Physician-scientist Ying-Xian Pan studies the mechanisms of opioid actions, providing the foundation of developing novel drugs for pain treatment.
The mu opioid receptors mediate the actions of morphine and most clinical analgesics, as well as drugs of abuse such as heroin. The goal of our research is to understand the mechanisms and functions of mu opioid receptor gene, OPRM1, gain insights into the pharmacological and physiological significance of the mechanisms and functions, and provide potential targets for developing novel drugs in control of pain and drug of abuse.
Most clinically used opioid analgesics, including morphine and fentanyl, as well as heroin, act primarily through mu opioid receptors generated from the single-copy mu opioid receptor gene (OPRM1). OPRM1 gene undergoes extensive alternative splicing, creating multiple splice variants that can be categorized into three major types based on the receptor structure: 1) Full-length 7 transmembrane (TM) C-terminal variants that are identical except for a different intracellular C-terminal tail due to 3’ splicing; 2) Truncated 6TM variants that lack the first TM due to 5’ splicing; and 3) Truncated single TM variants containing only the first TM due to exon skipping or insertion. We have demonstrated in vivo functions of several C-terminal splice variants using gene targeting mouse models. Different C-terminal sequences exhibited divergent effects on mu opioid actions, such as tolerance, physical dependence and reward. These divergent effects were mediated through different signaling mechanisms including G protein and β-arrestin2. Using our Oprm1 knockout mouse models and lentiviral rescue studies, we have showed that the truncated 6TM variants are essential for the analgesic action of a novel class of analgesics, such as 3’-iodobenzoyl-6β-naltrexamide (IBNtxA) that are potent against a broad spectrum of pain models without many side-effects associated with traditional opiates. Although unable to bind opioids, the truncated single TM variants can facilitate expression of the 7TM MOR-1 receptor as a molecular chaperone to enhance morphine analgesia. Our current research is mainly focused on the following areas:
Mechanisms and functions of Oprm1 alternative splicing: We are investigating region-specific and cell-specific regulation of alternative splicing and determining the role of alternative splicing in opioid responses. We are using several in vitro and in vivo techniques, such as mini-gene constructs, in vitro RNA splicing, RNA affinity purification and proteomic approaches, to identify potential cis-acting elements and trans-acting factors that involve region-specific splicing. We are also exploring functional single nucleotide polymorphisms (SNPs) of OPRM1 gene that can modulate alternative splicing.
In vivo function of Oprm1 splice variants in rat: Although mouse models are valuable, rats have many advantages both in behavioral modeling and in vivo manipulation. Also, some opioid responses in rat are different from those in mouse. We have generated two Oprm1 conditional rat models: one targeting exon 1 and the other exon 11. These rat models provide useful tools for further investigating region-specific or cell-specific functions of the rat Oprm1 variants.
Pharmacological functions of individual Oprm1 splice variants: Using a novel CRISPR/Cas9 approach, we are generating several gene targeting mouse models in which each individual variant is selectively expressed. These mouse models will be very useful to further explore in vivo function of individual variants.
Mu agonist-induced receptor-protein interactions: Protein-protein interactions (PPIs) or receptor–protein interactions (RPIs) are at the core of all cellular functions and dynamically change in response to intrinsic and extrinsic stimuli that regulate cellular signaling. Mu agonist-induced receptor signaling and function involves interactions of the receptor, particularly intracellular C-terminal tails, with multiple proteins in various pathways at different subcellular locations. We have used newly developed proximity-dependent biotin identification with an engineered ascorbate peroxidase (APEX2) coupled with tandem mass tag (TMT) proteomics approach (APEX2-TMT), to unbiasedly map these transient or dynamic RPIs under both basal state and activated conditions in response to different mu agonists.
We are also investigating biased signaling at different Oprm1 full-length 7TM variants and exploring molecular mechanisms of the truncated 6TM variants.