Together these results indicate that D1 receptors can indeed recycle very rapidly after endocytosis. We next searched for inhibitors of D1 receptor recycling to examine whether, similar to endocytosis, recycling also plays a causal role in promoting the acute D1 receptor-mediated cAMP response. As D1 receptors return to the plasma membrane via similar membrane pathway as transferrin receptors (Vickery and von Zastrow, 1999), we investigated the effect of a validated siRNA targeting Eps15 homology domain containing protein 3 (EHD3). EHD3 localizes to recycling

membrane structures (Galperin et al., 2002) and is required for efficient delivery of internalized transferrin receptors to the endocytic recycling compartment (Naslavsky et al., 2006). Knockdown of EHD3 was verified by immunoblot (Figure 7A). EHD3 siRNA significantly inhibited Dabrafenib surface recovery of tagged FD1 receptors 5 min after agonist washout (Figure 7B) but did not affect acute D1 receptor-mediated cAMP accumulation (Figure 7C). Bafilomycin A1 is a specific inhibitor of the vacuolar H+-ATPase that inhibits recycling of a number of signaling receptors (Johnson et al., 1993 and Presley et al., 1997). Pretreatment of FD1R-expressing HEK293 cells with

500 nM bafilomycin A1 significantly inhibited surface receptor recovery compared to cells pretreated with vehicle (Figure 7D). Nevertheless, bafilomycin A1 also did not produce Ceritinib in vitro any detectable effect on acute D1 receptor-mediated cAMP accumulation in HEK293 cells (Figure 7E) or striatal neurons (Figure 7F). These results indicate that the ability of D1 receptor endocytosis to augment the acute cAMP signal does not require subsequent receptor recycling to the plasma

membrane. Instead, the results suggest that D1 receptors contribute to the acute signaling response upon entry to, or in transit through, an early endocytic intermediate. To assess whether it is possible for D1 receptors to promote acute cAMP accumulation from an endocytic intermediate, L-NAME HCl we investigated the physical organization of internalized D1 receptors relative to the relevant downstream cAMP transduction machinery. D1 receptors stimulate agonist-dependent cAMP production in striatal neurons by coupling to Golf, a trimeric G protein closely related to Gs, whose liberated α-subunit stimulates adenylyl cyclase V (ACV) (Neve et al., 2004). ACV is an integral membrane protein, whereas Gs/olf α-subunits are membrane-tethered by palmitoylation and associate noncovalently with membrane-embedded βγ subunits. Gs/olf α-subunits dissociate from βγ and turn over their palmitoyl tether in response to receptor-mediated activation, allowing them to transiently redistribute to the cytoplasm (Marrari et al., 2007). Thus, for signaling to occur from activated receptors entering the endocytic pathway, both ACV and Gs/olf would need to exist in close proximity to D1 receptors.