Optimized genetic code expansion technology for time‐dependent induction of adhesion GPCR‐ligand engagement

The introduction of an engineered aminoacyl–tRNA synthetase/tRNA pair enables site‐specific incorporation of unnatural amino acids (uAAs) with functionalized side chains into proteins of interest. Genetic Code Expansion (GCE) via amber codon suppression confers functionalities to proteins but can also be used to temporally control the incorporation of genetically encoded elements into proteins. Here, we report an optimized GCE system (GCEXpress) for efficient and fast uAA incorporation. We demonstrate that GCEXpress can be used to efficiently alter the subcellular localization of proteins within living cells. We show that click labeling can resolve co‐labeling problems of intercellular adhesive protein complexes. We apply this strategy to study the adhesion G protein‐coupled receptor (aGPCR) ADGRE5/CD97 and its ligand CD55/DAF that play central roles in immune functions and oncological processes. Furthermore, we use GCEXpress to analyze the time course of ADGRE5‐CD55 ligation and replenishment of mature receptor‐ligand complexes. Supported by fluorescence recovery after photobleaching (FRAP) experiments our results show that ADGRE5 and CD55 form stable intercellular contacts that may support transmission of mechanical forces onto ADGRE5 in a ligand‐dependent manner. We conclude that GCE in combination with biophysical measurements can be a useful approach to analyze the adhesive, mechanical and signaling properties of aGPCRs and their ligand interactions.