Journal Article

Direct GPCR-EGFR interaction enables synergistic membrane-to-nucleus information transfer

Robaa, Dina, Dubourg, Virginie, Mildenberger, Sigrid, Eckenstaler, Robert, Benndorf, Ralf, Braun, Heike, Olgac, Abdurrahman, Schreier, Barbara, Sippl, Wolfgang, Gekle, Michael

2024

Overview

We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or –mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.

• Publisher: Springer International Publishing,Springer Nature B.V
• Subject: Angiotensin AT1 receptors / Angiotensin II / Angiotensin II - metabolism / Angiotensin II - pharmacology / Biochemistry / Biomedical and Life Sciences / Biomedicine / blood serum / Cell Biology / Cell Membrane - metabolism / Cell morphology / Cell Nucleus - metabolism / cell structures / computer simulation / Cytology / Energy transfer / Epidermal growth factor / Epidermal Growth Factor - metabolism / Epidermal growth factor receptors / ErbB Receptors - metabolism / Extracellular signal-regulated kinase / Fluorescence Resonance Energy Transfer / G protein-coupled receptors / G-proteins / Gene expression / Growth factors / HEK293 Cells / Humans / Immunoprecipitation / information exchange / Information transfer / Kinases / Life Sciences / Ligands / Microscopy / Mutants / Original / Original Article / Phosphorylation / precipitin tests / Protein Binding / protein synthesis / Proteins / Receptor, Angiotensin, Type 1 - genetics / Receptor, Angiotensin, Type 1 - metabolism / Receptors / Receptors, G-Protein-Coupled - genetics / Receptors, G-Protein-Coupled - metabolism / Serum response factor / Serum Response Factor - genetics / Serum Response Factor - metabolism / Synergism / transcriptional activation / Tyrosine