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

我们继续研究了GPCR-G蛋白结合的表面等离子体共振（SPR）测量和GPCR催化的鸟嘌呤核苷酸核苷酸的基本机制（GPCR）激活GPCR-G蛋白结合和GPCR催化鸟嘌呤核苷酸核苷酸核苷酸的体外测定的组合。在过去的一年中，我们完成了对α-I家族与牛Rhodopsin的相互作用的调查。与内源性视网膜G蛋白Transducin（GT）相比，我们从E.Coli产生了和纯化的同质肉豆蔻酰化的α-I1和α-O。所有这些G-Alpha亚基都是与结构相关的α-I家族的成员，但除了细胞表达外，它们还显示出明显的内源GTP汇率，β-Gamma二聚体亲和力和受体特异性。我们的最初发现证实了已发表的报告，该发现表明，视紫红质可以在所有三个α亚基上催化鸟嘌呤核苷酸交换，并在体外几乎相同。然而，与固定的牛视紫红质一起检查，与α-T相比，α-i1和α-O的分离率显着降低了分离率。 alpha-t与视网膜Beta1-Gamma1相结合，半场小于5秒，而Alpha-O的半衰期约为60-90秒，而Alpha-I1的半衰期为1000秒。我们确定这种明显的差异源于Myr-Alpha-I1的生化伪像，在脂质双层结构中自发地分解GDP时，这种差异得到了强烈增强。该蛋白质的GDP分离的实际贡献贡献是微不足道的，这与Rhodopsin-Alpha-I1复合物的寿命延长一致。对Alpha-T的视紫红质催化核苷酸交换的详细动力学检查表明，当前持有的GTP辅助解离从受体中解离的机制可能无法解释这些数据。作为对此的测试，我们在氨基酸残基上构建了α-i1的点突变，这些突变发生了GDP结合和GTP结合α之间的鸟嘌呤核苷酸相互作用的变化。我们对alpha-i1的数据表明，G203A突变体自发交换GDP类似于野生型，但在视紫红质催化的交换中受到了损害。 R208a的突变导致α交换类似于野生型的GDP，但对GTP的亲和力降低。 SPR研究发现，G203Aα-I1与持续时间延长的鸟氨基蛋白结合，与外源性鸟嘌呤核苷酸无关。总之，这些数据表明，从受体中解离活化的α并不需要GTP。而是，受体会催化未结合的α亚基的激活和解离，该亚基随后结合GTP。我们正在使用三种其他GPCR结构继续进行这些研究：结构上不同的头足类动蛋白，重组M1-摩苏丁氏酰乙酰胆碱受体以及人类5-羟色胺受体的5HT2C亚型。我们还继续研究了家族3 GPCR结构的独特特性，研究了代谢剂谷氨酸受体（MGLUR1）和钙敏感受体（CAR）的突变构建体以及金鱼味受体（5.24），据报道是一种精氨酸受体（ARG-R）。以前我们已经报道说，没有氨基末端钙结合域（ECD）的人类汽车（T903-RHOC）的七跨膜螺旋束（7tm）可以通过三个分裂阳离子的分射阳离子相互作用的位点来激活，用于分裂阳离子，多体阳离子，多体阳离子，多体酸，精子酸，五个酸ligigand ligigand inpst 5688888。 T903-RHOC的细胞外回路废除了NPS568，但没有钙激活的Polyarg协同作用。我们已经完成了在全长结构的背景下，在HCAR 7TM的外部序列中，第二个细胞外环和其他带电残基的其他位点的重要性。发现5-丙氨酸突变类同源物强烈激活。特别令人感兴趣的是，在第二个细胞外环（E767）中，单个残基的突变显示出较高的内在活性，并通过钙消除了HCAR的陡峭合作激活。第三个细胞外环的单个残基K831的突变是类似的，但并非如此强烈。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。但是，这对相对充满电的残基似乎并未形成内部盐桥。我们预计它们可能与HCAR的N末端细胞外域（ECD）中的带电残基相互作用。这些研究扩大了我们对HCAR 7TM核心内相互作用的变构位点的初步观察结果，这表明钙结合ECD通过该残基的接触与7TM核心相互作用。此外，这些数据强烈暗示ECD可能是对7TM核心活性的抑制作用。我们已经着手通过独立地表达MGLUR1，HCAR和5.24的ECD和7TM核心结构来测试这一建议。目前，我们对MGLUR1和5.24的分泌ECD蛋白的MGLUR1和HCAR 7TM核心和杆状病毒构建体具有高水平的表达。当我们完成分子试剂的补充后，我们将对三个ECD结构的均匀性进行分离，我们将检查这些构建体的配体结合和7TM核心调节性质。 5.24 ECD目前以足够的屈服来表达，可以娱乐该蛋白质的晶体结构，与大鼠MGLUR1 ECD的可用结构进行比较，这将引起极大的兴趣。但是，我们对全长5.24受体和自主表达的5.24的最初生化评估驳斥了该受体对L-精氨酸反应的已发表的报告。我们目前正在尝试识别该受体的配体。我们与NIDCD苏珊·沙利文（Susan Sullivan）博士合作的项目，试图确定由编码苦味味道受体的整个人类基因所认识到的味道化合物，在过去的一年中取得了长足的进步。 Sullivan博士从与已知的小鼠和大鼠苦味受体相似的人类遗传数据库中鉴定了23个候选基因，但不是嗅觉受体。目前，我们已经构建了用于表达这些的细菌病毒载体，并将其中的10个表征为指导细胞表面局部受体的表达。同时，也是NIDCD的Dennis Drayna博士，已经确定了编码苯基噻吩二酰胺（PTC）特征的人类基因基因座。他的研究揭示了人口中的五种等位基因变体（2种品尝，3种无味）。表达所有这些基因产物的细菌病毒载体也已经构建。我们正在继续开发一种基于细胞的新型筛查策略，以识别这些受体的配体。我们已经为富含味道的G-Beta-3和伽马13亚基，髓样α-15/16蛋白和水母水母蛋白构建了细菌病毒载体。通过使用这些味觉受体的这些病毒同时感染SF9细胞，我们期望将味道剂引发的G蛋白信号传导途径转移到化学发光的产生中。我们对此的初步测试已利用了几种特征良好的受体（5-HT1A，5-HT2C和GRP-R）来确认该策略可以成功。此外，用小鼠苦味受体T2R5感染成功地产生了化学发光SF9细胞，以响应环己酰胺。迄今为止，我们还没有成功地确定人类苦味受体的任何味道。但是，我们已经成功地采用了这些接受的体外测定的替代策略