Development of medium- to high-throughput GPCR phosphorylation assays for secondary screening of ligands and GRK inhibitors

• Applicants: Professor Dr. Carsten Hoffmann; Professor Dr. Stefan Schulz
• Subject Area: Pharmacology ; Pharmacy
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 569850315

G protein-coupled receptors (GPCRs) are important drug targets for many human diseases. Activation by agonists leads to conformational changes, which are recognized by a family of GPCR kinases (GRKs). The unique ability of GRKs to recognize activated receptors leads to agonist-dependent phosphorylation at intracellular serine and threonine residues. We have recently developed a quantitative GPCR phosphorylation assay that can be performed entirely in a multi-well format, eliminating the need for Western blot analysis. This assay is based on immunoprecipitation of affinity-tagged receptors using magnetic beads and subsequent protein detection with phosphorylation state-specific GPCR antibodies. We have also succeeded in generating eleven combinatorial HEK293 knockout cell clones lacking GRK2/3/5/6, including a single, double, triple and quadruple GRK knockout. One obvious limitation of our novel assay is that phosphosite-specific antibodies are not available for all GPCRs yet. 7TM Antibodies GmbH was founded specifically to fill this gap. Thus, 7TM Antibodies GmbH is the ideal Application Partner for this project. In this project, we will form a consortium to which Academic Partner 1 will contribute novel GPCR phosphorylation assay technologies, Academic Partner 2 will contribute a combinatorial library of GPCR kinase and phosphatase knockout cells, and the Application Partner will contribute a broad range of validated phosphosite-specific GPCR antibodies. Only this unique combination of partners will allow us to develop exemplary assay applications that will facilitate the identification of kinases and phosphatases involved in the phosphorylation and dephosphorylation of GPCRs. It will also facilitate the development of prototype assays for ligand profiling, screening of GRK inhibitors, and analysis of orphan GPCRs. Specifically, the consortium aims to (1) elucidate GRK specificity of GPCR phosphorylation, (2) develop selective cellular inhibitor assays for individual GRKs, (3) elucidate GPCR dephosphorylation by creating CRISPR/Cas9 protein phosphatase knockout cells, (4) develop GPCR phosphorylation assays for atypical 7TM receptors, (5) develop GPCR phosphorylation assays for orphan 7TM receptors, and (6) develop homogeneous 2nd generation NanoBRET-based GPCR phosphorylation assays. The work described here will not only facilitate knowledge transfer but also help to answer fundamental questions in basic GPCR research. However, for broad application in academic and pharmaceutical research, it is necessary to scale up this novel technology to the 384-well format and to produce monoclonal GPCR antibodies. It is anticipated that these methodological advances will be achieved by the application partner upon completion of this project.

• DFG Programme: Research Grants (Transfer Project)
• Application Partner: 7TM Antibodies GmbH