GPCR家族C成员VFTM的系统进化分析及靶向GB1-VFTM的荧光探针开发
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摘要
GPCR家族C成员是一类独具特色的GPCR,它们拥有大的细胞外结构域,其中包括捕蝇夹模块VFTM。VFTM主要负责配体的识别与结合,同时与细菌转运小分子氨基酸的PBP具有一定的序列相似性。由于家族C成员的VFTM具有结构相似性,而且其功能在不同亚组中发生了显著的歧化,因此通过系统进化分析以及正向选择性检测,研究家族C成员的VFTM,对于阐述其进化历程显得尤为重要。五个典型的家族C亚组,mGluRs、CaSR、T1R、GB1和GB2,其VFTM被用来检验正向选择压力条件下的功能歧化,从而对各个VFTM的选择性限制改变进行评价。另外,通过推测祖先VFTM的三维结构,从结构上也展示了VFTM在进化历程上的改变形式。
研究结果显示,GPCR家族C成员VFTM具有显著性的选择性限制改变,暗示在家族C的进化历程中,基因复制事件后功能歧化对于塑造各个VFTM的特性起关键性作用。同时,正向选择压力也可能介入到了这个进化历程中,驱动VFTM的功能歧化过程。系统进化也显示,在塑造五个典型的家族C亚组过程中,可能存在三次连续的基因复制事件。五个亚组成员通过各自不同位点的功能歧化,逐步改造和形成自身的结构与功能。因此,GPCR家族C成员VFTM的进化历程,即是正向选择压力驱动功能歧化,通过三次连续的基因复制事件,对VFTM进行结构与功能的改造过程。另外,参与家族C成员VFTM功能歧化的位点可能是潜在的药物靶点,对于进一步研究家族C成员VFTM的精细激活过程以及研发特异性的激动剂或拮抗剂,都将产生重要的研究意义。
为了在活细胞中标记GABA_BR,在拮抗剂CGP64213的基础上,设计、合成并鉴定了一种靶向GB1-VFTM的三元件活性荧光探针,该探针可以用来光标记CHO细胞中表达的GABA_BR。由于该探针能够特异性结合到GB1的VFTM中,并且具有高的光亲和特性,对于在活细胞上研究GABA_BR的定位和功能具有重要意义。
Class C G protein-coupled receptors (GPCRs) represent a distinct group among the large family of GPCRs, which structurally posses a large extracellular domain including a Venus flytrap module (VFTM). The VFTMs of class C GPCRs are responsible for the ligands recognizing and binding, and share sequence similarity with bacterial periplasmic amino acid binding proteins (PBPs) involved in the transport of small molecules like amino acids. Considering the structural homology and the functional divergence in the VFTMs, an extensively phylogenetic investigation based on the functional divergence analysis and the test for positive selection was conducted for five typical groups of class C GPCRs (mGluRs, CaSR, T1R, GB1 and GB2). The altered selective constraints were determined to identify the sites undergone functional divergence driven by the positive selections. In order to structurally demonstrate the change patterns during the evolutionary process, ancestral VFTMs were inferred and reconstructed with three-dimensional (3D) structures. The results indicate that functional divergences driven by positive selection are responsible for the evolutionary patterns via three continuous duplicate events of the VFTMs of class C GPCRs. The sites involved in functional divergence may provide more candidates for further researches on the relationship between structures and functions, and also shed lights on the activation mechanism of class C GPCRs.
In order to label the GABA_b receptors on living cells, a trimodular activity-based fluorescent probe was designed, synthesized and characterized based on the structure of CGP64213, an antagonist of GABAb receptor. This probe can be applied to photoaffinity label the GABAb receptor transiently expressed in Chinese hamster ovary cells. Moreover, it exhibits specific binding activity at the ligand-binding pocket of GB 1 subunits and high specificity of photoaffinity labeling, which makes the probe valuable for studying the localization and function of GABAb receptors on living cells.
研究结果显示,GPCR家族C成员VFTM具有显著性的选择性限制改变,暗示在家族C的进化历程中,基因复制事件后功能歧化对于塑造各个VFTM的特性起关键性作用。同时,正向选择压力也可能介入到了这个进化历程中,驱动VFTM的功能歧化过程。系统进化也显示,在塑造五个典型的家族C亚组过程中,可能存在三次连续的基因复制事件。五个亚组成员通过各自不同位点的功能歧化,逐步改造和形成自身的结构与功能。因此,GPCR家族C成员VFTM的进化历程,即是正向选择压力驱动功能歧化,通过三次连续的基因复制事件,对VFTM进行结构与功能的改造过程。另外,参与家族C成员VFTM功能歧化的位点可能是潜在的药物靶点,对于进一步研究家族C成员VFTM的精细激活过程以及研发特异性的激动剂或拮抗剂,都将产生重要的研究意义。
为了在活细胞中标记GABA_BR,在拮抗剂CGP64213的基础上,设计、合成并鉴定了一种靶向GB1-VFTM的三元件活性荧光探针,该探针可以用来光标记CHO细胞中表达的GABA_BR。由于该探针能够特异性结合到GB1的VFTM中,并且具有高的光亲和特性,对于在活细胞上研究GABA_BR的定位和功能具有重要意义。
Class C G protein-coupled receptors (GPCRs) represent a distinct group among the large family of GPCRs, which structurally posses a large extracellular domain including a Venus flytrap module (VFTM). The VFTMs of class C GPCRs are responsible for the ligands recognizing and binding, and share sequence similarity with bacterial periplasmic amino acid binding proteins (PBPs) involved in the transport of small molecules like amino acids. Considering the structural homology and the functional divergence in the VFTMs, an extensively phylogenetic investigation based on the functional divergence analysis and the test for positive selection was conducted for five typical groups of class C GPCRs (mGluRs, CaSR, T1R, GB1 and GB2). The altered selective constraints were determined to identify the sites undergone functional divergence driven by the positive selections. In order to structurally demonstrate the change patterns during the evolutionary process, ancestral VFTMs were inferred and reconstructed with three-dimensional (3D) structures. The results indicate that functional divergences driven by positive selection are responsible for the evolutionary patterns via three continuous duplicate events of the VFTMs of class C GPCRs. The sites involved in functional divergence may provide more candidates for further researches on the relationship between structures and functions, and also shed lights on the activation mechanism of class C GPCRs.
In order to label the GABA_b receptors on living cells, a trimodular activity-based fluorescent probe was designed, synthesized and characterized based on the structure of CGP64213, an antagonist of GABAb receptor. This probe can be applied to photoaffinity label the GABAb receptor transiently expressed in Chinese hamster ovary cells. Moreover, it exhibits specific binding activity at the ligand-binding pocket of GB 1 subunits and high specificity of photoaffinity labeling, which makes the probe valuable for studying the localization and function of GABAb receptors on living cells.
引文
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[4] Bockaert, J., Claeysen, S., Becamel, C, et al. G protein-coupled receptors: dominant players in cell-cell communication. Int Rev Cytol. 2002, 212: 63-132.
[5] Bockaert, J., Pin, J. P. Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO J. 1999, 18(7): 1723-1729.
[6] Houamed, K. M., Kuijper, J. L., Gilbert, T. L., et al. Cloning, expression, and gene structure of a G protein-coupled glutamate receptor from rat brain. Science. 1991, 252(5010): 1318-1321.
[7] Masu, M., Tanabe, Y., Tsuchida, K., et al. Sequence and expression of a metabotropic glutamate receptor. Nature. 1991, 349(6312): 760-765.
[8] Brown, E. M., Gamba, G., Riccardi, D., et al. Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature. 1993, 366(6455): 575-580.
[9] Kaupmann, K., Huggel, K., Heid, J., et al. Expression cloning of GABA(B) receptors uncovers similarity to metabotropic glutamate receptors. Nature. 1997, 386(6622): 239-246.
[10] Dulac, C., Axel, R. A novel family of genes encoding putative pheromone receptors in mammals. Cell. 1995, 83(2): 195-206.
[11] Cheng, Y., Lotan, R. Molecular cloning and characterization of a novel retinoic acid-inducible gene that encodes a putative G protein-coupled receptor. J Biol Chem. 1998, 273(52): 35008-35015.
[12] Pin, J. P., Galvez, T., Prezeau, L. Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors. Pharmacol Ther. 2003, 98(3): 325-354.
[13] O'Hara, P. J., Sheppard, P. O., Thogersen, H., et al. The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins. Neuron. 1993, 11(1): 41-52.
[14] Boudin, H., Doan, A., Xia, J., et al. Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site. Neuron. 2000, 28(2): 485-497.
[15] Dev, K. K., Nakajima, Y., Kitano, J., et al. PICK1 interacts with and regulates PKC phosphorylation of mGLUR7. J Neurosci. 2000, 20(19): 7252-7257.
[16] El Far, O., Airas, J., Wischmeyer, E., et al. Interaction of the C-terminal tail region of the metabotropic glutamate receptor 7 with the protein kinase C substrate PICK1.Eur J Neurosci. 2000, 12(12): 4215-4221.
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[18] Bouvier, M. Oligomerization of G-protein-coupled transmitter receptors. Nat Rev Neurosci. 2001, 2(4): 274-286.
[19] Pin, J.-P., Galvez, T., Prezeau, L. Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors. Pharmacology & Therapeutics. 2003, 98(3): 325-354.
[20] Kolakowski, L. F., Jr. GCRDb: a G-protein-coupled receptor database. Receptors Channels. 1994,2(1): 1-7.
[21] Fredriksson, R., Lagerstrom, M. C., Lundin, L. G., et al. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol. 2003, 63(6): 1256-1272.
[22] Bowery, N. G. GABA_B receptor: a site of therapeutic benefit. Curr Opin Pharmacol. 2006, 6(1): 37-43.
[23] Takahashi, K., Tsuchida, K., Tanabe, Y., et al. Role of the large extracellular domain of metabotropic glutamate receptors in agonist selectivity determination. J Biol Chem. 1993, 268(26): 19341-19345.
[24] Pin, J. P., Joly, C., Heinemann, S. F., et al. Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors. Embo J. 1994, 13(2): 342-348.
[25] Okamoto, T., Sekiyama, N., Otsu, M., et al. Expression and purification of the extracellular ligand binding region of metabotropic glutamate receptor subtype 1. J Biol Chem. 1998,273(21): 13089-13096.
[26] Parmentier, M. L., Joly, C., Restituito, S., et al. The G protein-coupling profile of metabotropic glutamate receptors, as determined with exogenous G proteins, is independent of their ligand recognition domain. Mol Pharmacol. 1998, 53(4): 778- 786.
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