Dopamine is a major neurotransmitter within the central nervous system (CNS). The D1 dopamine receptor is a member of a family of seven transmembrane spanning proteins called G-protein coupled receptors (GPCRs) and the principal mediator of excitatory dopaminergic transmission in the CNS. Disorders of dopaminergic signaling underlie a number of disease states including Parkinson’s disease, schizophrenia and drug addiction. As such, D1 signaling must be tightly regulated. Membrane trafficking is thought to have important consequences for GPCR signaling. Ligand-induced endocytosis physically removes GPCRs from the cell surface and contributes to the attenuation of receptor mediated signaling (desensitization). Following endocytosis, rapid recycling of GPCRs is thought to return functional receptors to plasma membrane and restore signaling (resensitization). We are currently investigating membrane trafficking and signaling of D1 on the cellular level in order to better understand how dopaminergic signaling is regulated within the brain. We have examined membrane trafficking of D1 in HEK 293 cells and in dissociated primary cultures of rat neurons using FLAG-epitope tagged version of the receptor. FLAG-D1 undergoes endocytosis following 10uM dopamine treatment and rapidly recycles to the plasma membrane after agonist washout. We have also investigated D1 mediated signaling using both a biochemical cAMP assay and a FRET-based cAMP reporter. Pretreatment of cells expressing D1 with 10uM dopamine for as little as 1 minute results in a reduced ability of the receptor to stimulate accumulation of cAMP upon subsequent stimulation with dopamine (desensitization). Preliminary results indicate that D1 mediated cAMP production recovers completely within 10 minutes after agonist washout in cells initially challenged with dopamine for 1 minute (resensitization). Cells pretreated with dopamine for 30 minutes do not exhibit this resensitization, even when assayed 60 minutes after agonist washout. This suggests that recovery of D1 signaling is highly sensitive to the duration of initial agonist challenge. We are currently examining the effects that directly inhibiting endocytosis and recycling of D1 have on dopamine stimulated cAMP production. We are also using total internal reflection fluorescence (TIRF) microscopy and a pH-sensitive ecliptic GFP version of the D1 receptor to examine dynamic signaling and trafficking events at the cell surface.