Fig. 2: Distinct binding mode of fenoldopam in D1R.

a Surface view of the fenoldopam-binding pocket. EM density map for fenoldopam is shown. b Detailed interactions between the two fenoldopam molecules and the receptor. Dashed lines represent hydrogen bonds. c cAMP accumulation assay for D1R wild-type (WT) and mutants activated by fenoldopam. The assay was performed in HEK293 cells stably expressing the cAMP GloSensor. EC50 values are determined from three independent experiments. Data are presented as mean values ± SEM. Statistical significances of D1R mutants were obtained using two-tailed Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). ND not determined, ns no significant difference. The exact p values are as follows, S198G, *p = 0.0104; S202A, *p = 0.0265; N292L, ***p = 0.0004; F288A, **p = 0.0042; F289A, **p = 0.0048; W321F, **p = 0.002; K81V, **p = 0.0059; D314S, p = 0.9475; S188I, p = 0.2013; L190A, ****p = 0.00003. d NanoBiT mini-Gα_s recruitment assay for D1R WT and mutants. Data are presented as mean values ± SEM from three independent experiments. p values were obtained by two-tailed Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). The exact p values are as follows, S198G, ***p = 0.0005; S202A, p = 0.0539; N292L, ***p = 0.001; F288A, **p = 0.0048; F289A, **p = 0.0052; W321F, *p = 0.042; K81V, **p = 0.0047; L190A, ***p = 0.0004. e Structural comparison of the fenoldopam-bound D1R complex between this study and previous studies (PDB ID: 7CKW) with the receptor aligned. f Close-up views of the conformational differences of the fenoldopam-binding pocket between different studies. Concentration-response curve for D1R WT and the K81V mutant activated by fenoldopam measured using cAMP accumulation assay (g), NanoBiT (h) and TANGO (i) β-arrestin recruitment assay. All data represent mean ± SEM from three independent experiments. Response of the cAMP assay is expressed as percentage of maximal response of WT. Source data are provided as a Source Date file.