دورية أكاديمية
Rapid elucidation of agonist-driven regulation of the neurokinin 1 receptor using a GPCR phosphorylation immunoassay.
| العنوان: | Rapid elucidation of agonist-driven regulation of the neurokinin 1 receptor using a GPCR phosphorylation immunoassay. |
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| المؤلفون: | Blum NK; Institut für Pharmakologie und Toxikologie, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Drackendorfer Str. 1, D-07747, Jena, Germany., Schaffner A; Institut für Pharmakologie und Toxikologie, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Drackendorfer Str. 1, D-07747, Jena, Germany., Drube J; Institut für Molekulare Zellbiologie, CMB - Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Hans-Knöll-Str. 2, D-07745, Jena, Germany., Nagel F; 7TM Antibodies GmbH, Hans-Knöll-Str. 6, D-07745, Jena, Germany., Reinscheid RK; Institut für Pharmakologie und Toxikologie, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Drackendorfer Str. 1, D-07747, Jena, Germany., Hoffmann C; Institut für Molekulare Zellbiologie, CMB - Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Hans-Knöll-Str. 2, D-07745, Jena, Germany., Schulz S; Institut für Pharmakologie und Toxikologie, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Drackendorfer Str. 1, D-07747, Jena, Germany; 7TM Antibodies GmbH, Hans-Knöll-Str. 6, D-07745, Jena, Germany. Electronic address: stefan.schulz@med.uni-jena.de. |
| المصدر: | European journal of pharmacology [Eur J Pharmacol] 2024 Jun 15; Vol. 973, pp. 176587. Date of Electronic Publication: 2024 Apr 19. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Elsevier Science Country of Publication: Netherlands NLM ID: 1254354 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1879-0712 (Electronic) Linking ISSN: 00142999 NLM ISO Abbreviation: Eur J Pharmacol Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: 2005- : Amsterdam : Elsevier Science Original Publication: Amsterdam, North Holland Pub. Co. |
| مواضيع طبية MeSH: | Receptors, Neurokinin-1*/metabolism , Receptors, Neurokinin-1*/agonists , Substance P*/pharmacology, Phosphorylation/drug effects ; Humans ; Animals ; Immunoassay/methods ; Cricetulus ; CHO Cells ; Mice ; Neurokinin-1 Receptor Antagonists/pharmacology ; Neurokinin A/pharmacology ; Neurokinin A/metabolism |
| مستخلص: | Agonist-induced phosphorylation is a crucial step in the activation/deactivation cycle of G protein-coupled receptors (GPCRs), but direct determination of individual phosphorylation events has remained a major challenge. We have recently developed a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in 96-well plates, thus eliminating the need for western blot analysis. In the present study, we adapted this assay to three novel phosphosite-specific antibodies directed against the neurokinin 1 (NK1) receptor, namely pS338/pT339-NK1, pT344/pS347-NK1, and pT356/pT357-NK1. We found that substance P (SP) stimulated concentration-dependent phosphorylation of all three sites, which could be completely blocked in the presence of the NK1 receptor antagonist aprepitant. The other two endogenous ligands of the tachykinin family, neurokinin A (NKA) and neurokinin B (NKB), were also able to induce NK1 receptor phosphorylation, but to a much lesser extent than substance P. Interestingly, substance P promoted phosphorylation of the two distal sites more efficiently than that of the proximal site. The proximal site was identified as a substrate for phosphorylation by protein kinase C. Analysis of GPCR kinase (GRK)-knockout cells revealed that phosphorylation was mediated by all four GRK isoforms to similar extents at the T344/S347 and the T356/T357 cluster. Knockout of all GRKs resulted in abolition of all phosphorylation signals highlighting the importance of these kinases in agonist-mediated receptor phosphorylation. Thus, the 7TM phosphorylation assay technology allows for rapid and detailed analyses of GPCR phosphorylation. Competing Interests: Declaration of competing interest Stefan Schulz is the founder and scientific advisor of 7TM Antibodies GmbH, Jena, Germany. Falko Nagel is an employee of 7TM Antibodies GmbH. All other authors declare no competing interests. (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.) |
| فهرسة مساهمة: | Keywords: Bead-based immunoassay; GPCR phosphorylation; Neurokinin 1 receptor |
| المشرفين على المادة: | 0 (Receptors, Neurokinin-1) 33507-63-0 (Substance P) 0 (Neurokinin-1 Receptor Antagonists) 86933-74-6 (Neurokinin A) |
| تواريخ الأحداث: | Date Created: 20240420 Date Completed: 20240506 Latest Revision: 20240524 |
| رمز التحديث: | 20240524 |
| DOI: | 10.1016/j.ejphar.2024.176587 |
| PMID: | 38642667 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1879-0712 |
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| DOI: | 10.1016/j.ejphar.2024.176587 |