Use of probes with fluorescence indicator distributed throughout the pharmacophore to examine the peptide agonist-binding environment of the family B G protein-coupled secretin receptor
Use of probes with fluorescence indicator distributed throughout the pharmacophore to examine the peptide agonist-binding environment of the family B G protein-coupled secretin receptor
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DOI：
10.1074/jbc.m509197200
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发表时间：
2006-02-03
影响因子：
4.8
通讯作者：
Miller, LJ
中科院分区：
文献类型：
--
作者：
Harikumar, KG;Hosohata, K;Miller, LJ
荧光技术可以深入了解位于配体内不同位点的荧光指示剂的环境，因为它与受体结合。在这里，我们开发了一系列27个氨基酸的激素分泌素的类似物，将荧光Alexa Fluor 488结合到氨基端，羧基端和位置13和22。每个探针都具有高亲和力和生物活性，在受体携带的中国仓鼠卵巢secr细胞中刺激充分的cAMP反应。用10 μ M鸟苷5′-（β， γ -酰亚胺）三磷酸（GppNHp）处理后，激动剂结合的受体进入G蛋白解偶联的低亲和力状态。探针在溶液中和与受体结合的荧光光谱显示，在481 nm激发后，最大发射波长为521 nm。荧光与碘化钾的碰撞猝灭表明，分泌素氨基端的Alexa比探针内其他三个位置更容易接近。值得注意的是，每个探针在活性状态下的猝灭常数都高于在G蛋白不偶联、受体低亲和力状态下的猝灭常数，其中最显著的变化发生在两个中间区域探针。各向异性值和荧光寿命证实了这一点，在解耦状态下与受体结合的位置13和22探针的各向异性和寿命比在活性状态下观察到的更长。这些观察结果表明，与受体对接的分泌素的氨基端最容易暴露于亲水的水环境中，并且主要的构象变化和暴露于介质发生在分泌素的中间区域。光亲和标记研究已经证明了这些配体残基与不同受体残基的近似。将荧光数据和光亲和标记数据相结合，可以深入了解与B族G蛋白偶联受体对接的天然肽配体的构象和动力学。
Fluorescence techniques can provide insight into the environment of fluorescence indicators situated at distinct sites within a ligand as it is bound to its receptor. Here, we have developed a series of analogues of the 27-amino acid hormone, secretin, that incorporate a fluorescent Alexa Fluor 488 into the amino terminus, the carboxyl terminus, and positions 13 and 22. Each probe bound with high affinity and was biologically active, stimulating full cAMP responses in receptor-bearing Chinese hamster ovary-SecR cells. Treatment with 10 mu M guanosine 5'-(beta, gamma-imido) triphosphate (GppNHp) shifted the agonist-bound receptor into a G protein-uncoupled low affinity state. Fluorescence spectra for the probes in solution and bound to the receptor demonstrated maximal emission at 521 nm after excitation at 481 nm. Collisional quenching of fluorescence with potassium iodide revealed that Alexa at the amino terminus of secretin was more accessible than at the other three positions within the probes. Of note, quenching constants for each probe were higher when bound in the active state than in the G protein-uncoupled, low affinity state of the receptor, with the most marked changes occurring for the two midregion probes. Anisotropy values and fluorescence lifetimes confirmed this, with higher anisotropy and longer lifetimes observed for position 13 and 22 probes bound to the receptor in its uncoupled state than in its active state. These observations suggest that the amino terminus of secretin as docked to the receptor is most exposed to the hydrophilic aqueous milieu, and that the major changes in conformation and exposure to the medium occur in the midregion of secretin. Photoaffinity labeling studies have demonstrated approximation of each of these ligand residues with distinct receptor residues. Combining the fluorescence data with photoaffinity labeling data provides insights into the conformation and dynamics of a natural peptide ligand docked to a Family B G protein-coupled receptor.