μ-Opioid receptors negatively regulate striatal dopamine D₁ receptor signaling through heteromerization

Integrating biophysical, computational, in situ, and in vivo approaches with selective heteromer-disrupting transmembrane peptides, this study demonstrates that μ-opioid receptors (MORs) and dopamine D₁ receptors (D₁Rs) assemble into functional heteromers. Furthermore, real-time in vivo fiber photometry using a genetically encoded cAMP biosensor directly demonstrates that MORs regulate striatal D₁Rs via heteromerization in the intact mouse brain. Our findings support a tetrameric structure composed of Gi-coupled MOR and Gs-coupled D₁R homodimers, which enables both canonical Gi-Gs antagonistic interaction at the level of adenylyl cyclase and receptor-level cross-antagonism, by which MOR agonists and antagonists counteract D₁R agonist-induced signaling. Located predominantly within striosomal GABAergic projection neurons, MOR-D₁R heteromers constitute mechanistically defined signaling units that integrate dopaminergic and opioid neurotransmission governing motor control and reward processing. Collectively, these findings establish MOR-D₁R heteromers as a previously unrecognized therapeutic target for the treatment of opioid withdrawal-associated restlessness and restless legs syndrome.