A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus -: Evidence for Gαi and Gαo coupling to a human leukotriene B4 receptor
A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus -: Evidence for Gαi and Gαo coupling to a human leukotriene B4 receptor
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DOI：
10.1074/jbc.m302801200
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发表时间：
2003-07-04
影响因子：
4.8
通讯作者：
Hamakubo, T
中科院分区：
文献类型：
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作者：
Masuda, K;Itoh, H;Hamakubo, T
为了研究G蛋白和G蛋白偶联受体（GPCR）的偶联选择性，我们建立了由GPCR和异源三聚体G蛋白在细胞外杆状病毒颗粒（出芽病毒（BV））上的重组系统。编码人白三烯B-4受体（BLT1） cDNA的重组杆状病毒感染Sf9细胞释放的BV显示出高水平的BLT1表达（27.3 pmol/mg蛋白）和特异性的[H-3]白三烯B-4结合活性（K-d = 3.67 nM）。表达的BLT1明显的低亲和力被认为是由于在BV中与BLT1偶联的Galpha(i)异构体相对不可用。异源三聚体G蛋白重组病毒共感染导致BLT1和G蛋白亚基在BV上共表达。在共表达Galpha(i1)beta(1)gamma(2) （K-d = 0.17 nM）的BLT1 BV中，观察到鸟苷-5/-（β， γ -亚胺）三磷酸敏感、高亲和力的配体结合。在共表达的BV部分中，回收了相对较多的高亲和力受体蛋白（6.81 pmol/mg蛋白）。没有Gbeta(1)和gamma(2)的BLT1和Galpha（i1）组合在BV上没有表现出高亲和力的配体结合，这表明GPCR-G蛋白偶联在BV重构系统中的背景环境较低。为了检测其他G蛋白的偶联性，我们将不同的Galpha亚基与BLT1和Gbeta(1)gamma(2)在BV中组合表达。共表达Galpha(oA)beta(1)gamma(2)的BLT1 BV表现出较高亲和力的配体结合以及配体刺激的鸟苷5′-3- o-（硫代）三磷酸与Galpha(i1)beta(1)gamma(2)的结合。Galpha(s)、Galpha（11）、Galpha（14）、Galpha（16）、Galpha（12）或Galpha（13）等其他同种异构体的共表达对该系统中配体结合亲和力没有明显影响。这些结果表明，BLT1和偶联的三聚体G蛋白在BV上功能重构，并且Galpha(o)和Galpha(i)与BLT1偶联。该表达系统将被证明对药理学表征、生物传感器芯片应用以及针对膜受体蛋白高度重要靶点的药物发现非常有用。
To investigate the coupling selectivity of G proteins and G protein-coupled receptors (GPCRs), we developed a reconstitution system made up of GPCR and heterotrimeric G proteins on extracellular baculovirus particles (budded virus (BV)). BV released from Sf9 cells infected with a recombinant baculovirus coding for human leukotriene B-4 receptor (BLT1) cDNA exhibited a high level of BLT1 expression (27.3 pmol/mg of protein) and specific [H-3]leukotriene B-4 binding activity (K-d = 3.67 nM). The apparent low affinity of the expressed BLT1 is thought to be due to relative non-availability of the Galpha(i) isoform, which couples to BLT1, in BV. Co-infection of heterotrimeric G protein recombinant viruses led to co-expression of BLT1 and G protein subunits on BV. A guanosine-5/-(beta,gamma-imido)triphosphate-sensitive, high affinity ligand binding was observed in the BLT1 BV co-expressing Galpha(i1)beta(1)gamma(2) (K-d = 0.17 nM). A relatively large amount of high affinity receptor protein was recovered in the co-expressing BV fraction (6.81 pmol/mg of protein). A combination of BLT1 and Galpha(i1) without Gbeta(1)gamma(2) did not exhibit high affinity ligand binding on BV, indicating the low background environment for the GPCR-G protein coupling in this BV reconstitution system. To test other G proteins for coupling, various Galpha subunits were combinatorially expressed in BV with BLT1 and Gbeta(1)gamma(2). The BLT1 BV co-expressing Galpha(oA)beta(1)gamma(2) exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5'-3-O-(thio)triphosphate binding to Galpha(i1)beta(1)gamma(2). Co-expression of other Galpha isoforms such as Galpha(s), Galpha(11), Galpha(14), Galpha(16), Galpha(12), or Galpha(13) did not exhibit any significant effects on ligand binding affinity in this system. These results reveal that BLT1 and coupled trimeric G proteins were functionally reconstituted on BV and that Galpha(o) as well as Galpha(i) couples to BLT1. This expression system should prove highly useful for pharmacological characterization, biosensor chip applications, and also drug discovery directed at highly important targets of the membrane receptor proteins.