Structure And Functions Of Signal-transducing G-proteins

信号转导 G 蛋白的结构和功能

• 批准号: 6814180
• 负责人: John K Northup
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 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/6814180
• 关键词: 3T3 cells; G protein; G protein coupled receptor kinase; Sf9 cell line; allosteric site; biological signal transduction; chemoreceptors; conformation; cyclic GMP; glutamate receptor; neuropeptide receptor; phosphodiesterases; protein reconstitution; protein structure function; receptor coupling; receptor sensitivity; rhodopsin; serotonin inhibitor; serotonin receptor; structural biology; surface plasmon resonance; taste; visual perception

We have continued our investigations of the basic mechanisms of receptor (GPCR) activation of G-proteins using a combination of surface plasmon resonance (SPR) measurement of GPCR-G-protein binding and in vitro assay of GPCR-catalyzed guanine nucleotide turnover. In this past year we have completed investigations of the interactions of the alpha-i family with bovine rhodopsin. We produced and purified homogeneously myristoylated alpha-i1 and alpha-o from e.coli in comparison with the endogenous retinal G-protein, transducin (Gt). All of these G-alpha subunits are members of the structurally-related alpha-i family, but they display distinct endogenous GTP exchange rates, beta-gamma dimer affinities, and receptor specificities in addition to cellular expression. Our initial findings, which confirmed published reports, suggested rhodopsin could catalyze guanine nucleotide exchange on all three alpha subunits with nearly identical kinetics in vitro. However, examined by SPR with immobilized bovine rhodopsin, the alpha-i1 and alpha-o show dramatically decreased dissociation rates in comparison to alpha-t. Whereas alpha-t combined with the retinal beta1-gamma1 dissociates with a half-time less than 5 sec, the half-life of alpha-o is about 60-90 sec, while that for alpha-i1 is on the order of 1000 sec. We determined that this apparent discrepancy arises from a biochemical artifact of the myr-alpha-i1, which is strongly enhanced in spontaneous GDP-dissociation by lipid bilayer structures. The actual rhodopsin contribution to GDP-dissociation of this protein is marginal, consistent with the very prolonged lifetime of the rhodopsin-alpha-i1 complex. Detailed kinetic examination of the rhodopsin-catalyzed nucleotide exchange on alpha-t suggests that the currently held mechanism of GTP-assisted dissociation of activated alpha from receptor may not explain these data. As a test of this, we constructed point mutations of alpha-i1 at amino acid residues that undergo changes in guanine nucleotide interaction between GDP-bound and GTP-bound conformations of alpha. Our data for alpha-i1 show that the G203A mutant spontaneously exchanges GDP similar to wild-type, but is impaired in rhodopsin-catalyzed exchange. Mutation of R208A leads to an alpha that exchanges GDP similar to wild-type, but shows diminished affinity for GTP. SPR studies find that G203A alpha-i1 binds to rhodopsin with a prolonged life-time, independent of exogenous guanine nucleotide. Together, these data suggest that GTP is not required for dissociation of activated alpha from receptor; rather, the receptor catalyzes the activation and dissociation of an un-liganded alpha subunit, which subsequently binds GTP. We are continuing these studies using three additional GPCR structures: a structurally distinct cephalopod rhodopsin, recombinant M1-muscarinic acetylcholine receptors, and the 5HT2c subtype of human serotonin receptors. We have also continued examination of the unique properties of the family3 GPCR structures, examining mutant constructs of metabotropic glutamate receptors (mGluR1) and calcium-sensing receptors (CaR) as well as a gold-fish taste receptor (5.24), reportedly an arginine receptor (Arg-R). Previously we have reported that the seven-transmembrane helix bundle (7TM) of the human CaR (t903-rhoC) without the amino-terminal calcium binding domain (ECD) can be activated by three allosterically interacting sites for divalent cations, polyvalent organic cations (poly-Arg, spermine) and the synthetic ligand NPS 568. Mutation of all five acidic residues in the second extracellular loop of t903-RhoC abrogated the NPS568, but not PolyArg synergy of calcium activation. We have completed examination of the importance of the second extracellular loop and other loci of charged residues in the extracelluar sequences of the hCaR 7TM in the context of the full-length structure. The 5-alanine mutant homolog was found to be strongly activating. Of particular interest, mutation of a single residue in the second extracellular loop (E767) displays a high intrinsic activity, and abrogates the steeply cooperative activation of the hCaR by calcium. Mutation of the single residue K831 of the third extracellular loop is similarly, but not so strongly activating. Analysis of various amino acid substitutions at these two positions clearly identifies them as participating in ionic interactions. However, this pair of oppositely charged residues does not appear to form an internal salt-bridge. We expect that they may be interacting with charged residues within the N-terminal extracellular domain (ECD) of the hCaR. These studies amplify on our initial observations of the interacting allosteric sites within the 7TM core of the hCaR, suggesting that the calcium-binding ECD interacts with the 7TM core through a contact at this residue. Further, these data strongly imply that the ECD may be an inhibitory constraint on the activity of the 7TM core. We have set out to test this suggestion by independently expressing the ECD and 7TM core structures for mGluR1, hCaR, and 5.24. Currently, we have high level expression of the mGluR1 and hCaR 7TM cores and baculoviral constructs for secreted ECD proteins from mGluR1 and 5.24. When our complement of molecular reagents is completed, we will undertake the isolation to homogeneity of the three ECD structures, and we will examine the ligand binding and 7TM core regulating properties of these constructs. The 5.24 ECD is currently expressed with sufficient yield to entertain obtaining a crystal structure for this protein, which would be of immense interest to compare with the available structure of the rat mGluR1 ECD. However, our initial biochemical evaluation of both the full-length 5.24 receptor and the autonomously expressed ECD of 5.24 has refuted the published report that this receptor responds to L-arginine. We are currently trying to identify the ligand for this receptor. Our project in collaboration with Dr. Susan Sullivan, NIDCD seeking to identify the tastant compounds recognized by the entire repertoire of human genes encoding bitter taste receptors has made considerable progress this past year. Dr. Sullivan has identified 23 candidate genes from the human genetic databases with high similarity to the known mouse and rat bitter taste receptors but which are not olfactory receptors. At present we have constructed baculoviral vectors for expressing these, and have characterized 10 of them as directing the expression of a cell-surface localized receptor. In parallel, Dr. Dennis Drayna, also of NIDCD, has identified the human gene locus encoding the phenylthiocarbimide (PTC) trait. His studies revealed five allelic variants (2 tasting, 3 non-tasting) within the human population. Baculoviral vectors expressing all of these gene products have also been constructed. We are continuing the development of a novel cell-based screening strategy to identify the ligands for these receptors. We have constructed baculoviral vectors for the taste-enriched G-beta-3 and gamma-13 subunits, the myeloid alpha-15/16 proteins and jellyfish aquorin. By simultaneous infection of Sf9 cells with these and viruses encoding the taste receptors, we expect to re-direct the G-protein signaling pathways initiated by the tastants to the production of chemiluminescence. Our initial tests of this have utilized several well-characterized receptors (5-HT1A, 5-HT2C and GRP-R) to confirm that the strategy can succeed. Further, infection with the mouse bitter taste receptor T2R5 succeeds in producing chemiluminescent Sf9 cells in response to cyclohexamide. To date, we have not succeeded with this strategy to identify any of the tastants for the human bitter taste receptors. We have, however, succeeded with an alternative strategy of in vitro assay for these recept

我们结合使用表面等离振子共振 (SPR) 测量 GPCR-G 蛋白结合和体外检测 GPCR 催化的鸟嘌呤核苷酸转换，继续研究 G 蛋白受体 (GPCR) 激活的基本机制。在过去的一年里，我们完成了 α-i 家族与牛视紫红质相互作用的研究。我们从大肠杆菌中生产并纯化了均一的肉豆蔻酰化 α-i1 和 α-o，与内源性视网膜 G 蛋白转导蛋白 (Gt) 进行比较。所有这些 G-α 亚基都是结构相关的 α-i 家族的成员，但除了细胞表达之外，它们还表现出不同的内源性 GTP 交换率、β-γ 二聚体亲和力和受体特异性。我们的初步研究结果证实了已发表的报告，表明视紫红质可以催化所有三个 α 亚基上的鸟嘌呤核苷酸交换，并且在体外具有几乎相同的动力学。然而，通过使用固定化牛视紫红质的 SPR 检查，与 α-t 相比，α-i1 和 α-o 显示解离率显着降低。 α-t 与视网膜 beta1-gamma1 结合的半衰期不到 5 秒，而 α-o 的半衰期约为 60-90 秒，而 alpha-i1 的半衰期约为 1000 秒。我们确定这种明显的差异是由 myr-alpha-i1 的生化伪影引起的，它在脂质双层结构的自发 GDP 解离中得到强烈增强。视紫红质对该蛋白 GDP 解离的实际贡献很小，这与视紫红质-α-i1 复合物的非常长的寿命一致。对视紫红质催化的 α-t 核苷酸交换的详细动力学检查表明，目前持有的 GTP 辅助激活的 α 与受体解离的机制可能无法解释这些数据。作为对此的测试，我们在氨基酸残基处构建了α-i1的点突变，这些氨基酸残基经历了α的GDP结合和GTP结合构象之间的鸟嘌呤核苷酸相互作用的变化。我们的 alpha-i1 数据表明，G203A 突变体自发地交换 GDP，与野生型相似，但视紫红质催化的交换受到损害。 R208A 的突变导致 α 与野生型类似地交换 GDP，但显示出与 GTP 的亲和力降低。 SPR 研究发现，G203A α-i1 与视紫红质的结合时间较长，不依赖于外源鸟嘌呤核苷酸。总之，这些数据表明 GTP 不是激活 α 从受体解离所必需的；相反，受体催化未配体的 α 亚基的激活和解离，随后与 GTP 结合。我们正在使用另外三种 GPCR 结构继续进行这些研究：结构不同的头足类动物视紫红质、重组 M1-毒蕈碱乙酰胆碱受体和人类血清素受体的 5HT2c 亚型。我们还继续检查 family3 GPCR 结构的独特特性，检查代谢型谷氨酸受体 (mGluR1) 和钙敏感受体 (CaR) 以及金鱼味觉受体 (5.24)（据报道是一种精氨酸受体）的突变结构(精氨酸-R)。之前我们报道过，没有氨基末端钙结合域（ECD）的人CaR（t903-rhoC）的七跨膜螺旋束（7TM）可以被二价阳离子、多价有机阳离子的三个变构相互作用位点激活。聚精胺）和合成配体 NPS 568。第二个细胞外环中所有五个酸性残基的突变t903-RhoC 消除了 NPS568，但没有消除 PolyArg 的钙激活协同作用。我们已经完成了对全长结构背景下 hCaR 7TM 细胞外序列中第二个细胞外环和其他带电残基位点的重要性的检查。 5-丙氨酸突变体同系物被发现具有强烈的激活作用。特别令人感兴趣的是，第二个细胞外环（E767）中单个残基的突变表现出高内在活性，并消除了钙对 hCaR 的急剧协同激活。第三个细胞外环的单个残基 K831 的突变也类似，但激活不那么强烈。对这两个位置的各种氨基酸取代的分析清楚地表明它们参与了离子相互作用。然而，这对带相反电荷的残基似乎没有形成内部盐桥。我们预计它们可能与 hCaR N 端胞外域 (ECD) 内的带电残基相互作用。这些研究放大了我们对 hCaR 7TM 核心内相互作用变构位点的初步观察结果，表明钙结合 ECD 通过该残基处的接触与 7TM 核心相互作用。此外，这些数据强烈暗示 ECD 可能是对 7TM 核心活性的抑制性约束。我们已经开始通过独立表达 mGluR1、hCaR 和 5.24 的 ECD 和 7TM 核心结构来测试这一建议。目前，我们拥有高水平表达的 mGluR1 和 hCaR 7TM 核心以及用于 mGluR1 和 5.24 分泌的 ECD 蛋白的杆状病毒构建体。当我们的分子试剂补充完成后，我们将进行三种 ECD 结构的同质分离，并且我们将检查这些构建体的配体结合和 7TM 核心调节特性。目前，5.24 ECD 的表达产量足以获得该蛋白质的晶体结构，与大鼠 mGluR1 ECD 的现有结构进行比较将非常有意义。然而，我们对全长 5.24 受体和自主表达的 5.24 ECD 的初步生化评估驳斥了已发表的该受体对 L-精氨酸有反应的报告。我们目前正在尝试鉴定该受体的配体。我们与 NIDCD 的 Susan Sullivan 博士合作的项目旨在识别编码苦味受体的整个人类基因库所识别的促味剂化合物，该项目在过去的一年中取得了相当大的进展。 Sullivan 博士从人类基因数据库中鉴定出 23 个候选基因，这些基因与已知的小鼠和大鼠苦味受体高度相似，但它们不是嗅觉受体。目前，我们已经构建了杆状病毒载体来表达这些载体，并鉴定了其中 10 个可指导细胞表面局部受体表达的杆状病毒载体。与此同时，NIDCD 的 Dennis Drayna 博士也确定了编码苯硫碳酰亚胺 (PTC) 特征的人类基因座。他的研究揭示了人类中的五种等位基因变异（2种有味，3种无味）。还构建了表达所有这些基因产物的杆状病毒载体。我们正在继续开发一种新的基于细胞的筛选策略来鉴定这些受体的配体。我们构建了富含味道的 G-β-3 和 γ-13 亚基、髓样 α-15/16 蛋白和水母水母蛋白的杆状病毒载体。通过用这些和编码味觉受体的病毒同时感染 Sf9 细胞，我们期望将促味剂启动的 G 蛋白信号传导途径重新引导至化学发光的产生。我们对此的初步测试利用了几种特征明确的受体（5-HT1A、5-HT2C 和 GRP-R）来确认该策略可以成功。此外，小鼠苦味受体 T2R5 的感染成功地产生了对环己酰胺作出反应的化学发光 Sf9 细胞。迄今为止，我们还没有成功地利用这一策略来识别人类苦味受体的任何促味剂。然而，我们已经成功地采用了针对这些受体的体外测定的替代策略