Flow cytometric analysis of surface accessibility of FLAG epitope-tagged D1 DA receptors (FD1R) in HEK293 cells verified robust internalization in response to DA. Internalization was dose-dependent and rapid, approaching the steady state value with an estimated t1/2 of 3.9 min (Figure 1A). For greater temporal resolution, we employed live imaging by total internal reflection fluorescence (TIRF) microscopy and the pH-sensitive GFP variant superecliptic pHluorin (SpH, or SEP) fused to the N-terminal extracellular region of the D1 receptor (SpH-D1R). SpH is highly

fluorescent at neutral pH, facilitating detection when in contact with the extracellular media. This fluorescence is rapidly quenched in the acidic SCH727965 clinical trial environment of the endocytic pathway (Miesenböck et al., 1998 and Sankaranarayanan et al., 2000). We used these properties to observe individual endocytic events

Autophagy Compound Library screening in SpH-D1R expressing HEK293 cells. In the absence of DA, SpH-D1R fluorescence was visible on the plasma membrane (Figure 1B, left). Bath application of DA caused rapid clustering of SpH-D1Rs into puncta that subsequently endocytosed (Figure 1B, right; see Movie S1 available online). Strikingly, an initial wave of SpH-D1R clustering and endocytosis occurred as soon as 30 s after agonist addition (Movie S1). Analysis of individual puncta by fluorescence intensity tracing verified their disappearance within 30 s to 1 min after formation (Figure 1C). These properties are consistent with previous data indicating that D1 receptors endocytose primarily via clathrin-coated

pits in HEK293 cells (Vickery and von Zastrow, 1999), and with descriptions of clathrin-mediated endocytosis of signaling receptors imaged by TIRF microscopy (Puthenveedu and von Zastrow, 2006 and Yudowski Carnitine dehydrogenase et al., 2006). Rapid endocytosis was further verified by the rate of DA-dependent reduction in integrated SpH-D1R surface fluorescence intensity (Figure 1D). Given that significant endocytosis of D1 receptors occurred within ∼1 min of DA addition, we examined D1 receptor-mediated signaling over a similar time scale. We employed the FRET-based cAMP biosensor, Epac1-cAMPs, to measure DA stimulated cAMP production in real time, in individual cells, without phosphodiesterase inhibitors (Nikolaev et al., 2004). Cells expressing FD1R and Epac1-cAMPs showed a robust decrease in the normalized FRET emission ratio after DA addition, indicating elevated cytoplasmic cAMP concentration (Figure 1E). DA application produced both a rapid decrease of YFP emission and a corresponding increase in CFP emission, verifying that the observed changes were indeed due to decreased FRET (Figure S1A).