Abstract
<jats:p>G protein-coupled receptors (GPCRs) signal across a continuum of activation states, yet how ligands encode distinct signaling efficacies remains poorly understood. Here, we define the molecular mechanism underlying graded activation of the dopamine D1 receptor (D1R), a major therapeutic target for neuropsychiatric disorders. Functional analyses reveal that two widely used pharmacological tools, LE300 and SCH23390, possess intrinsic efficacy as an inverse agonist and a weak partial agonist, respectively, rather than the efficacy-silent neutral antagonists. Structural, molecular dynamics and mutagenesis analyses capture previously unrecognized inactive and intermediate receptor activation states that bridge known active conformations, and reveal that ligand efficacy is encoded through the progressive engagement of a conserved activation pathway centered on the W6.48 toggle switch. Guided by this mechanism, a single chemical modification markedly increases the agonist efficacy of SCH23390. Comparison with dopamine D2 receptor structures further reveals a conserved mechanism of inverse agonism despite distinct subtype-specific recognition. Together, these findings establish a structural framework for graded agonism at D1R and provide general principles for the rational design of efficacy-tuned therapeutics at dopamine receptors and related GPCRs.</jats:p>