Figure 1. Rapid agonist-stimulated D₁ receptor endocytosis and real-time examination of receptor-mediated regulation of cellular cAMP.

(A) Agonist-induced effects on surface receptor number as analyzed by fluorescence flow cytometry. Surface fluorescence of labeled FD1Rs measured at time 0 was defined as 100%. Data = mean fluorescence values +/− SEM, n = 3, 10,000 cells/condition, each time point in duplicate. (B) Representative images of SpH-D1R surface fluorescence visualized by TIRF microscopy prior to (left, inset) and 120 sec. after DA addition (right, inset). (C) Maximum intensity trace of representative SpH-D1R endocytic cluster vs. time. Trace represents area outlined in blue in (B), measurements taken every 4 sec. (D) Normalized integrated surface fluorescence of SpH-D1R measured every 3 sec. in the absence of agonist (Bleaching Control, n = 5 cells) or in response to DA (10μM DA, n = 20 cells). Integrated fluorescence value at time 0 was defined as 100%. Data = mean +/− SEM. (E) Representative pseudo-color image of Epac1-cAMP prior to (left) or 60 sec. after 10μM DA addition (right). Lookup table is scaled linearly to YFP/CFP emission ratio. (F) Change in normalized Epac1-cAMPs FRET signal in response to stimulation over a range of DA concentrations. Data = mean +/− SEM normalized FRET emission ratio at each time point, n = 8-16 cells per dose. Scale bars = 5μm. See also Figure S1.