G protein coupled receptors (GPCRs) represent the largest gene family identified in the human genome and respond to a diversity of stimuli including odors, light, peptides, and neurotransmitters. Activation of GPCRs by their cognate ligands induces a conformational change in the receptor that activates heterotrimeric G proteins that in turn signal to a variety of effector molecules for second messenger production. Termination of the signaling cascade is accomplished by a process called desensitization whereby GPCRs display reduced activity in the continued presence of ligand stimulation. Desensitization is generally thought to be initiated by phosphorylation of the activated receptor by G protein-coupled receptor kinases (GRKs) and/or second messenger kinases (PKA or PKC) followed by arrestin binding and internalization. We have previously demonstrated that the G&#945;s-linked D1 dopamine receptor (DAR) can be phosphorylated by both classes of kinases. In FY2008, using mutational analyses, we have now mapped out all of the GRK and PKC phosphorylation sites within the cytoplasmic regions of the D1 DAR. Using phosphorylation null receptor mutants, we find that the D1 DAR displays efficient G protein coupling and cAMP accumulation; however, receptor expression is impaired in some mutants. Truncation at residue 347 or substitution of all phospho-acceptor sites by site-directed mutagenesis within the carboxyl tail (Tail TOTAL mutant) of the D1 DAR results in complete abolishment of receptor phosphorylation in the presence or absence of agonist; however, these phosphorylation null mutants still exhibit efficient desensitization as measured by both cAMP accumulation assays and 35SGTP&#947;S binding experiments. We are currently assesssing the phosphorylation null mutant interactions with arrestin using arrestin-GFP translocation assays. Although abolition of D1 DAR phosphorylation results in decreased receptor expression, when receptor levels are taken into account, phosphorylation null D1 DAR mutants actually display increased cAMP accumulation and 35SGTP&#947;S binding, suggesting that basal phosphorylation regulates G protein coupling. Interestingly, inhibition of basal receptor phosphorylation by PKC, using selective PKC inhibitors, also results in increased agonist-mediated 35SGTP&#947;S binding and cAMP accumulation. We conclude that receptor phosphorylation is not required for agonist-induced desenstization, although arrestin interactions and receptor internalization remains to be determined. Furthermore, we conclude that constitutive phosphorylation of the D1 DAR, primarily by PKC, regulates receptor expression and G protein coupling. [unreadable] [unreadable] In FY2008, we also completed a study which pharmacologically characterized a novel PET agonist probe of the D2 receptor. Dopaminergic signaling pathways have been extensively investigated using PET imaging, primarily with antagonist radioligands of D2 and D3 dopamine receptors (DARs). Recently, agonist radioligands of D2/D3 DARs have begun to be developed and employed. One such agonist is (R)-2-11CH3O-N-n-propylnorapomorphine (MNPA). We performed a pharmacological characterization of MNPA using recombinant D2 and D3 DARs expressed in HEK293 cells. MNPA was found to robustly inhibit forskolin-stimulated cAMP accumulation to the same extent as dopamine in D2 or D3 DAR-transfected cells, indicating that it is a full agonist at both receptors. MNPA is 50-fold more potent than dopamine at the D2 DAR, but equally potent as dopamine at the D3 DAR. MNPA competition binding curves in membrane preparations expressing D2 DARs revealed two binding states of high and low-affinity. In the presence of GTP, only one binding state of low affinity was observed. Direct saturation binding assays using 3HMNPA revealed similar results as with the competition experiments leading to the conclusion that MNPA binds to the D2 DAR in an agonist-specific fashion. In contrast to membrane preparations, using intact cell binding assays, only one site of low affinity was observed for MNPA and other agonists binding to the D2 DAR. MNPA was also found to induce D2 DAR internalization to an even greater extent than dopamine as determined using both cell surface receptor binding assays and confocal fluorescence microscopy. Taken together, our data indicate that the PET tracer, MNPA, is a full and potent agonist at both D2 and D3 receptors.[unreadable] [unreadable] In FY2008, we also initiated a drug discovery project for allosteric ligands of the D2 receptor. G protein-coupled receptors (GPCRs) represent the largest family of therapeutic drug targets and account for the mechanisms of action of >60% of all FDA-approved drugs. Receptors for the neurotransmitter dopamine are members of this GPCR super-family and are involved in the etiology and/or therapy of a number of neuropsychiatric and endocrine disorders. In fact, amongst the dopamine receptors (DARs), the D2 subtype is arguably one of the most validated drug targets in neurology and psychiatry. For instance, all receptor-based antiparkinsonian drugs work via stimulating the D2 DAR whereas all FDA-approved antipsychotic agents are antagonists of this receptor. The D2 DAR is also therapeutically targeted in other disorders such as restless legs syndrome, tardive dyskinesia, Tourettes syndrome, and hyperprolactinemia. Most drugs targeting the D2 DAR are problematic, however, either being less efficacious as desired or possessing limiting side effects, most of which are due to cross-GPCR reactivity. One pharmacological approach towards improved target specificity is to identify allosteric ligands which bind to less conserved regions of receptors and therefore have the potential to be much more selective. Ligands that bind to such allosteric sites may promote conformation changes in the receptor that can produce positive or negative effects with respect to activation by the endogenous agonist, or in some cases can exhibit functional efficacy (agonist or inverse agonist) of their own. The goal of this project is to use high throughput screening (HTS) approaches to identify and develop novel small molecule allosteric modulators of the D2 DAR for use as in vitro and in vivo pharmacological tools and in proof-of-concept experiments in animal models of neuropsychiatric disease. To this end, we propose to develop two assays capable of large-scale, high throughput screening of small molecule libraries. One assay involves the cellular co-expression of the D2 DAR with a chimeric Gq protein thus enabling the receptor to stimulate Ca2+ mobilization, which is detected through the activation of an intracellular fluorescent dye. The second assay measures the ability of D2 DARs to promote the flux of thallium ion through G protein-regulated inward rectifying potassium (GIRK) channels, again measured through the activation of an intracellular dye. These assays will be configured into HTS formats and evaluated through the generation/calculation of Z parameters. The superior assay will be submitted for consideration by the Molecular Libraries Screening Centers Network (MLSCN) program for interrogation of the NIH Molecular Libraries small molecule repository. Secondary and counter-screening assays for other DAR subtypes will also be developed and implemented as necessary to confirm and validate MLSCN-generated hits. If needed, limited medicinal chemistry efforts will be performed to enhance the potency or efficacy, or the bioavailability of the most promising hit compounds. Future studies will be directed at evaluating the therapeutic potential of D2 DAR allosteric modulators using proof-of-concept efficacy tests in animal models of Parkinsons disease and schizophrenia.