Molecular And Pharmacological Studies Of Dopamine Receptors

多巴胺受体的分子和药理学研究

基本信息

批准号：
7594645
负责人：
DAVID R. SIBLEY
金额：
$ 137.91万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7594645
关键词：
ADRBK1 gene Accounting Address Adenylate Cyclase Agonist Agreement Alcohol consumption Alcohols Attenuated Binding Biochemical Biological Assay Brain Cell membrane Cells Cellular biology Class Condition Corpus striatum structure Coupling Cyclic AMP Data Depressed mood Dopamine Dopamine D1 Receptor Dopamine D2 Receptor Dopamine Receptor Ethanol Ethanol dependence Exhibits Forskolin G protein coupled receptor kinase G-Protein-Coupled Receptors GTP-Binding Proteins Gene Family Goals Heterotrimeric GTP-Binding Proteins Human Genome Immune Sera Immunoprecipitation In Vitro Investigation Isoenzymes Ligands Light Lipids Mass Spectrum Analysis Measures Mediating Membrane Molecular Molecular Cloning Mus Mutagenesis Mutation Neurons Neurotransmitter Receptor Neurotransmitters Odors Pathway interactions Peptides Phosphorylation Phosphotransferases Play Process Production Property Proteins Rate Rattus Reading Receptor Activation Receptor Signaling Recycling Regulation Reporting Research Role Second Messenger Systems Serine Signal Transduction Site Site-Directed Mutagenesis Slice Stimulus Structure System Tail Thinking Threonine Time alcohol effect alcohol exposure attenuation desensitization drug of abuse in vivo inhibitor/antagonist mutant radioligand receptor receptor coupling receptor expression receptor function receptor recycling research study response second messenger trafficking

项目摘要

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. 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. 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.

G 蛋白偶联受体 (GPCR) 代表人类基因组中发现的最大的基因家族。 GPCR 对多种刺激做出反应，包括气味、光、肽和启动多种细胞反应途径的神经递质。 GPCR 的同源配体激活会诱导受体发生构象变化，从而激活异三聚体 G 蛋白，进而向多种效应分子发出信号以产生第二信使。信号级联的终止是通过称为脱敏的过程来完成的，在该过程中，GPCR 在持续存在配体刺激的情况下表现出活性降低。通常认为脱敏是由 G 蛋白受体激酶 (GRK) 和第二信使激酶 (PKC) 激活的受体磷酸化介导的。我们之前证明 D1 多巴胺受体 (D1 DAR) 可以被两类激酶磷酸化。我们现在提供的数据表明，D1 DAR 在没有受体磷酸化的情况下表现出有效的脱敏作用。 D1 DAR 与 Gα 偶联，并在配体刺激后激活腺苷酸环化酶 (AC)，产生 cAMP。通过 D1 DAR 羧基尾部内的定点诱变，在残基 347 处截断或替换所有磷酸受体位点（丝氨酸或苏氨酸），导致在存在或不存在激动剂的情况下完全消除受体磷酸化；然而，通过 cAMP 积累测定和 35SGTPγS 结合实验测量，这些磷酸化无效突变体仍然表现出有效的脱敏作用。根据放射性配体结合测定的测量，通过截短或诱变消除总 D1 DAR 磷酸化导致受体表达降低，与野生型 (WT) 对照相比，但当考虑受体表达水平时，磷酸化无效的 D1 DAR 突变体表现出 cAMP 积累增加和 35SGTPγS绑定。 D1 DAR 磷酸化确实会减弱受体的 G 蛋白偶联和 cAMP 积累特性，因为抑制受体的 PKC 磷酸化会导致 35SGTPγS 结合和第二信使产生增加；然而，这对于减弱受体激活来说并不是必需的。我们提出 WT D1 DAR 的受体磷酸化通过调节 G 蛋白偶联和伴随的 cAMP 产生的效率来调节基础受体信号传导潜力。此外，D1 DAR 磷酸化有助于有效的受体表达、脱敏和再敏化；然而，这些过程并不需要它。多巴胺 (DA) D2 受体 (D2DAR) 在激动剂刺激下也会被磷酸化，主要是通过 G 蛋白偶联受体激酶 (GRK) 2/3。我们之前表明，第三个细胞质环内的 6 个丝氨酸和 2 个苏氨酸残基的同时突变降低了该受体突变体 -GRK(-) 的基础并完全消除了 DA 刺激和 GRK2 介导的磷酸化。我们现在研究了 GRK2 介导的磷酸化使用 HEK293T 细胞中表达的 GRK(-) 和 WT 受体对 D2DAR 信号传导和运输中的受体磷酸化。为了评估 D2DAR 信号传导，我们检查了受体减弱 cAMP 积累的能力。与WT受体相比，GRK(-)突变体在DA抑制毛喉素刺激的cAMP积累方面没有差异。有趣的是，GRK2 与 WT 受体的过度表达使 D2DAR 信号传导减少了 50%。令人惊讶的是，GRK(-)突变体的功能也因GRK2的过度表达而减弱。然而，随着激酶缺陷突变体（GRK2K220R）的过度表达，没有观察到WT或GRK（-）受体功能的抑制。总而言之，这些数据表明 GRK2 不仅可以通过受体磷酸化，还可以通过另一种（相互作用？）蛋白质的磷酸化来减弱 D2 受体信号传导。为了评估磷酸化在 D2DAR 脱敏中的作用，我们检查了激动剂刺激的 35S-GTPγS 结合作为功能读数。 DA 预处理通过促进激动剂刺激的 35S-GTPγS 结合以时间依赖性方式减少，使 WT 受体脱敏。 GRK(-)突变受体的脱敏作用延迟，但最大脱敏作用与WT受体相同。这些结果表明激动剂刺激的D2受体磷酸化促进D2受体脱敏的速率，但不是最大受体脱敏所必需的。我们还通过使用完整细胞 3Hsulpiride 结合测定来检查 D2 受体运输。在GRK2过表达不存在或存在的情况下，GRK(-)突变体内化程度与WT受体相同，以响应DA处理。有趣的是，我们发现与WT受体相比，GRK(-)突变体在内化后对质膜的受体循环减少(50%)。这些结果表明，GRK2 引起的 D2 受体磷酸化可能在受体再循环中发挥更重要的作用，而不是在受体脱敏和内化中发挥更重要的作用。受体磷酸化调节 D2DAR 循环的机制正在研究中。大脑多巴胺信号传导是许多滥用药物（包括酒精）的目标。乙醇消耗增强了多巴胺能信号传导，这部分是通过 D1 多巴胺受体的激活介导的。我们之前报道过，D1 受体转染的 HEK293T 细胞的乙醇预处理增强了多巴胺刺激的 cAMP 积累，同时 D1 受体磷酸化降低。为了检查乙醇预处理对大脑中 D1 受体信号传导的影响，在大鼠纹状体制备的切片中评估了 DARPP-32 的磷酸化。使用磷酸特异性抗血清来检测 PKA 介导的 DARPP-32 在 Thr-34 上的磷酸化，这种磷酸化是响应 D1 受体刺激而发生的。与我们之前的数据一致，乙醇预处理增强了 D1 受体介导的 DARPP-32 磷酸化，支持乙醇预处理增强体内 D1 受体信号传导的观点。 D1 受体信号传导的乙醇依赖性调节由 PKC 以同工酶特异性方式介导。 PKC 抑制剂模仿乙醇对 HEK293T 细胞中多巴胺刺激的 cAMP 水平和 D1 受体磷酸化的影响，表明乙醇抑制受体的基础 PKC 磷酸化。乙醇和 PKC 抑制剂治疗促进 D1 受体磷酸化和活性的非加和效应的观察结果支持了这一点。使用体外激酶测定直接评估乙醇对特定 PKC 同工酶的调节。乙醇预处理减弱了脂质激活的PKCgamma的膜激酶活性；和 PKCdelta；，但不是 PKCalpha、beta 或 epsilon。乙醇调节 PKCgamma 和 PKCdelta 的机制尚不清楚。我们假设乙醇预处理改变了 PKC 同工酶特异性相互作用蛋白与其附属 PKC 同工酶的关联。为了解决这个问题，HEK293T 细胞表达 PKCgamma；用培养基或乙醇处理，然后从膜组分中选择性免疫沉淀 PKCgamma。使用质谱法鉴定每种处理（乙醇）的 PKCgamma 相互作用蛋白。重要的是，我们已经确定了乙醇依赖性 PKCgamma 相互作用蛋白，它们可能介导脂质激活 PKCgamma 的乙醇依赖性调节。综上所述，这些结果表明 PKCgamma 和 PKCdelta 在基础条件下磷酸化 D1 受体，从而抑制 D1 受体信号传导。乙醇暴露会抑制这些 PKC 同工酶的活性，导致基础受体磷酸化降低并增强 D1 受体介导的信号传导。