Molecular And Pharmacological Studies Of Dopamine Receptors

Neurological Disorders And Stroke

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Abstract

G protein coupled receptors (GPCRs) represent the largest gene family identified in the human genome. GPCRs respond to a diversity of stimuli including odors, light, peptides, and neurotransmitters that initiate a variety of cellular response pathways. 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 mediated by phosphorylation of the activated receptor by both G protein receptor kinases (GRKs) and second messenger kinases (PKC). We previously demonstrated that the D1 dopamine receptor (D1 DAR) can be phosphorylated by both classes of kinases. We now present data that show that D1 DAR displays efficient desensitization in the absence of receptor phosphorlation. D1 DAR couples to Gαs and upon ligand stimulation results in the activation of adenylyl cyclase (AC) to produce cAMP. Truncation at residue 347 or substitution of all phospho-acceptor sites (serine or threonine) by site-directed mutagenesis within the carboxyl tail of the D1 DAR results in a 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 35SGTPγS binding experiments. Abolition of total D1 DAR phosphorylation by truncation or mutagenesis resulted in decreased receptor expression compared to wildtype (WT) controls as measured by radioligand binding assays, yet when receptor expression levels are taken into account, phosphorylation null D1 DAR mutants displayed increased cAMP accumulation and 35SGTPγS binding. D1 DAR phosphorylation does attenuate both G protein coupling and cAMP accumulation properties of the receptor, since inhibition of PKC phosphorylation of the receptor resulted in increased 35SGTPγS binding and second messenger production; however, it is not necessary for attenuation of receptor activation. We propose that receptor phosphorylation of the WT D1 DAR regulates basal receptor signaling potential by regulating the efficiency of G protein coupling and concomitant cAMP production. Furthermore, D1 DAR phosphorylation facilitates efficient receptor expression, desensitization, and resensitization; however, it is not required for these processes.[unreadable] [unreadable] Dopamine (DA) D2 receptors (D2DARs) are also phosphorylated upon agonist stimulation, mainly by G-protein coupled receptor kinases (GRKs) 2/3. We previously showed that simultaneous mutation of 6 serine and 2 threonine residues within the 3rd cytoplasmic loop decreased basal and completely abolished DA-stimulated and GRK2-mediated phosphorylation of this receptor mutant−GRK(-).We now investigated the role of GRK2-mediated receptor phosphorylation in D2DAR signaling and trafficking using the GRK(-) and WT receptors expressed in HEK293T cells. To assess D2DAR signaling, we examined the ability of the receptors to attenuate cAMP accumulation. The GRK(-) mutant showed no difference in DA inhibition of forskolin-stimulated cAMP accumulation compared to the WT receptor. Interestingly, over-expression of GRK2 with the WT receptor decreased D2DAR signaling by 50%. Surprisingly, the function of the GRK(-) mutant was also attenuated by over-expression of GRK2. Inhibition of either WT or GRK(-) receptor function was not observed, however, with over-expression of the kinase-defective mutant (GRK2K220R). Taken together, these data suggest that GRK2 can attenuate D2 receptor signaling not only by receptor phosphorylation but also by phosphorylation of another (interacting?) protein. To assess the role of phosphorylation in D2DAR desensitization, we examined agonist-stimulated 35S-GTPγS binding as a functional read-out. Pretreatment with DA desensitized the WT receptor by promoting a decrease in agonist-stimulated 35S-GTPγS binding in a time-dependent fashion. Desensitization onset was delayed with the GRK(-) mutant receptor, but the maximum desensitization was the same as with the WT receptor. These results suggest that agonist-stimulated D2 receptor phosphorylation facilitates the rate of D2 receptor desensitization, but is not required for maximum receptor desensitization. We also examined D2 receptor trafficking by using intact cell 3Hsulpiride binding assays. The GRK(-) mutant internalized to the same extent as the WT receptor in response to DA treatment in the absence or presence of GRK2 over-expression. Interestingly, we found that the GRK(-) mutant showed diminished (50%) receptor recycling to the plasma membrane after internalization when compared to the WT receptor. These results suggest that D2 receptor phosphorylation by GRK2 may play a more important role in receptor recycling rather than in receptor desensitization and internalization. The mechanism by which receptor phosphorylation regulates D2DAR recycling is under investigation.[unreadable] [unreadable] Brain dopamine signaling is the target of many drugs of abuse, including alcohol. Ethanol consumption potentiates dopaminergic signaling and this is mediated partially by activation of the D1 dopamine receptor. We previously reported that ethanol pretreatment of D1 receptor-transfected HEK293T cells potentiates dopamine-stimulated cAMP accumulation with a concurrent decrease in D1 receptor phosphorylation. To examine the effect of ethanol pretreatment on D1 receptor signaling in the brain, the phosphorylation of DARPP-32 was evaluated in slices prepared from rat striatum. A phospho-specific antiserum was used to detect PKA-mediated phosphorylation of DARPP-32 on Thr-34, which occurs in response to D1 receptor stimulation. In agreement with our previous data, ethanol pretreatment potentiated D1 receptor-mediated DARPP-32 phosphorylation supporting the notion that ethanol pretreatment enhances D1 receptor signaling in vivo. The ethanol-dependent modulation of D1 receptor signaling is mediated by PKC in an isozyme specific fashion. PKC inhibitors mimicked the effects of ethanol on both dopamine-stimulated cAMP levels and D1 receptor phosphorylation in HEK293T cells, suggesting that ethanol inhibits basal PKC phosphorylation of the receptor. This is supported by the observation that treatment with both ethanol and PKC inhibitors promoted non-additive effects on D1 receptor phosphorylation and activity. Specific PKC isozyme modulation by ethanol was directly assessed using in vitro kinase assays. Ethanol pretreatment attenuated the membrane kinase activities of lipid-activated PKCgamma; and PKCdelta;, but not PKCalpha, beta, or epsilon. The mechanism responsible for the ethanol modulation of PKCgamma and PKCdelta is unclear. We hypothesized that ethanol pretreatment altered the association of PKC-isozyme specific interacting proteins with their affiliated PKC isozyme. To address this, HEK293T cells expressing PKCgamma; were treated with media or ethanol followed by selective immunoprecipitation of PKCgamma from membrane fractions. PKCgamma-interacting proteins for each treatment (ethanol) were identified using mass spectrometry. Importantly, we have identified ethanol-dependent PKCgamma interacting proteins that may mediate the ethanol-dependent modulation of lipid-activated PKCgamma. Taken together, these results suggest that PKCgamma and PKCdelta phosphorylate the D1 receptor under basal conditions and that this depresses D1 receptor signaling. Ethanol exposure inhibits the activity of these PKC isozymes resulting in decreased basal receptor phosphorylation and enhanced D1 receptor-mediated signaling.