and S.K. (Soochong Kim); methodology, P.K.C. and S.K. (Sanggu Kim); validation, S.P.K. and S.K. (Soochong Kim); investigation, P.K.C. and S.K. (Soochong Kim); writing—original draft, P.K.C.; writing—review and editing, P.K.C., S.K. (Sanggu Kim), S.P.K., and S.K. (Soochong Kim); supervision, S.K. (Soochong Kim). All the authors have read and agreed to the published version of the manuscript.




Publication History

Received: 03 June 2024

Accepted: 16 October 2024

Accepted Manuscript online:
17 October 2024

Article published online:
11 November 2024

© 2024. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Gibbins JM, Okuma M, Farndale R, Barnes M, Watson SP. Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain. FEBS Lett 1997; 413 (02) 255-259
    CrossrefPubMedGoogle Scholar
  • 2 Du X. Signaling and regulation of the platelet glycoprotein Ib-IX-V complex. Curr Opin Hematol 2007; 14 (03) 262-269
    CrossrefPubMedGoogle Scholar
  • 3 Daniel JL, Dangelmaier C, Jin J, Ashby B, Smith JB, Kunapuli SP. Molecular basis for ADP-induced platelet activation. I. Evidence for three distinct ADP receptors on human platelets. J Biol Chem 1998; 273 (04) 2024-2029
    CrossrefPubMedGoogle Scholar
  • 4 Jin J, Kunapuli SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci U S A 1998; 95 (14) 8070-8074
    CrossrefPubMedGoogle Scholar
  • 5 Kim S, Foster C, Lecchi A. et al. Protease-activated receptors 1 and 4 do not stimulate G(i) signaling pathways in the absence of secreted ADP and cause human platelet aggregation independently of G(i) signaling. Blood 2002; 99 (10) 3629-3636
    CrossrefPubMedGoogle Scholar
  • 6 Coughlin SR. How the protease thrombin talks to cells. Proc Natl Acad Sci U S A 1999; 96 (20) 11023-11027
    CrossrefPubMedGoogle Scholar
  • 7 Paul BZ, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors. J Biol Chem 1999; 274 (41) 29108-29114
    CrossrefPubMedGoogle Scholar
  • 8 Offermanns S, Laugwitz KL, Spicher K, Schultz G. G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets. Proc Natl Acad Sci U S A 1994; 91 (02) 504-508
    CrossrefPubMedGoogle Scholar
  • 9 Shenker A, Goldsmith P, Unson CG, Spiegel AM. The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel Gq family. J Biol Chem 1991; 266 (14) 9309-9313
    CrossrefPubMedGoogle Scholar
  • 10 de Chaffoy de Courcelles D, Roevens P, Van Belle H, De Clerck F. The synergistic effect of serotonin and epinephrine on the human platelet at the level of signal transduction. FEBS Lett 1987; 219 (02) 283-288
    CrossrefPubMedGoogle Scholar
  • 11 Shenoy SK, Lefkowitz RJ. Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling. Biochem J 2003; 375 (Pt 3): 503-515
    CrossrefPubMedGoogle Scholar
  • 12 Gurevich EV, Gurevich VV. Arrestins: ubiquitous regulators of cellular signaling pathways. Genome Biol 2006; 7 (09) 236
    CrossrefPubMedGoogle Scholar
  • 13 Latorraca NR, Wang JK, Bauer B. et al. Molecular mechanism of GPCR-mediated arrestin activation. Nature 2018; 557 (7705) 452-456
    CrossrefPubMedGoogle Scholar
  • 14 Paing MM, Stutts AB, Kohout TA, Lefkowitz RJ, Trejo J. beta -Arrestins regulate protease-activated receptor-1 desensitization but not internalization or Down-regulation. J Biol Chem 2002; 277 (02) 1292-1300
    CrossrefPubMedGoogle Scholar
  • 15 Chaudhary PK, Kim S. The GRKs reactome: role in cell biology and pathology. Int J Mol Sci 2021; 22 (07) 3375
    CrossrefPubMedGoogle Scholar
  • 16 Lefkowitz RJ. The superfamily of heptahelical receptors. Nat Cell Biol 2000; 2 (07) E133-E136
    CrossrefPubMedGoogle Scholar
  • 17 Kobilka BK. G protein coupled receptor structure and activation. Biochim Biophys Acta 2007; 1768 (04) 794-807
    CrossrefPubMedGoogle Scholar
  • 18 Sorkin A, von Zastrow M. Endocytosis and signalling: intertwining molecular networks. Nat Rev Mol Cell Biol 2009; 10 (09) 609-622
    CrossrefPubMedGoogle Scholar
  • 19 Goodman Jr OB, Krupnick JG, Santini F. et al. Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor. Nature 1996; 383 (6599) 447-450
    CrossrefPubMedGoogle Scholar
  • 20 Laporte SA, Oakley RH, Zhang J. et al. The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc Natl Acad Sci U S A 1999; 96 (07) 3712-3717
    CrossrefPubMedGoogle Scholar
  • 21 Goodman Jr OB, Krupnick JG, Santini F. et al. Role of arrestins in G-protein-coupled receptor endocytosis. Adv Pharmacol 1998; 42: 429-433
    CrossrefPubMedGoogle Scholar
  • 22 García-Sevilla JA, Alvaro-Bartolomé M, Díez-Alarcia R. et al. Reduced platelet G protein-coupled receptor kinase 2 in major depressive disorder: antidepressant treatment-induced upregulation of GRK2 protein discriminates between responder and non-responder patients. Eur Neuropsychopharmacol 2010; 20 (10) 721-730
    CrossrefPubMedGoogle Scholar
  • 23 Penela P, Ribas C, Sánchez-Madrid F, Mayor Jr F. G protein-coupled receptor kinase 2 (GRK2) as a multifunctional signaling hub. Cell Mol Life Sci 2019; 76 (22) 4423-4446
    CrossrefPubMedGoogle Scholar
  • 24 Chen X, Gupta S, Cooper M. et al. GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets. Blood Adv 2020; 4 (01) 76-86
    CrossrefPubMedGoogle Scholar
  • 25 Li C, Malloy M, Ture SK. et al. G protein-coupled receptor kinase 5 regulates thrombin signaling in platelets. Res Pract Thromb Haemost 2024; 8 (06) 102556
    CrossrefPubMedGoogle Scholar
  • 26 Downes K, Zhao X, Gleadall NS. et al. G protein-coupled receptor kinase 5 regulates thrombin signaling in platelets via PAR-1. Blood Adv 2022; 6 (07) 2319-2330
    CrossrefPubMedGoogle Scholar
  • 27 Zhao X, Cooper M, Michael JV. et al. GRK2 regulates ADP signaling in platelets via P2Y1 and P2Y12. Blood Adv 2022; 6 (15) 4524-4536
    CrossrefPubMedGoogle Scholar
  • 28 Chaudhary PK, Kim S, Kim S. The predominant role of arrestin3 in general GPCR desensitization in platelets. J Clin Med 2021; 10 (20) 4743
    CrossrefPubMedGoogle Scholar
  • 29 Zeiler M, Moser M, Mann M. Copy number analysis of the murine platelet proteome spanning the complete abundance range. Mol Cell Proteomics 2014; 13 (12) 3435-3445
    CrossrefPubMedGoogle Scholar
  • 30 Hardy AR, Conley PB, Luo J, Benovic JL, Poole AW, Mundell SJ. P2Y1 and P2Y12 receptors for ADP desensitize by distinct kinase-dependent mechanisms. Blood 2005; 105 (09) 3552-3560
    CrossrefPubMedGoogle Scholar
  • 31 Chaudhary PK, Kim S, Jee Y, Lee SH, Park KM, Kim S. Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization. Int J Mol Sci 2020; 21 (11) 3932
    CrossrefPubMedGoogle Scholar
  • 32 Premont RT, Macrae AD, Aparicio SA, Kendall HE, Welch JE, Lefkowitz RJ. The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation. J Biol Chem 1999; 274 (41) 29381-29389
    CrossrefPubMedGoogle Scholar
  • 33 Tiruppathi C, Yan W, Sandoval R. et al. G protein-coupled receptor kinase-5 regulates thrombin-activated signaling in endothelial cells. Proc Natl Acad Sci U S A 2000; 97 (13) 7440-7445
    CrossrefPubMedGoogle Scholar
  • 34 Penela P, Murga C, Ribas C, Lafarga V, Mayor Jr F. The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets. Br J Pharmacol 2010; 160 (04) 821-832
    CrossrefPubMedGoogle Scholar
  • 35 Zhou H, Yan F, Tai HH. Phosphorylation and desensitization of the human thromboxane receptor-alpha by G protein-coupled receptor kinases. J Pharmacol Exp Ther 2001; 298 (03) 1243-1251
    PubMedGoogle Scholar
  • 36 Kim J, Ahn S, Ren XR. et al. Functional antagonism of different G protein-coupled receptor kinases for beta-arrestin-mediated angiotensin II receptor signaling. Proc Natl Acad Sci U S A 2005; 102 (05) 1442-1447
    CrossrefPubMedGoogle Scholar
  • 37 Ishii K, Chen J, Ishii M. et al. Inhibition of thrombin receptor signaling by a G-protein coupled receptor kinase. Functional specificity among G-protein coupled receptor kinases. J Biol Chem 1994; 269 (02) 1125-1130
    CrossrefPubMedGoogle Scholar
  • 38 Ren XR, Reiter E, Ahn S, Kim J, Chen W, Lefkowitz RJ. Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor. Proc Natl Acad Sci U S A 2005; 102 (05) 1448-1453
    CrossrefPubMedGoogle Scholar
  • 39 Alhosaini K, Azhar A, Alonazi A, Al-Zoghaibi F. GPCRs: The most promiscuous druggable receptor of the mankind. Saudi Pharm J 2021; 29 (06) 539-551
    CrossrefPubMedGoogle Scholar
  • 40 Li Y, Fan Y, Xu J. et al. GRK3 is a poor prognosticator and serves as a therapeutic target in advanced gastric adenocarcinoma. J Exp Clin Cancer Res 2022; 41 (01) 257
    CrossrefPubMedGoogle Scholar
  • 41 Benovic JL, Stone WC, Huebner K, Croce C, Caron MG, Lefkowitz RJ. cDNA cloning and chromosomal localization of the human beta-adrenergic receptor kinase. FEBS Lett 1991; 283 (01) 122-126
    CrossrefPubMedGoogle Scholar
  • 42 Kim S, Jin J, Kunapuli SP. Relative contribution of G-protein-coupled pathways to protease-activated receptor-mediated Akt phosphorylation in platelets. Blood 2006; 107 (03) 947-954
    CrossrefPubMedGoogle Scholar
  • 43 Gurevich EV, Tesmer JJ, Mushegian A, Gurevich VV. G protein-coupled receptor kinases: more than just kinases and not only for GPCRs. Pharmacol Ther 2012; 133 (01) 40-69
    CrossrefPubMedGoogle Scholar
  • 44 Steury MD, Lucas PC, McCabe LR, Parameswaran N. G-protein-coupled receptor kinase-2 is a critical regulator of TNFα signaling in colon epithelial cells. Biochem J 2017; 474 (14) 2301-2313
    CrossrefPubMedGoogle Scholar
  • 45 Yarman Y, Zhao X, Cooper M, Thomson H, Ma P. A common variant in GRK5 reveals a PAR1-specific mechanism for its regulation of platelet function. Blood 2022; 140: 5514-5515
    CrossrefPubMedGoogle Scholar
  • 46 DebBurman SK, Ptasienski J, Benovic JL, Hosey MM. G protein-coupled receptor kinase GRK2 is a phospholipid-dependent enzyme that can be conditionally activated by G protein betagamma subunits. J Biol Chem 1996; 271 (37) 22552-22562
    CrossrefPubMedGoogle Scholar
  • 47 Pitcher JA, Freedman NJ, Lefkowitz RJ. G protein-coupled receptor kinases. Annu Rev Biochem 1998; 67: 653-692
    CrossrefPubMedGoogle Scholar
  • 48 Zidar DA, Violin JD, Whalen EJ, Lefkowitz RJ. Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands. Proc Natl Acad Sci U S A 2009; 106 (24) 9649-9654
    CrossrefPubMedGoogle Scholar
  • 49 Choi M, Staus DP, Wingler LM. et al. G protein-coupled receptor kinases (GRKs) orchestrate biased agonism at the β2-adrenergic receptor. Sci Signal 2018; 11 (544) 11
    CrossrefPubMedGoogle Scholar
  • 50 Vinge LE, Andressen KW, Attramadal T. et al. Substrate specificities of g protein-coupled receptor kinase-2 and -3 at cardiac myocyte receptors provide basis for distinct roles in regulation of myocardial function. Mol Pharmacol 2007; 72 (03) 582-591
    CrossrefPubMedGoogle Scholar
  • 51 Kahner BN, Shankar H, Murugappan S, Prasad GL, Kunapuli SP. Nucleotide receptor signaling in platelets. J Thromb Haemost 2006; 4 (11) 2317-2326
    CrossrefPubMedGoogle Scholar
  • 52 Bynagari-Settipalli YS, Lakhani P, Jin J. et al. Protein kinase C isoform ε negatively regulates ADP-induced calcium mobilization and thromboxane generation in platelets. Arterioscler Thromb Vasc Biol 2012; 32 (05) 1211-1219
    CrossrefPubMedGoogle Scholar