Multipronged Approach to Investigate the Mu‐Delta Opioid Receptor Heteromer Interface
Authors: Alongkronrusmee Doungkamol , Ghomi Hamed T. , Tang Shiqi , Lill Markus A. , van Rijn Richard M. 
Published: November 2023

Opioid receptors are originally classified into four major subtypes including mu (MOR), delta (DOR) and kappa, with nociceptin receptor being the least characterized. Previous studies have shown that opioid receptors can interact with other members of their receptor family to form multimeric complexes, so‐called heteromers. Substantial evidence has revealed that the heteromeric complexes display unique functions relative to homomeric complexes. For example, the heteromerization of MOR and DOR may contribute to negative side effects of opioid‐mediated analgesia. The disruption of heteromeric complexes may therefore be useful to lessen the side effects of long‐term opioid use. Yet, there is still a lack of tools to selectively investigate the role of MOR‐DOR heteromers in vivo, stifling our full understanding of the therapeutic potential of these MOR‐DOR heteromers. Our ultimate goal is to develop compounds that can either stabilize or disrupt the MOR‐DOR heteromers, as those types of compounds will be invaluable in studying MOR‐DOR pharmacology in vivo. Here, we present data detailing the first step in this process: Determining which amino acids in the MOR‐DOR heteromer interface are important in forming and maintaining the heteromers.We used a multipronged approach including heteromer‐selective functional assay, biomolecular fluorescence complementation (BiFC), co‐immunoprecipitation (Co‐IP) and homology modeling to investigate the impact of single or double amino acid substitutions on the stability and function of the MOR‐DOR heteromers. Based on previously published studies, we focused our mutagenesis efforts on transmembrane domains five and six (TM5 and TM6). We created nearly thirty mutants spanning TM5 and TM6 of the DOR. The heteromer‐selective functional assay which we created by fusing a chimeric Gqi protein to a truncated DOR and co‐expressing it with a wild‐type MOR (van Rijn et al., JPET 2013) revealed that single mutations at amino acid positions 208, 209, 222, 260, 271, 279 or 288 of the DOR inhibited MOR‐DOR heteromer function. Using the BiFC assay, for which we fused MOR and DOR to complementary fragments of a fluorescent protein, we confirmed the importance of amino acid positions 222, 242, 243, 244 and 288. The obtained results were used to optimize a computational model of the MOR‐DOR heteromers, created using the available crystal structures. The computational model was used to predict additional amino acids that could be important in the formation of the MOR‐DOR heteromers. Using this strategy, we further identified amino acid positions 239, 241 and 253 to be involved in the heteromer interface stability. We will continue to validate these results by creating reciprocal mutations in the MOR. Eventually, we aim to use the computational model to design compounds that can prevent or disrupt the heteromer formation, which can be used to probe the physiological role of the MOR‐DOR heteromers.Support or Funding InformationSupport by the Ralph W. and Grace M. Showalter Research Trust and a NIH Pathway to Independent Award from the National Institute for Alcohol Abuse and Alcoholism (NIAA, AA020359). None of the authors has a conflict of interest related to this research.