Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses. Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

G protein-coupled receptors (GPCRs) regulate processes ranging from immune responses to neuronal signaling. However, ligands for many GPCRs remain unknown, suffer from off-target effects or have poor bioavailability. Additionally, dissecting cell type-specific responses is challenging when the same GPCR is expressed on different cells within a tissue. Here, we overcome these limitations by engineering DREADD-based GPCR chimeras that bind their agonist clozapine-N-oxide (CNO) and mimic a GPCR-of-interest. We show that the chimeric DREADD-β2AR triggers responses comparable to levalbuterol-stimulated β2AR on second messenger and kinase activity, post-translational modifications, and protein-protein interactions. Moreover, we successfully recapitulate β2AR-mediated filopodia formation in primary microglia, an immune cell capable of driving central nervous system inflammation. When dissecting microglial inflammation, we utilized the microglia-like HMC3 cell line and included two additionally designed DREADD-based chimeras mimicking GPR65 and GPR109A. DREADD-β2AR and DREADD-GPR65 modulate the inflammatory response with high similarity to endogenous β2AR, while DREADD-GPR109A had no impact. Our DREADD-based approach allows investigation of cell type-dependent pathways without known endogenous ligands. Overall design: We used lentiviral transduction and generated HMC3 cell lines with stable expression of DREADD-β2AR, DREADD-GPR65, or DREADD-GPR109A, respectively. Non-transduced cells represent unaltered (wild type) HMC3 cells, which endogenously express β2AR that can be stimulated with levalbuterol (LB). We induced inflammation in HMC3 cells through simultaneous challenge with the recombinant cytokines IFNγ and IL1β. Concomitantly, we applied levalbuterol or CNO to study how GPCR signaling can modulate inflammatory gene expression. In total, we sequenced 30 samples with three replicates for each of the following 10 experimental conditions: Non-transduced: Untreated; Non-transduced: IFNγ/IL1β; Non-transduced: IFNγ/IL1β+LB; Non-transduced: IFNγ/IL1β+CNO; DREADD-β2AR: IFNγ/IL1β; DREADD-β2AR: IFNγ/IL1β+CNO; DREADD-GPR65: IFNγ/IL1β; DREADD-GPR65: IFNγ/IL1β+CNO; DREADD-GPR109A: IFNγ/IL1β; DREADD-GPR109A: IFNγ/IL1β+CNO.