Jessica Ullrich, Philip Jonas Göhmann, Anne Zemella, Stefan Kubick

• Understanding the assembly mechanism and function of membrane proteins is a fundamental problem in biochemical research. Among the membrane proteins, G protein-coupled receptors (GPCRs) represent the largest class in the human body and have long been considered to function as monomers. Nowadays, the oligomeric assembly of GPCRs is widely accepted, although the functional importance and therapeutic intervention remain largely unexplored. This is partly due to difficulties in the heterologous production of membrane proteins. Cell-free protein synthesis (CFPS) with its endogenous endoplasmic reticulum-derived structures has proven as a technique to address this issue. In this study, we investigate for the first time the conceptual CFPS of a heteromeric GPCR, the gamma-aminobutyric acid receptor type B (GABA(B)), from its protomers BR1 and BR2 using a eukaryotic cell-free lysate. Using a fluorescence-based proximity ligation assay, we provide evidence for colocalization and thus suggesting heterodimerization. We prove theUnderstanding the assembly mechanism and function of membrane proteins is a fundamental problem in biochemical research. Among the membrane proteins, G protein-coupled receptors (GPCRs) represent the largest class in the human body and have long been considered to function as monomers. Nowadays, the oligomeric assembly of GPCRs is widely accepted, although the functional importance and therapeutic intervention remain largely unexplored. This is partly due to difficulties in the heterologous production of membrane proteins. Cell-free protein synthesis (CFPS) with its endogenous endoplasmic reticulum-derived structures has proven as a technique to address this issue. In this study, we investigate for the first time the conceptual CFPS of a heteromeric GPCR, the gamma-aminobutyric acid receptor type B (GABA(B)), from its protomers BR1 and BR2 using a eukaryotic cell-free lysate. Using a fluorescence-based proximity ligation assay, we provide evidence for colocalization and thus suggesting heterodimerization. We prove the heterodimeric assembly by a bioluminescence resonance energy transfer saturation assay providing the manufacturability of a heterodimeric GPCR by CFPS. Additionally, we show the binding of a fluorescent orthosteric antagonist, demonstrating the feasibility of combining the CFPS of GPCRs with pharmacological applications. These results provide a simple and powerful experimental platform for the synthesis of heteromeric GPCRs and open new perspectives for the modelling of protein-protein interactions. Accordingly, the presented technology enables the targeting of protein assemblies as a new interface for pharmacological intervention in disease-relevant dimers.…