Structure And Functions Of Signal-transducing G-proteins

信号转导 G 蛋白的结构和功能

• 批准号: 6513789
• 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/6513789
• 关键词: 3T3 cells; G protein; Sf9 cell line; allosteric site; biological signal transduction; bombesin; conformation; cyclic GMP; ketanserin; neuropeptide receptor; phosphodiesterases; piperazines; protein reconstitution; protein structure function; receptor coupling; receptor sensitivity; serotonin inhibitor; serotonin receptor; structural biology; surface plasmon resonance; visual perception

We have completed establishing the methods for measuring G-protein-coupled receptor (GPCR) interactions with G-proteins by surface plasmon resonance spectroscopy (SPR). For these studies we have utilized the binding of extracellular carbohydrates of bovine rhodopsin by mitogenic lectins (concanavalin A, ConA) to immobilize a prototype GPCR on the metal surface substrate for SPR spectroscopy. Rhodopsin immobilized in this manner retained full biochemical activity to catalyze the activation of retinal transducin (Gt). The binding interactions of retinal Galpha and Gbeta-gamma subunits with rhodopsin were profoundly synergistic. Binding of Gbeta-gamma dimers with distinct gamma subunits to rhodopsin, independent of alpha subunit, was readily observable by SPR. Further, these dimers displayed dramatically different binding affinities and kinetics. The physiologically appropriate retinal dimer displayed rapid association and dissociation kinetics, while the other beta-gamma dimers dissociated at more than 100-fold slower rates. These data suggest that the duration of a G-protein-coupled receptor-signaling event is an intrinsic property of the G-protein coupling partners, in particular, the beta-gamma dimer. These findings are being followed up by analysis of the binding interactions of mutant Gt alpha subunits with alterred guanine nucleotide binding properties and of beta-gamma dimers with chimeric gamma subunit chains, as described below. We have continued to explore the structural determinants of Gbeta-gamma selectivity of receptors by analysis of the independent contributions of isoprenoid modification and primary protein structure of gamma subunits. Dimers of beta1gamma1 proteins have lowered affinity for rhodopsin than that for beta1gamma2. However, gamma1 and gamma2 proteins also differ in isoprenoid modification. To test the relative contributions of protein and isoprenoid structures, we constructed mutants of gamma1 and gamma2 subunits encoding the C-terminal recognition sequences for both farnesyl and geranylgeranyl transferases to introduce C15 (farnesyl) or C20 (geranylgeranyl) isoprenoids into gamma1 and gamma2 proteins. Further, we constructed a set of chimeric protein structures with gamma1 and gamma2 sequences replacing homologous sequence all with the identical C-terminal geranylgeranyl transferase recognition sequence. All proteins were expressed as dimers with the beta1 gene product using baculoviral vectors in Sf9 cells, and the expressed beta-gamma dimmers were purified to near homogeneity for in vitro biochemical and biophysical analyses. The biochemical activity tests reveal that all constructs display equivalent interaction with the retinal G-protein alpha-t, expect a mutant constructed to contain no isoprenoid modification, which is severely reduced in affinity for alpha. However, both isoprenoid and protein modifications result in differing affinities for rhodopsin interaction, tested either for activation of the alpha-t or by surface plasmon resonance measurement of binding to rhodopsin. The isoprenoid modifications of the expressed mutant and chimeric gamma chains were confirmed by mass spectrometry, revealing that the prenyltransferase specificities are not uniquely determined by the carboxyl terminal 3 amino acids (CAAX motif). However, the products were sufficiently correctly modified to allow the assignment of the C-terminal third of the gamma chain as a major determinant of rhodopsin affinity of the beta-gamma dimmer. In addition, they reveal that isoprenoid modification has a more significant impact on gamma1 affinity for rhodopsin than for gamma2. This past year we have extended our characterization of the mechanism of activation of class3 GPCR using the metabotropic glutamate receptor (mGluR1a) and the calcium sensing receptor (CaR). Our initial experiments were designed to address the function(s) of the large amino-terminal domains of these receptors, by expressing chimeric receptors (mGluR1a/CaR and CaR/mGluR1a) bearing the N-terminal domain of the rat mGluR1a replacing homologous sequence of the human CaR and the converse. We also constructed a soluble, secreted N-terminal domain of mGluR1a (mGluR-N-term) and a membrane anchored form of the N-terminal domain consisting of the amino-terminal domain and the first transmembrane helix of the mGluR1a (mGluR1a-tm). The properties of the mGluR1a, CaR, mGluR1a/CaR and mGluR1a-tm were examined by expression in HEK293 cells either transiently or stably transformed. Quantitative examination of the binding and signaling properties of these receptors reveals that the chimeric receptors are considerably less efficient in signaling, with an EC50/Kd ratio some 7-fold higher that the wild-type mGluR1a. Further, maximal PI-hydrolysis response in HEK293 cells expressing equivalent abundances of mGluR1a/CaR or CaR/mGluR1a are some 5-fold lower than for mGluR1a or CaR parent structures. These data indicate that signaling initiated by binding of ligand to a class3 GPCR is transmitted by amino-terminal domain interaction with the transmembrane helix bundle of the receptors. In addition, the contact sites for this interaction are sufficiently, but not completely, conserved between mGluR1a and CaR such that the domain swaps produce a functional receptor with reduced signaling efficiency. To examine the intramolecular mechanism(s) of class3 GPCR, we have expressed the transmembrane helix bundles of the CaR and mGluR1a without their amino-terminal domains. The signaling activities of these structures are readily measured in membranes from HEK293 cells expressing them as measured by catalytic activation of Gq protein. Further, the CaR 7TM structure contains multiple, interactive ligand regulatory sites for divalent metal ion, basic amino acids and the novel allosteric ligand (NPS568). These allosteric sites are functioning within the full-length structure, since the metal and NPS regulation also is exhibited by the mGluR1a/CaR, but not the CaR/mGluR1a chimera. These data indicate that the regulation of class3 GPCR is more complex than that for rhodopsin-homologous receptors, with the structures exhibiting unique allosteric regulatory sites and properties.

我们已经完成了通过表面等离子体等离子体共振光谱（SPR）与G蛋白偶联受体（GPCR）相互作用的方法的方法。在这些研究中，我们利用有丝分裂凝集素（康那肽A，CONA）将牛ropopsin的细胞外碳水化合物结合，以将原型GPCR固定在金属表面底物上以进行SPR光谱。以这种方式固定的视紫红素保留了全面的生化活性，以催化视网膜转丁蛋白（GT）的激活。视网膜galpha和Gbeta-gamma亚基与视紫红质的结合相互作用是深刻的协同作用。 SPR可以很容易地观察到具有独立于α亚基的Rhopopsin的Gbeta-Gamma二聚体与独立于Alpha亚基的视紫红质的结合。此外，这些二聚体表现出截然不同的结合亲和力和动力学。生理上适当的视网膜二聚体显示出快速的关联和解离动力学，而其他β-gamma二聚体以速度慢100倍以上的速率解离。这些数据表明，G蛋白偶联受体信号事件的持续时间是G蛋白耦合伙伴，尤其是Beta-Gamma二聚体的内在特性。这些发现是通过分析突变GT Alpha亚基与鸟嘌呤核苷酸结合特性的结合相互作用以及具有嵌合伽玛亚基链的β-gamma二聚体的结合相互作用，如下所述。 我们继续通过分析类异丙定义的独立贡献和γ亚基的一级蛋白质结构来探索受体GBETA-GAMMA选择性的结构决定因素。 β1GAMMA1蛋白的二聚体比对β1GAMMA2的二聚体降低了对视紫红质的亲和力。然而，γ1和γ2蛋白在类药物修饰方面也有所不同。为了测试蛋白质和异丙裔结构的相对贡献，我们构建了编码Farnesyl和geranylgeranyl转移酶的C末端识别序列的γ1和γ2亚基的突变体，以将C15（Farnesyl）或C20（farnesyl）或C20（geranylgeranyl）（geranylgeranyl）（geranylgeranyl）引入γ1和gamma1和gamma2 poteins。此外，我们用Gamma1和γ2序列构建了一组嵌合蛋白结构，用相同的C末端黄凝酶转移酶识别序列代替同源序列。所有蛋白质均用SF9细胞中的杆状病毒载体用beta1基因产物表示为二聚体，并将表达的β-gamma调光剂纯化为几乎均匀性，以用于体外生化和生物物理分析。生化活性测试表明，所有构建体都与视网膜G蛋白α-T相同的相互作用，期望构成的突变体含有不含类异型的变化，这对α的亲和力大大降低。然而，类异丙因和蛋白质的修饰都导致视紫红蛋白相互作用的亲和力不同，以激活α-T或通过表面等离子体的等离子体等离子体共振测量与杜鹃蛋白的结合。通过质谱法证实了表达突变体和嵌合伽马链的异普尼修饰，表明前转移酶特异性不是由羧基末端3氨基酸（CAAX基序）唯一确定的。但是，对产品进行了充分的正确修改，以使C末端三分之一的伽马链分配为Beta-Gamma Dimmer的Rhodopsin亲和力的主要决定因素。此外，他们揭示了类类化的修饰对Rhodopsin的γ1亲和力的影响比对γ2的影响更大。 在过去的一年中，我们使用代谢型谷氨酸受体（MGLUR1A）和钙传感受体（CAR）扩展了Class3 GPCR激活机制的表征。我们的最初实验旨在通过表达带有大鼠MGlUR1A的N末端结构域的嵌合受体（MGLUR1A/CAR和CAR/MGLUR1A）来解决这些受体的大型氨基末端结构域的功能。我们还构建了MGLUR1A（MGLUR-N-TERM）的可溶的，分泌的N末端结构域，并构建了由氨基末端结构域和MGLUR1A（MGLUR1A-TM）组成的N末端结构域的膜锚固形式。 MGLUR1A，CAR，MGLUR1A/CAR和MGLUR1A-TM的特性通过瞬时或稳定转化的HEK293细胞中的表达进行了检查。对这些受体的结合和信号传导特性的定量检查表明，嵌合受体在信号传导方面的效率较低，EC50/kD的比率比野生型MGlUR1A高出7倍。此外，在HEK293细胞中，最大的pi-hydrolsysis反应表达了MGlUR1A/CAR或CAR/MGLUR1A等效丰度的比较，比MGLUR1A或CAR父结构低5倍。这些数据表明，通过配体与Class3 GPCR结合引发的信号传导是通过氨基末端结构域与受体的跨膜螺旋束的相互作用来传递的。另外，这种相互作用的接触位点足够，但不是完全，在MGlUR1a和CAR之间保守，以使域交换产生具有信号传导效率降低的功能受体。 为了检查3类GPCR的分子内机理，我们表达了汽车和mglur1a的跨膜螺旋束，而没有其氨基末端结构域。这些结构的信号传导活性很容易在HEK293细胞的膜中测量，这些细胞通过GQ蛋白的催化激活来测量它们。此外，CAR 7TM结构包含多个用于二价金属离子，碱性氨基酸和新型变构配体的相互作用配体调节位点（NPS568）。这些变构位点在全长结构内起作用，因为MGLUR1A/CAR也表现出金属和NPS调节，但不显示CAR/MGLUR1A嵌合体。这些数据表明，3类GPCR的调节比Rhopopsin同源受体更复杂，其结构表现出独特的变构调节位点和特性。