Demonstration of the specificity of receptor receptor interaction

证明受体受体相互作用的特异性

基本信息

批准号：
10267550
负责人：
Amina Woods
金额：
$ 34.5万
依托单位：
NATIONAL INSTITUTE ON DRUG ABUSE
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/10267550
关键词：
3-Dimensional Adenylate Cyclase Affect Affinity Amino Acid Sequence Amino Acids Arginine Astrocytes Automobile Driving Base Sequence Binding Binding Sites Bioinformatics Brain CNR1 gene Cannabinoids Cell membrane Charge Cleaved cell Collaborations Complex Complex Mixtures Consensus Corpus striatum structure Coupled Cyclic AMP-Dependent Protein Kinases Dancing Data Dimerization Disease Dissociation Docking Dopamine Dopamine D2 Receptor Dopamine Receptor Electron Transport Electrostatics Epitopes G-Protein-Coupled Receptors Goals Human Investigation Journals Ligand Binding Membrane Molecular Molecular Conformation Mosaicism Multiple Partners Nervous system structure Neurosciences Neurotransmitters Pathway interactions Phase Phosphopeptides Phosphorylation Play Post-Translational Protein Processing Process Protein Region Proteins Publications Rest Role Sampling Site Specificity Spectrometry, Mass, Electrospray Ionization Structure Synaptic Transmission Techniques Transmembrane Domain Universities Wood material Work addiction casein kinase I computerized tools covalent bond extracellular flexibility functional plasticity hydrophilicity inorganic phosphate ionization molecular recognition neural network neurotransmission professor receptor tandem mass spectrometry three dimensional structure

项目摘要

Our collaboration on receptors with professor Luigi F Agnati from the University of Modena, resulted in two publications. Agnati LF, Marcoli M, Maura G, Woods A, Guidolin D. The brain as a hyper network: the key role of neural networks as main producers of the integrated brain actions especially via the broadcasted neuroconnectomics Journal of Neural Transmission. 125, 883897 (2018) and Cervetto C, Venturini A, Passalacqua M, Guidolin D, Genedani S, Fuxe K, Borroto-Escuela DO, Cortelli P, Woods A, Maura G, Marcoli M and Agnati LF. A2A-D2 receptor-receptor interaction in modulates of glliotransmitter release from striatal astrocyte processes., J. Neurochem. 140, 268-279. (2017). Our work targets the receptors involved in addiction, to explain how their structure dictates how they interact with other receptors and what makes these interactions specific. Our data suggest that heteromer formation, mainly involves linear motifs found in disordered regions of proteins. Local disorder imparts plasticity to linear motifs. Many molecular recognition of proteins occur between short linear segments, known as LMs. Interaction of short continuous epitopes are not constrained by sequence and have the advantage of resulting in interactions with micromolar affinities which suites transient, reversible complexes such as receptor heteromers. Electrostatic Interactions between epitopes of the GPCR involved, is the Key step in driving heteromer formation forward. The first step in heteromerization, involves phosphorylating the Ser/Thr, in an epitope containing a casein kinase 1/2 (CK1/2)-consensus site. Our data suggests that dopaminergic neurotransmission, through cAMP dependent Protein kinase A (PKA) slows down heteromerization. The negative charge, acquired by the phosphorylation of a Ser/Thr in a PKA consensus site in the Arg rich epitope affects the activity of the receptors involved in heteromerization by causing allosteric conformational changes, due to the repulsive effect generated by the negatively charged phosphate. In addition to modulating heteromerization, it affects the stability of the heteromers interactions and heir binding affinity. So here we have an instance where phosphorylation is not just an on/off switch, instead by weakening the noncovalent bond, heteromerization acts like a rheostat that controls the stability of the heteromer through activation or inhibition of adenylate cyclase by the neurotransmitter Dopamine depending on which Dopamine receptor it docks at. Complex molecular and cellular mechanisms regulate G protein-coupled receptors (GPCRs). It is suggested that proteins intrinsically disordered regions (IDRs) are to play a role in GPCRsintra and extracellular regions plasticity, due to their potential for post-translational modification and interaction with other proteins. These regions are defined as lacking a stable three-dimensional (3D) structure. They are rich in hydrophilic cand charged, amino acids and are capable to assume different conformations which allow them to interact with multiple partners. In this study we analyzed 75 GPCR involved in synaptic transmission using computational tools for sequence-based prediction of IDRs within a protein. We also evaluated putative ligand-binding motifs using receptor sequences. The disorder analysis indicated that neurotransmitter GPCRs have a significant amount of disorder in their N-terminus, third intracellular loop (3IL) and C-terminus. About 31%, 39% and 53% of human GPCR involved in synaptic transmission are disordered in these regions. Thirty-three percent of receptors show at least one predicted PEST motif, this being statistically greater than the estimate for the rest of human GPCRs. About 90% of the receptors had at least one putative site for dimerization in their 3IL or C-terminus. ELM instances sampled in these domains were 14-3-3, SH3, SH2 and PDZ motifs. In conclusion, the increased flexibility observed in GPCRs, added to the enrichment of linear motifs, PEST and heteromerization sites, maybe critical for the nervous systems functional plasticity. Certain amino acid residues and posttranslational modifications play an important role in the formation of noncovalent complexes (NCXs) by electrostatic interactions. Electrospray ionization mass spectrometry(ESI-MS) is the most widely used MS technique for the study of NCXs, due to its softer ionization process and compatibility with the solution phase of NCX mixtures. In order to locate the site where interactions are forming in the NCXs involving phosphopeptides and adjacent arginines, tandem mass spectrometry studies using collision-induced dissociation (CID) and electron transfer dissociation (ETD) were performed on NCXs at different charge states. CID fragmentation revealed two dissociation pathways:one in which the electrostatic interaction is disrupted and another in which the covalent bond attaching the phosphate group to the amino acid residue is cleaved, while the electrostatic interaction is maintained. ETD and sequential ETD/ETD, and CID/ETD allow the determination of the NCX interaction site. These results confirmed the involvement of the phosphorylated amino acid and at least two adjacent arginines as the binding site. The concept of Receptor Mosaic (RM) is discussed; hence the integrative functions of the assemblage of Gprotein coupled receptors physically interacting in the plane of the plasma membrane. The main focus is on a heterotrimer of G-protein coupled receptors, namely the A2A-D2-CB1 receptor trimer. A bioinformatics analysis was carried out on the amino acid sequence of these receptors to indicate domains possibly involved in the receptor-receptor interactions. Such a bioinformatic analysis was also carried out on the RM formed by mGLU R5, D2 and A2A. The importance of topology, i.e., of the reciprocal localisation of the three interacting receptors in the plan of the membrane for the RM integrative functions is underlined. However, it is also pointed out that this fundamental aspect still waits techniques capable of an appropriate investigation. Finally, it is discussed how RM topology can give hints for a structural definition of the concept of hub receptor. Thus, just as in any network, the receptor operating as a hub is the one that in the molecular network formed by the receptors has the highest number of inputs.

我们与摩德纳大学（University of Modena）与Luigi F Agnati教授进行的受体合作导致了两份出版物。 Agnati LF，Marcoli M，Maura G，Woods A，GuidolinD。大脑作为一个超级网络：神经网络作为综合大脑动作的主要生产者的关键作用，尤其是通过广播的神经性神经传播杂志。 125，883897（2018）和Cervetto C，Venturini A，Passalacqua M，Guidolin D，Genedani S，Fuxe K，Borroto-Escuela DO，Cortelli P，Woods A，Woods A，Maura G，Marcoli M，Marcoli M和Agnati LF。 Glliotterter释放的A2A-D2受体受体相互作用从纹状体星形胶质细胞过程中释放出来。 140，268-279。（2017）。我们的工作针对成瘾所涉及的受体，以解释它们的结构如何决定它们与其他受体的相互作用以及使这些相互作用具体的原因。我们的数据表明，异构体形成主要涉及在蛋白质无序区域中发现的线性基序。局部障碍将可塑性赋予线性基序。短线性片段（称为LMS）之间发生了许多分子识别蛋白。短连续表位的相互作用不受序列的限制，并且具有与微摩尔亲和力相互作用的优势，这些微摩尔亲和力相互作用，这些亲和力是套件瞬时，可逆的复合物，例如受体异构体。涉及的GPCR表位之间的静电相互作用是驱动异构体形成向前的关键步骤。异质化的第一步涉及在含有酪蛋白激酶1/2（CK1/2） - consensus位点的表位中磷酸化Ser/Thr。我们的数据表明，通过cAMP依赖性蛋白激酶A（PKA）减慢异质化，多巴胺能神经传递会减慢。通过在PKA共识位点中Ser/THR在ARG富集位置中获得的Ser/Thr磷酸化获得的负电荷会影响异化构象变化所涉及的受体的活性，这是由于负电荷的磷酸磷酸盐产生的排斥作用。除了调节异构化外，它还会影响异构体相互作用和继承人结合亲和力的稳定性。因此，在这里，我们有一个实例，不仅磷酸化不仅是开/关开关，还可以通过削弱非共价键来进行异构化的作用，就像是一种变性，它通过激活或抑制腺苷酸环磷酸酶通过神经素抗氨基甲酸胺的多巴胺来控制异构体的稳定性，而这些腺苷酸环化酶在哪些多巴胺中依赖于多发性氨基胺受体。复杂的分子和细胞机制调节G蛋白偶联受体（GPCR）。据建议，蛋白质本质上无序的区域（IDR）将在GPCRSintra和细胞外区域发挥作用，这是由于它们在翻译后修饰和与其他蛋白质的相互作用的潜力，因此它们具有可塑性。这些区域被定义为缺乏稳定的三维（3D）结构。它们富含充电，氨基酸的亲水性CAND，并能够假设不同的构象使它们与多个伴侣相互作用。在这项研究中，我们使用计算工具分析了75个GPCR，该GPCR使用计算工具用于基于蛋白质内IDR的序列预测。我们还使用受体序列评估了推定的配体结合基序。该疾病分析表明，神经递质GPCR在其N末端，第三个细胞内环（3IL）和C-末端中具有相当多的疾病。在这些地区，参与突触传播的人GPCR的31％，39％和53％是无序的。 33％的受体显示至少一个预测的害虫基序，从统计学上讲，这大于其他人GPCR的估计值。大约90％的受体在其3IL或C末端至少有一个假定位点用于二聚化。在这些域中采样的ELM实例为14-3-3，SH3，SH2和PDZ图案。总之，在GPCR中观察到的灵活性的提高，增加了线性基序，害虫和异质化位点的富集，这对于神经系统功能可塑性至关重要。通过静电相互作用，某些氨基酸残基和翻译后修饰在非共价复合物（NCX）中起重要作用。电喷雾电离质谱法（ESI-MS）是NCX研究最广泛使用的MS技术，因为其较软的电离工艺和与NCX混合溶液相的兼容性。为了定位在涉及磷酸肽和相邻精氨酸的NCX中形成相互作用的位置，使用碰撞引起的解离（CID）和电子转移解离（ETD）在不同电荷状态下对NCXS进行了串联质谱研究。 CID碎片揭示了两种解离途径：一种破坏静电相互作用，而另一个将磷酸基团连接到氨基酸残基的共价键被切割到静电相互作用的同时保持静电相互作用。 ETD和顺序ETD/ETD以及CID/ETD允许确定NCX相互作用位点。这些结果证实了磷酸化的氨基酸和至少两个相邻精氨酸作为结合位点的参与。讨论了受体马赛克（RM）的概念；因此，Gprotein的组合的综合功能耦合受体在质膜平面上物理相互作用。主要重点是异三聚体 G蛋白偶联受体的，即A2A-D2-CB1受体三聚体。进行了生物信息学分析在这些受体的氨基酸序列上表明可能与受体受体相互作用有关的结构域。这种生物信息学分析也对由MGLU R5，D2和A2A形成的RM进行了。拓扑的重要性，即三个相互作用受体在膜计划中的相互定位的重要性对于RM集成函数，下划线。但是，还指出这个基本方面仍然等待能够进行适当调查的技术。最后，讨论了RM拓扑如何为一个提示集线器受体概念的结构定义。因此，就像在任何网络中一样，作为集线器运行的受体是一个在受体形成的分子网络中，输入数量最多。