Investigating molecular details of the µ-opioid receptor complex with arrestin using genetically encoded crosslinkers in living cells

• Applicant: Professorin Dr. Irene Coin
• Subject Area: Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 568338957

Opioid drugs targeting the Gi-coupled μ-opioid receptor (MOR) are the most important analgesics for management of severe pain. However, they induce tolerance and addiction and can have life-threatening side effects. It is possible that these effects are related to the interaction of the MOR with arrestin, which mediates its internalization and is therefore involved in the process of desensitization. It is known that agonists of varying efficacy lead to the recruitment of different GPCR kinases (GRKs) to the MOR, resulting in varying degrees of arrestin recruitment. However, high-resolution structures of the MOR bound to either GRK or arrestin are lacking, and whether the MOR-arrestin complexes induced by pharmacologically distinct agonists exhibit structural differences is still unknown. The interaction between MOR and arrestin is transient, hampers its structural characterization. We have developed a novel method to elucidate the structural details of GPCR-arrestin complexes directly in living cells. This method is based on the systematic genetic incorporation of non-canonical amino acids for photo- and chemical crosslinking across the protein-protein interaction surface, combined with molecular modeling and MD simulations. Using this approach, we recently characterized the PTH1R-arrestin2 complex in high detail, which included information about flexible parts of the complex that are not accessible to conventional methods. Importantly, we have demonstrated that this method is applicable to both long-lived and transient complexes, particularly MOR-arrestin complexes (preliminary work). In this project, we aim to elucidate the interactions between MOR, arrestins, and GRKs, and uncover the functional implications of specific structural elements, with the ultimate goal of advancing our understanding of MOR signaling regulation and our ability to manipulate it. We will apply our crosslinking platform to characterize MOR-arrestin complexes induced by both high-efficacy (e.g., DAMGO) and low-efficiency agonists (e.g., Morphine). We will integrate the experimental data into energy-based molecular modeling to build accurate models of the MOR-arrestin complex. We will analyze molecular interactions in the context of existing literature and investigate selected elements using complementary methods (e.g. structure-activity relationship studies, manipulation of phosphorylation states). Moreover, we will examine whether and how different GRKs induce the formation of structurally distinct complexes. Eventually, we aim to extend our crosslinking approach to investigate interactions between GRKs and both MOR and other GPCRs. This project targets a molecular interaction of extremely high biomedical relevance, with profound implications for society (Opioid epidemics). Our experimental strategy can provide insights directly from the transient complex formed in the cellular environment, which is not accessible to reductionist approaches.

• DFG Programme: Research Grants