DIFFERENTIAL ACTIVATION OF G-PROTEIN SUBTYPES BY DOPAMINE D1 RECEPTOR ALLOSTERIC MODULATION

Allosteric modulation of G protein-coupled receptors (GPCR) is a rapidly advancing area of drug discovery with considerable therapeutic potential. In this study, we used various bioluminescence resonance energy transfer (BRET) assay configurations to investigate the differential activity of LY3154207, a dopamine D1 receptor (D1R)-positive allosteric modulator (PAM), in potentiating D1R-mediated signaling pathways. Our data showed that LY3154207 demonstrated clear bias toward β-arrestin recruitment over G-protein engagement. Among the G-protein subtypes, LY3154207 preferentially potentiated Gα _s signaling induced by DA. Interestingly, when tested for its effect on the Gα _s -selective D1R agonist DHX and the Gα _olf -selective agonist TAV, LY3154207 further enhanced the selectivity of these compounds, suggesting a probe-dependent effect. We also studied the structural mechanism underlying this subtype selectivity and found that W123 and R130 in the intracellular loop 2 (ICL2) of D1R were crucial for the activity of LY3154207. Furthermore, the αN domain of the G-protein appeared to make minor contributions to this G-protein subtype selectivity of LY3154207. Lastly, our data demonstrated that LY3154207 induced cAMP production in the absence of the orthosteric ligand, indicating that this compound is an ago-PAM.