, 1995). We are not aware of previous evidence that endocytosis augments the acute cAMP response mediated by any signaling receptor. Our study focused on cells in which D1 receptors are primarily thought to endocytose via clathrin-coated pits. There is evidence that caveolae mediate a slower component of D1 receptor endocytosis in other

cells, but this is not thought to affect the acute cAMP signal (Kong et al., 2007). As such, we believe that the presently identified role of endocytosis in supporting acute D1 receptor-mediated signaling is unique. What is the mechanism by which rapid endocytosis contributes to dopaminergic signaling? We initially favored the hypothesis that this augmentation might occur Alisertib mw by rapid cycling of receptors back to the plasma membrane. This was motivated by analogy with the resensitization paradigm established for several other GPCRs. As presently understood, however, the resensitization paradigm explains recovery of signal responsiveness after prolonged or repeated activation, and does

not affect the acute signaling response (Lefkowitz, 1998 and Pippig et al., 1995). Nevertheless, given the rapid kinetics with which D1 receptors were found to traverse Temozolomide solubility dmso the recycling pathway, we considered the hypothesis that D1 receptors might recycle so rapidly that their resensitization might have been missed by the previous paradigm. We rejected this hypothesis because genetic (EHD3 knockdown) and chemical (bafilomycin A1) inhibition of the recycling pathway did not affect D1 receptor-mediated cAMP accumulation, in contrast to the pronounced inhibition produced by various endocytic inhibitors. Our results thus support the alternative hypothesis that endocytosis augments acute dopaminergic signaling by facilitating direct D1 receptor-mediated signaling from a membrane domain in the early endocytic pathway (Figure 8E). This is supported by immunocytochemical localization data showing close proximity between D1 receptors and downstream transduction machinery upon initial entry

to the endocytic pathway. While there nearly is no doubt that the plasma membrane is a major site of GPCR signaling, there is emerging evidence that signaling can also occur from the endocytic pathway, and there is presently no compelling reason to rule out endosomal signaling via trimeric G proteins (Calebiro et al., 2010 and Sorkin and von Zastrow, 2009). Trimeric G proteins have previously been detected on endomembrane compartments in mammalian cells and tissues (Marrari et al., 2007). Further, G protein α-subunits can mediate functionally significant signaling from endosomes in yeast (Slessareva and Dohlman, 2006). Recent evidence suggests that two other mammalian GPCRs (the TSH and PTH receptors) signal via G protein-linked activation of AC directly from the endosome membrane (Calebiro et al., 2009 and Ferrandon et al., 2009).