Molecular And Pharmacological Studies Of Dopamine Receptors

多巴胺受体的分子和药理学研究

• 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
• 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

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）代表了人类基因组中鉴定的最大基因家族。 GPCR对启动多种细胞反应途径的各种刺激做出了反应。通过其同源配体激活GPCR，会引起受体的构象变化，该构象会激活异三聚体G蛋白，进而信号向多种效应子分子进行第二递Messenger的产生。信号级联的终止是通过称为脱敏的过程来完成的，从而在持续存在配体刺激的情况下，GPCR显示出降低的活性。通常认为脱敏是由G蛋白受体激酶（GRKS）和第二信使激酶（PKC）对活化受体的磷酸化介导的。我们先前证明了D1多巴胺受体（D1 DAR）可以被两种类型的激酶磷酸化。现在，我们提出的数据表明，在没有受体磷脂的情况下，D1 DAR显示出有效的脱敏。 D1 dar伴侣与Gαs和配体刺激后，导致腺苷环酶（AC）的激活产生cAMP。在残基347处的截断或通过位置定向的诱变在D1 DAR的羧基尾部中取代所有磷酸化的位点（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废除受体磷酸化；然而，这些磷酸化零突变体仍然表现出有效的脱敏化，如cAMP积累测定和35SGTPγs结合实验所测量的。与野生型（WT）对照相比，通过截断或诱变消除总D1 DAR磷酸化导致受体表达降低，如通过放射性体形结合测定所测量的，但是当考虑到受体表达水平时，磷酸化的NULL D1 DAR突变体显示出表现出的cAMP积累和35SGTPγ的结合。 D1 DAR磷酸化确实减弱了受体的G蛋白偶联和cAMP积累特性，因为受体的PKC磷酸化抑制会导致35SGTPγS的结合和第二递Messenger产生增加。但是，没有必要衰减受体激活。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磷酸化促进了有效的受体表达，脱敏和敏化。但是，这些过程并不需要。 多巴胺（DA）D2受体（D2DAR）也会在激动剂刺激下磷酸化，主要是通过G蛋白偶联受体激酶（GRKS）2/3磷酸化。我们先前表明，在第三个细胞质环内同时突变的6个丝氨酸和2个苏氨酸残基减少了基础，并完全废除了该受体突变体 - GRK的DA刺激和GRK2介导的磷酸化（ - ）。我们现在研究了GRK2介导的受体磷酸化和GRK磷酸化的作用，并使用了D22D的传播（GRK）的作用（在HEK293T细胞中。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cAMP积累的抑制作用上没有差异。有趣的是，WT受体的GRK2过表达使D2DAR信号传导降低了50％。 令人惊讶的是，GRK2的过表达也会减弱GRK（ - ）突变体的功能。但是，没有观察到抑制WT或GRK（ - ）受体功能，但是，激酶缺陷突变体（GRK2K220R）过表达。综上所述，这些数据表明，GRK2不仅可以通过受体磷酸化，而且可以通过另一种（相互作用？）蛋白的磷酸化来减弱D2受体信号传导。 为了评估磷酸化在D2DAR脱敏化中的作用，我们检查了激动剂刺激的35S-GTPγs结合作为功能读数。用DA进行预处理，通过以时间依赖性方式促进了激动剂刺激的35S-GTPγS结合的降低WT受体。用GRK（ - ）突变受体延迟脱敏开始，但最大脱敏与WT受体相同。这些结果表明，激动剂刺激的D2受体磷酸化促进了D2受体脱敏的速率，但最大受体脱敏并不需要。我们还通过使用完整的3Hs硫硫硫化物结合测定法检查了D2受体运输。在不存在或存在GRK2过表达的情况下，GRK（ - ）突变体与WT受体相同的程度与WT受体相同。有趣的是，我们发现与WT受体相比，内在化后，GRK（ - ）突变体在内在化后向质膜回收减少（50％）。这些结果表明，GRK2的D2受体磷酸化可能在受体回收中起着更重要的作用，而不是受体脱敏和内在化。研究受体磷酸化调节D2DAR回收的机制正在研究中。 脑多巴胺信号传导是包括酒精在内的许多滥用药物的目标。 乙醇消耗增强了多巴胺能信号传导，这是通过激活D1多巴胺受体来部分介导的。 我们先前报道说，D1受体转染的HEK293T细胞对乙醇预处理增强了多巴胺刺激的cAMP积累，并同时降低D1受体磷酸化。 为了检查乙醇预处理对大脑中D1受体信号传导的影响，在用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DARPP-32在THR-34上的PKA介导的磷酸化，这是响应于D1受体刺激而发生的。与我们先前的数据一致，乙醇预处理增强了D1受体介导的DARPP-32磷酸化，这支持了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是由PKC以同工酶特异性的。 PKC抑制剂模仿了乙醇对HEK293T细胞中多巴胺刺激的cAMP水平和D1受体磷酸化的影响，这表明乙醇抑制受体的基础PKC磷酸化。这是通过观察到的，即乙醇和PKC抑制剂的治疗促进了对D1受体磷酸化和活性的非添加作用。 使用体外激酶测定直接评估乙醇的特定PKC同工酶调节。 乙醇预处理减弱了脂质激活PKCGAMMA的膜激酶活性；和pkcdelta;，但不是pkcalpha，beta或epsilon。 负责PKCGAMMA和PKCDELTA乙醇调节的机制尚不清楚。 我们假设乙醇预处理改变了PKC-异杂因素相互作用蛋白与其关联PKC同工酶的关联。 为了解决这个问题，HEK293T表达PKCGAMMA的细胞；用培养基或乙醇处理，然后从膜级分对PKCGAMMA进行选择性免疫沉淀。 使用质谱法鉴定出每种处理的PKCGAMMA相互作用蛋白（乙醇）。 重要的是，我们已经确定了依赖乙醇的PKCGAMMA相互作用蛋白，这些蛋白可能介导脂肪激活的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，并且降低了D1受体信号传导。 乙醇暴露抑制了这些PKC同工酶的活性，导致基底受体磷酸化降低并增强了D1受体介导的信号传导。