{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T15:59:05Z","timestamp":1790351945868,"version":"4.1.0"},"reference-count":74,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T00:00:00Z","timestamp":1768521600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2026,2,19]],"date-time":"2026-02-19T00:00:00Z","timestamp":1771459200000},"content-version":"vor","delay-in-days":34,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Commun Biol"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>G protein-coupled receptor (GPCR) signaling is regulated by four ubiquitously expressed GPCR kinase isoforms (GRKs), namely GRK2, GRK3, GRK5, and GRK6. Overexpression of individual GRKs occurs in diseases like cancer and heart failure, prompting a search for potent GRK inhibitors. While various in silico and in vitro approaches exist, few methods assess inhibitor efficacy in cellular systems. To address this, we used HEK293 cell lines co-expressing the \u03b22 adrenergic receptor (\u03b22) and one GRK isoform on a quadruple GRK2\/3\/5\/6 knockout background (\u0394Q-GRK). We evaluated the inhibition of isoproterenol (ISO)-induced T360\/S364-\u03b22 phosphorylation using the 7TM phosphorylation assay. This combination allowed comprehensive evaluation of commercially available GRK inhibitors. We conclude that compound 8h (GRK2\/3 inhibitor) and compound 18 (GRK5\/6 inhibitor) are highly recommendable tools for the study of GPCR phosphorylation and function in cellular systems. Together, these cell-based GRK inhibitor assays can facilitate medium- to high-throughput screening of future GRK-targeted drug candidates.<\/jats:p>","DOI":"10.1038\/s42003-026-09568-0","type":"journal-article","created":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T18:04:55Z","timestamp":1768586695000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Cell-based and isoform-selective G protein-coupled receptor kinase assays for comprehensive inhibitor evaluation"],"prefix":"10.1038","volume":"9","author":[{"given":"Nina K.","family":"Blum","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manuela C.","family":"Kiefer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angelika","family":"Decker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-3956-6059","authenticated-orcid":false,"given":"Laura","family":"Klement","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7760-5900","authenticated-orcid":false,"given":"Edda S. F.","family":"Matthees","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Verena","family":"Weitzel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Falko","family":"Nagel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Babu","family":"Joseph","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6188-2279","authenticated-orcid":false,"given":"Julia","family":"Drube","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Uehling","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0884-9300","authenticated-orcid":false,"given":"Carsten","family":"Hoffmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5997-8885","authenticated-orcid":false,"given":"Stefan","family":"Schulz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,1,16]]},"reference":[{"key":"9568_CR1","doi-asserted-by":"crossref","first-page":"1256","DOI":"10.1124\/mol.63.6.1256","volume":"63","author":"R Fredriksson","year":"2003","unstructured":"Fredriksson, R., Lagerstrom, M. C., Lundin, L. G. & Schioth, H. B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharm. 63, 1256\u20131272 (2003).","journal-title":"Mol. Pharm."},{"key":"9568_CR2","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1146\/annurev.physiol.69.022405.154712","volume":"69","author":"CA Moore","year":"2007","unstructured":"Moore, C. A., Milano, S. K. & Benovic, J. L. Regulation of receptor trafficking by GRKs and arrestins. Annu. Rev. Physiol. 69, 451\u2013482 (2007).","journal-title":"Annu. Rev. Physiol."},{"key":"9568_CR3","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1146\/annurev.physiol.69.022405.154731","volume":"69","author":"RT Premont","year":"2007","unstructured":"Premont, R. T. & Gainetdinov, R. R. Physiological roles of G protein-coupled receptor kinases and arrestins. Annu. Rev. Physiol. 69, 511\u2013534 (2007).","journal-title":"Annu. Rev. Physiol."},{"key":"9568_CR4","volume":"9","author":"ESF Matthees","year":"2021","unstructured":"Matthees, E. S. F., Haider, R. S., Hoffmann, C. & Drube, J. Differential regulation of GPCRs-are GRK expression levels the key? Front Cell Dev. Biol. 9, 687489 (2021).","journal-title":"Front Cell Dev. Biol."},{"key":"9568_CR5","doi-asserted-by":"crossref","first-page":"12457","DOI":"10.1073\/pnas.0803153105","volume":"105","author":"JS Martini","year":"2008","unstructured":"Martini, J. S. et al. Uncovering G protein-coupled receptor kinase-5 as a histone deacetylase kinase in the nucleus of cardiomyocytes. Proc. Natl. Acad. Sci. USA 105, 12457\u201312462 (2008).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"9568_CR6","doi-asserted-by":"crossref","first-page":"952","DOI":"10.1038\/nm1289","volume":"11","author":"S Liu","year":"2005","unstructured":"Liu, S., Premont, R. T., Kontos, C. D., Zhu, S. & Rockey, D. C. A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension. Nat. Med 11, 952\u2013958 (2005).","journal-title":"Nat. Med"},{"key":"9568_CR7","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1097\/FJC.0000000000000483","volume":"70","author":"SM Schumacher","year":"2017","unstructured":"Schumacher, S. M. & Koch, W. J. Noncanonical roles of G protein-coupled receptor kinases in cardiovascular signaling. J. Cardiovasc. Pharm. 70, 129\u2013141 (2017).","journal-title":"J. Cardiovasc. Pharm."},{"key":"9568_CR8","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.phrs.2016.05.008","volume":"110","author":"J Hullmann","year":"2016","unstructured":"Hullmann, J., Traynham, C. J., Coleman, R. C. & Koch, W. J. The expanding GRK interactome: Implications in cardiovascular disease and potential for therapeutic development. Pharm. Res. 110, 52\u201364 (2016).","journal-title":"Pharm. Res."},{"key":"9568_CR9","doi-asserted-by":"crossref","first-page":"857","DOI":"10.4049\/jimmunol.1300790","volume":"191","author":"Z Liu","year":"2013","unstructured":"Liu, Z. et al. TLR4 Signaling augments monocyte chemotaxis by regulating G protein-coupled receptor kinase 2 translocation. J. Immunol. 191, 857\u2013864 (2013).","journal-title":"J. Immunol."},{"key":"9568_CR10","volume":"8","author":"SZ Lutz","year":"2018","unstructured":"Lutz, S. Z. et al. Single nucleotide polymorphisms in the G-protein coupled receptor kinase 5 (GRK5) gene are associated with plasma LDL-cholesterol levels in humans. Sci. Rep. 8, 7745 (2018).","journal-title":"Sci. Rep."},{"key":"9568_CR11","doi-asserted-by":"crossref","first-page":"7478","DOI":"10.1073\/pnas.112198299","volume":"99","author":"AM Fong","year":"2002","unstructured":"Fong, A. M. et al. Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6-deficient mice. Proc. Natl. Acad. Sci. USA 99, 7478\u20137483 (2002).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"9568_CR12","doi-asserted-by":"crossref","first-page":"e90597","DOI":"10.1371\/journal.pone.0090597","volume":"9","author":"Z Xia","year":"2014","unstructured":"Xia, Z., Yang, T., Wang, Z., Dong, J. & Liang, C. GRK5 intronic (CA)n polymorphisms associated with type 2 diabetes in Chinese Hainan Island. PLoS One 9, e90597 (2014).","journal-title":"PLoS One"},{"key":"9568_CR13","volume":"5","author":"MR Ahmed","year":"2015","unstructured":"Ahmed, M. R., Bychkov, E., Li, L., Gurevich, V. V. & Gurevich, E. V. GRK3 suppresses L-DOPA-induced dyskinesia in the rat model of Parkinson\u2019s disease via its RGS homology domain. Sci. Rep. 5, 10920 (2015).","journal-title":"Sci. Rep."},{"key":"9568_CR14","doi-asserted-by":"crossref","first-page":"1873","DOI":"10.1016\/j.neurobiolaging.2006.08.013","volume":"28","author":"Z Suo","year":"2007","unstructured":"Suo, Z. et al. GRK5 deficiency leads to early Alzheimer-like pathology and working memory impairment. Neurobiol. Aging 28, 1873\u20131888 (2007).","journal-title":"Neurobiol. Aging"},{"key":"9568_CR15","doi-asserted-by":"crossref","first-page":"112","DOI":"10.3389\/fphar.2019.00112","volume":"10","author":"C Murga","year":"2019","unstructured":"Murga, C. et al. G protein-coupled receptor kinase 2 (GRK2) as a potential therapeutic target in cardiovascular and metabolic diseases. Front Pharm. 10, 112 (2019).","journal-title":"Front Pharm."},{"key":"9568_CR16","volume":"9","author":"LP Jiang","year":"2018","unstructured":"Jiang, L. P. et al. GRK5 functions as an oncogenic factor in non-small-cell lung cancer. Cell Death Dis. 9, 295 (2018).","journal-title":"Cell Death Dis."},{"key":"9568_CR17","doi-asserted-by":"crossref","first-page":"1594","DOI":"10.1182\/blood-2009-09-243980","volume":"115","author":"RE Tiedemann","year":"2010","unstructured":"Tiedemann, R. E. et al. Kinome-wide RNAi studies in human multiple myeloma identify vulnerable kinase targets, including a lymphoid-restricted kinase, GRK6. Blood 115, 1594\u20131604 (2010).","journal-title":"Blood"},{"key":"9568_CR18","doi-asserted-by":"crossref","first-page":"767","DOI":"10.1016\/j.biopsych.2014.01.021","volume":"76","author":"L Gluck","year":"2014","unstructured":"Gluck, L. et al. Loss of morphine reward and dependence in mice lacking G protein-coupled receptor kinase 5. Biol. Psychiatry 76, 767\u2013774 (2014).","journal-title":"Biol. Psychiatry"},{"key":"9568_CR19","doi-asserted-by":"crossref","DOI":"10.1038\/s41467-018-08162-1","volume":"10","author":"A Kliewer","year":"2019","unstructured":"Kliewer, A. et al. Phosphorylation-deficient G-protein-biased mu-opioid receptors improve analgesia and diminish tolerance but worsen opioid side effects. Nat. Commun. 10, 367 (2019).","journal-title":"Nat. Commun."},{"key":"9568_CR20","doi-asserted-by":"crossref","first-page":"ra231","DOI":"10.1126\/scitranslmed.aaa0154","volume":"7","author":"SM Schumacher","year":"2015","unstructured":"Schumacher, S. M. et al. Paroxetine-mediated GRK2 inhibition reverses cardiac dysfunction and remodeling after myocardial infarction. Sci. Transl. Med 7, ra231 (2015).","journal-title":"Sci. Transl. Med"},{"key":"9568_CR21","volume":"7","author":"Q Wang","year":"2017","unstructured":"Wang, Q. et al. Paroxetine alleviates T lymphocyte activation and infiltration to joints of collagen-induced arthritis. Sci. Rep. 7, 45364 (2017).","journal-title":"Sci. Rep."},{"key":"9568_CR22","first-page":"2065","volume":"8","author":"J Tang","year":"2015","unstructured":"Tang, J., Dong, J., Yang, L., Gao, L. & Zheng, J. Paroxetine alleviates rat limb post-ischemia induced allodynia through GRK2 upregulation in superior cervical ganglia. Int J. Clin. Exp. Med 8, 2065\u20132076 (2015).","journal-title":"Int J. Clin. Exp. Med"},{"key":"9568_CR23","doi-asserted-by":"crossref","first-page":"1448","DOI":"10.18632\/oncotarget.27562","volume":"11","author":"T Pham","year":"2020","unstructured":"Pham, T., Robinson, K., Vleeshouwer-Neumann, T., Annis, J. E. & Chen, E. Y. Characterization of GRK5 as a novel regulator of rhabdomyosarcoma tumor cell growth and self-renewal. Oncotarget 11, 1448\u20131461 (2020).","journal-title":"Oncotarget"},{"key":"9568_CR24","doi-asserted-by":"crossref","first-page":"620","DOI":"10.1002\/art.41152","volume":"72","author":"T Sueishi","year":"2020","unstructured":"Sueishi, T. et al. GRK5 inhibition attenuates cartilage degradation via decreased NF-kappaB signaling. Arthritis Rheumatol. 72, 620\u2013631 (2020).","journal-title":"Arthritis Rheumatol."},{"key":"9568_CR25","doi-asserted-by":"crossref","DOI":"10.3389\/fcell.2021.719351","volume":"9","author":"GK Adzika","year":"2021","unstructured":"Adzika, G. K. et al. Amlexanox and forskolin prevents isoproterenol-induced cardiomyopathy by subduing cardiomyocyte hypertrophy and maladaptive inflammatory responses. Front Cell Dev. Biol. 9, 719351 (2021).","journal-title":"Front Cell Dev. Biol."},{"key":"9568_CR26","doi-asserted-by":"crossref","first-page":"1830","DOI":"10.1021\/cb3003013","volume":"7","author":"DM Thal","year":"2012","unstructured":"Thal, D. M. et al. Paroxetine is a direct inhibitor of g protein-coupled receptor kinase 2 and increases myocardial contractility. ACS Chem. Biol. 7, 1830\u20131839 (2012).","journal-title":"ACS Chem. Biol."},{"key":"9568_CR27","doi-asserted-by":"publisher","unstructured":"Carlson, E. L. et al. Paroxetine-mediated GRK2 inhibition is a disease-modifying treatment for osteoarthritis. Sci. Transl. Med. 13, https:\/\/doi.org\/10.1126\/scitranslmed.aau8491 (2021).","DOI":"10.1126\/scitranslmed.aau8491"},{"key":"9568_CR28","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1124\/mol.115.098293","volume":"88","author":"JD Lowe","year":"2015","unstructured":"Lowe, J. D. et al. Role of G protein-coupled receptor kinases 2 and 3 in mu-opioid receptor desensitization and internalization. Mol. Pharm. 88, 347\u2013356 (2015).","journal-title":"Mol. Pharm."},{"key":"9568_CR29","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.ejphar.2018.04.022","volume":"831","author":"Q Yu","year":"2018","unstructured":"Yu, Q. et al. Inhibition of prostatic smooth muscle contraction by the inhibitor of G protein-coupled receptor kinase 2\/3, CMPD101. Eur. J. Pharm. 831, 9\u201319 (2018).","journal-title":"Eur. J. Pharm."},{"key":"9568_CR30","doi-asserted-by":"crossref","DOI":"10.1096\/fj.202100704RR","volume":"35","author":"R Agren","year":"2021","unstructured":"Agren, R. & Sahlholm, K. G protein-coupled receptor kinase-2 confers isoform-specific calcium sensitivity to dopamine D(2) receptor desensitization. FASEB J. 35, e22013 (2021).","journal-title":"FASEB J."},{"key":"9568_CR31","doi-asserted-by":"crossref","DOI":"10.1016\/j.bcp.2023.115595","volume":"213","author":"S Michinaga","year":"2023","unstructured":"Michinaga, S., Nagata, A., Ogami, R., Ogawa, Y. & Hishinuma, S. Differential regulation of histamine H(1) receptor-mediated ERK phosphorylation by G(q) proteins and arrestins. Biochem. Pharm. 213, 115595 (2023).","journal-title":"Biochem. Pharm."},{"key":"9568_CR32","doi-asserted-by":"crossref","first-page":"8031","DOI":"10.1523\/JNEUROSCI.0653-18.2018","volume":"38","author":"AD Abraham","year":"2018","unstructured":"Abraham, A. D. et al. Estrogen regulation of GRK2 inactivates kappa opioid receptor signaling mediating analgesia, but not aversion. J. Neurosci. 38, 8031\u20138043 (2018).","journal-title":"J. Neurosci."},{"key":"9568_CR33","doi-asserted-by":"publisher","unstructured":"Pollard, C. M. et al. GRK2-mediated crosstalk between beta-adrenergic and angiotensin II receptors enhances adrenocortical aldosterone production in vitro and in vivo. Int. J. Mol. Sci. 21, https:\/\/doi.org\/10.3390\/ijms21020574 (2020).","DOI":"10.3390\/ijms21020574"},{"key":"9568_CR34","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1124\/mol.119.118661","volume":"97","author":"RA Bouley","year":"2020","unstructured":"Bouley, R. A. et al. A new paroxetine-based GRK2 inhibitor reduces internalization of the mu-opioid receptor. Mol. Pharm. 97, 392\u2013401 (2020).","journal-title":"Mol. Pharm."},{"key":"9568_CR35","volume":"30","author":"G Xu","year":"2020","unstructured":"Xu, G. et al. Hit-to-lead optimization and discovery of a potent, and orally bioavailable G protein coupled receptor kinase 2 (GRK2) inhibitor. Bioorg. Med. Chem. Lett. 30, 127602 (2020).","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"9568_CR36","doi-asserted-by":"crossref","first-page":"566","DOI":"10.1021\/acs.jmedchem.0c01522","volume":"64","author":"RA Rowlands","year":"2021","unstructured":"Rowlands, R. A. et al. Generation of highly selective, potent, and covalent G protein-coupled receptor kinase 5 inhibitors. J. Med Chem. 64, 566\u2013585 (2021).","journal-title":"J. Med Chem."},{"key":"9568_CR37","doi-asserted-by":"crossref","first-page":"11129","DOI":"10.1021\/acs.jmedchem.1c00506","volume":"64","author":"DE Uehling","year":"2021","unstructured":"Uehling, D. E. et al. Design, synthesis, and characterization of 4-aminoquinazolines as potent inhibitors of the G protein-coupled receptor kinase 6 (GRK6) for the treatment of multiple myeloma. J. Med Chem. 64, 11129\u201311147 (2021).","journal-title":"J. Med Chem."},{"key":"9568_CR38","doi-asserted-by":"crossref","first-page":"20577","DOI":"10.1074\/jbc.M513605200","volume":"281","author":"JD Violin","year":"2006","unstructured":"Violin, J. D., Ren, X. R. & Lefkowitz, R. J. G-protein-coupled receptor kinase specificity for beta-arrestin recruitment to the beta2-adrenergic receptor revealed by fluorescence resonance energy transfer. J. Biol. Chem. 281, 20577\u201320588 (2006).","journal-title":"J. Biol. Chem."},{"key":"9568_CR39","doi-asserted-by":"crossref","first-page":"4155","DOI":"10.1021\/bi952961+","volume":"35","author":"L Menard","year":"1996","unstructured":"Menard, L. et al. Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration. Biochemistry 35, 4155\u20134160 (1996).","journal-title":"Biochemistry"},{"key":"9568_CR40","doi-asserted-by":"crossref","first-page":"1206","DOI":"10.1038\/s42003-022-04135-9","volume":"5","author":"J Kaufmann","year":"2022","unstructured":"Kaufmann, J. et al. A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening. Commun. Biol. 5, 1206 (2022).","journal-title":"Commun. Biol."},{"key":"9568_CR41","doi-asserted-by":"crossref","DOI":"10.1038\/s41467-022-28152-8","volume":"13","author":"J Drube","year":"2022","unstructured":"Drube, J. et al. GPCR kinase knockout cells reveal the impact of individual GRKs on arrestin binding and GPCR regulation. Nat. Commun. 13, 540 (2022).","journal-title":"Nat. Commun."},{"key":"9568_CR42","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1161\/01.CIR.87.2.454","volume":"87","author":"M Ungerer","year":"1993","unstructured":"Ungerer, M., Bohm, M., Elce, J. S., Erdmann, E. & Lohse, M. J. Altered expression of beta-adrenergic receptor kinase and beta 1-adrenergic receptors in the failing human heart. Circulation 87, 454\u2013463 (1993).","journal-title":"Circulation"},{"key":"9568_CR43","doi-asserted-by":"crossref","first-page":"7000","DOI":"10.1073\/pnas.95.12.7000","volume":"95","author":"HA Rockman","year":"1998","unstructured":"Rockman, H. A. et al. Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice. Proc. Natl. Acad. Sci. USA 95, 7000\u20137005 (1998).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"9568_CR44","doi-asserted-by":"crossref","first-page":"17223","DOI":"10.1074\/jbc.272.27.17223","volume":"272","author":"DJ Choi","year":"1997","unstructured":"Choi, D. J., Koch, W. J., Hunter, J. J. & Rockman, H. A. Mechanism of beta-adrenergic receptor desensitization in cardiac hypertrophy is increased beta-adrenergic receptor kinase. J. Biol. Chem. 272, 17223\u201317229 (1997).","journal-title":"J. Biol. Chem."},{"key":"9568_CR45","doi-asserted-by":"crossref","first-page":"9954","DOI":"10.1073\/pnas.93.18.9954","volume":"93","author":"HA Rockman","year":"1996","unstructured":"Rockman, H. A. et al. Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice. Proc. Natl. Acad. Sci. USA 93, 9954\u20139959 (1996).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"9568_CR46","doi-asserted-by":"crossref","first-page":"1350","DOI":"10.1126\/science.7761854","volume":"268","author":"WJ Koch","year":"1995","unstructured":"Koch, W. J. et al. Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor. Science 268, 1350\u20131353 (1995).","journal-title":"Science"},{"key":"9568_CR47","doi-asserted-by":"crossref","first-page":"1320","DOI":"10.1016\/S0003-4975(00)01754-9","volume":"71","author":"EP Chen","year":"2001","unstructured":"Chen, E. P., Bittner, H. B., Akhter, S. A., Koch, W. J. & Davis, R. D. Myocardial function in hearts with transgenic overexpression of the G protein-coupled receptor kinase 5. Ann. Thorac. Surg. 71, 1320\u20131324 (2001).","journal-title":"Ann. Thorac. Surg."},{"key":"9568_CR48","doi-asserted-by":"crossref","first-page":"510","DOI":"10.1038\/nm1750","volume":"14","author":"SB Liggett","year":"2008","unstructured":"Liggett, S. B. et al. A GRK5 polymorphism that inhibits beta-adrenergic receptor signaling is protective in heart failure. Nat. Med. 14, 510\u2013517 (2008).","journal-title":"Nat. Med."},{"key":"9568_CR49","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1016\/S1995-7645(13)60027-9","volume":"6","author":"YP Li","year":"2013","unstructured":"Li, Y. P. GRK6 expression in patients with hepatocellular carcinoma. Asian Pac. J. Trop. Med 6, 220\u2013223 (2013).","journal-title":"Asian Pac. J. Trop. Med"},{"key":"9568_CR50","doi-asserted-by":"crossref","first-page":"54227","DOI":"10.18632\/oncotarget.17203","volume":"8","author":"LQ Xu","year":"2017","unstructured":"Xu, L. Q. et al. G protein-coupled receptor kinase 6 is overexpressed in glioma and promotes glioma cell proliferation. Oncotarget 8, 54227\u201354235 (2017).","journal-title":"Oncotarget"},{"key":"9568_CR51","doi-asserted-by":"crossref","first-page":"5329","DOI":"10.4049\/jimmunol.1202058","volume":"190","author":"SK Raghuwanshi","year":"2013","unstructured":"Raghuwanshi, S. K. et al. G protein-coupled receptor kinase 6 deficiency promotes angiogenesis, tumor progression, and metastasis. J. Immunol. 190, 5329\u20135336 (2013).","journal-title":"J. Immunol."},{"key":"9568_CR52","doi-asserted-by":"crossref","DOI":"10.1016\/j.pep.2021.105890","volume":"185","author":"TL Olson","year":"2021","unstructured":"Olson, T. L. et al. Protein expression and purification of G-protein coupled receptor kinase 6 (GRK6), toward structure-based drug design and discovery for multiple myeloma. Protein Expr. Purif. 185, 105890 (2021).","journal-title":"Protein Expr. Purif."},{"key":"9568_CR53","first-page":"4253","volume":"16","author":"Z Zhang","year":"2018","unstructured":"Zhang, Z., Li, Z. & Chen, W. Down-regulated G protein-coupled receptor kinase 6 leads to apoptosis in multiple myeloma MM1R cells. Exp. Ther. Med 16, 4253\u20134259 (2018).","journal-title":"Exp. Ther. Med"},{"key":"9568_CR54","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1021\/cb5006323","volume":"10","author":"KT Homan","year":"2015","unstructured":"Homan, K. T. et al. Identification and structure-function analysis of subfamily selective G protein-coupled receptor kinase inhibitors. ACS Chem. Biol. 10, 310\u2013319 (2015).","journal-title":"ACS Chem. Biol."},{"key":"9568_CR55","doi-asserted-by":"crossref","first-page":"3052","DOI":"10.1021\/acs.jmedchem.7b00112","volume":"60","author":"HV Waldschmidt","year":"2017","unstructured":"Waldschmidt, H. V. et al. Structure-based design of highly selective and potent G protein-coupled receptor kinase 2 inhibitors based on paroxetine. J. Med Chem. 60, 3052\u20133069 (2017).","journal-title":"J. Med Chem."},{"key":"9568_CR56","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1124\/mol.117.110130","volume":"92","author":"R Bouley","year":"2017","unstructured":"Bouley, R. et al. Structural determinants influencing the potency and selectivity of indazole-paroxetine hybrid G protein-coupled receptor kinase 2 inhibitors. Mol. Pharm. 92, 707\u2013717 (2017).","journal-title":"Mol. Pharm."},{"key":"9568_CR57","volume":"264","author":"Y Chen","year":"2024","unstructured":"Chen, Y. et al. Development of a new class of potent and highly selective G protein-coupled receptor kinase 5 inhibitors and structural insight from crystal structures of inhibitor complexes. Eur. J. Med Chem. 264, 115931 (2024).","journal-title":"Eur. J. Med Chem."},{"key":"9568_CR58","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1124\/mol.65.1.196","volume":"65","author":"TM Tran","year":"2004","unstructured":"Tran, T. M. et al. Characterization of agonist stimulation of cAMP-dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the beta2-adrenergic receptor using phosphoserine-specific antibodies. Mol. Pharm. 65, 196\u2013206 (2004).","journal-title":"Mol. Pharm."},{"key":"9568_CR59","volume":"14","author":"J Chen","year":"2024","unstructured":"Chen, J. et al. Astragaloside IV protects brain cells from ischemia-reperfusion injury by inhibiting ryanodine receptor expression and reducing the expression of P-Src and P-GRK2. Sci. Rep. 14, 17497 (2024).","journal-title":"Sci. Rep."},{"key":"9568_CR60","doi-asserted-by":"crossref","first-page":"20649","DOI":"10.1074\/jbc.M115.647370","volume":"290","author":"KT Homan","year":"2015","unstructured":"Homan, K. T. et al. Crystal structure of G protein-coupled receptor kinase 5 in complex with a rationally designed inhibitor. J. Biol. Chem. 290, 20649\u201320659 (2015).","journal-title":"J. Biol. Chem."},{"key":"9568_CR61","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1042\/CS20191032","volume":"134","author":"CC Han","year":"2020","unstructured":"Han, C. C. et al. CP-25 inhibits PGE2-induced angiogenesis by down-regulating EP4\/AC\/cAMP\/PKA-mediated GRK2 translocation. Clin. Sci. (Lond.) 134, 331\u2013347 (2020).","journal-title":"Clin. Sci. (Lond.)"},{"key":"9568_CR62","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1016\/j.canlet.2022.01.004","volume":"529","author":"H Wang","year":"2022","unstructured":"Wang, H. et al. Decreased CXCR2 expression on circulating monocytes of colorectal cancer impairs recruitment and induces Re-education of tumor-associated macrophages. Cancer Lett. 529, 112\u2013125 (2022).","journal-title":"Cancer Lett."},{"key":"9568_CR63","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1038\/s42003-020-0752-4","volume":"3","author":"M Patrone","year":"2020","unstructured":"Patrone, M. et al. Combinatorial allosteric modulation of agonist response in a self-interacting G-protein coupled receptor. Commun. Biol. 3, 27 (2020).","journal-title":"Commun. Biol."},{"key":"9568_CR64","doi-asserted-by":"crossref","first-page":"6195","DOI":"10.1007\/s12035-024-04683-7","volume":"62","author":"M Yan","year":"2025","unstructured":"Yan, M. et al. Microcystin-LR exposure damages neurons by inducing alpha-syn aggregation via MAPK4\/GATA2\/SNCA and PP2A\/GRKs pathways. Mol. Neurobiol. 62, 6195\u20136211 (2025).","journal-title":"Mol. Neurobiol."},{"key":"9568_CR65","doi-asserted-by":"publisher","DOI":"10.1038\/s41366-025-01712-w","author":"ME Seramur","year":"2025","unstructured":"Seramur, M. E. et al. GRK5 is required for adipocyte differentiation through ERK activation. Int J. Obes. (Lond.) https:\/\/doi.org\/10.1038\/s41366-025-01712-w (2025).","journal-title":"Int J. Obes. (Lond.)"},{"key":"9568_CR66","doi-asserted-by":"crossref","first-page":"482","DOI":"10.4062\/biomolther.2020.129","volume":"28","author":"JH Lee","year":"2020","unstructured":"Lee, J. H. et al. KR-39038, a novel GRK5 inhibitor, attenuates cardiac hypertrophy and improves cardiac function in heart failure. Biomol. Ther. (Seoul.) 28, 482\u2013489 (2020).","journal-title":"Biomol. Ther. (Seoul.)"},{"key":"9568_CR67","doi-asserted-by":"crossref","first-page":"933","DOI":"10.1038\/s42003-024-06571-1","volume":"7","author":"O Underwood","year":"2024","unstructured":"Underwood, O. et al. Key phosphorylation sites for robust beta-arrestin2 binding at the MOR revisited. Commun. Biol. 7, 933 (2024).","journal-title":"Commun. Biol."},{"key":"9568_CR68","doi-asserted-by":"crossref","first-page":"7157","DOI":"10.1073\/pnas.95.12.7157","volume":"95","author":"J Zhang","year":"1998","unstructured":"Zhang, J. et al. Role for G protein-coupled receptor kinase in agonist-specific regulation of mu-opioid receptor responsiveness. Proc. Natl. Acad. Sci. USA 95, 7157\u20137162 (1998).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"9568_CR69","doi-asserted-by":"crossref","DOI":"10.1016\/j.jbc.2023.105293","volume":"299","author":"V Burghi","year":"2023","unstructured":"Burghi, V. et al. Galphas is dispensable for beta-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by beta2-adrenergic receptor. J. Biol. Chem. 299, 105293 (2023).","journal-title":"J. Biol. Chem."},{"key":"9568_CR70","doi-asserted-by":"crossref","first-page":"1448","DOI":"10.1073\/pnas.0409534102","volume":"102","author":"XR Ren","year":"2005","unstructured":"Ren, X. R. et al. Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor. Proc. Natl. Acad. Sci. USA 102, 1448\u20131453 (2005).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"9568_CR71","doi-asserted-by":"crossref","first-page":"5767","DOI":"10.1074\/jbc.M110647200","volume":"277","author":"KR Watterson","year":"2002","unstructured":"Watterson, K. R. et al. Dual regulation of EDG1\/S1P(1) receptor phosphorylation and internalization by protein kinase C and G-protein-coupled receptor kinase 2. J. Biol. Chem. 277, 5767\u20135777 (2002).","journal-title":"J. Biol. Chem."},{"key":"9568_CR72","doi-asserted-by":"crossref","first-page":"1898","DOI":"10.1126\/science.1208248","volume":"333","author":"TI Arnon","year":"2011","unstructured":"Arnon, T. I. et al. GRK2-dependent S1PR1 desensitization is required for lymphocytes to overcome their attraction to blood. Science 333, 1898\u20131903 (2011).","journal-title":"Science"},{"key":"9568_CR73","volume":"16","author":"ESF Matthees","year":"2025","unstructured":"Matthees, E. S. F. et al. Helix-bundle and C-terminal GPCR domains differentially influence GRK-specific functions and beta-arrestin-mediated regulation. Nat. Commun. 16, 5430 (2025).","journal-title":"Nat. Commun."},{"key":"9568_CR74","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","volume":"9","author":"J Schindelin","year":"2012","unstructured":"Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676\u2013682 (2012).","journal-title":"Nat. Methods"}],"container-title":["Communications Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s42003-026-09568-0","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s42003-026-09568-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s42003-026-09568-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T01:05:02Z","timestamp":1771549502000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s42003-026-09568-0"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,16]]},"references-count":74,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,12]]}},"alternative-id":["9568"],"URL":"https:\/\/doi.org\/10.1038\/s42003-026-09568-0","relation":{},"ISSN":["2399-3642"],"issn-type":[{"value":"2399-3642","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,16]]},"assertion":[{"value":"10 July 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"11 January 2026","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"16 January 2026","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"S.S. is the founder and scientific advisor of 7TM Antibodies GmbH, Jena, Germany. F.N. is an employee of 7TM Antibodies. All other authors declare no competing interests. An invention disclosure related to the assay described in this work has been filed at the University Hospital Jena.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"287"}}