Regulation of substance P receptor affinity by guanine nucleotide-binding proteins.
Regulation of substance P receptor affinity by guanine nucleotide-binding proteins.
复制标题
鸟嘌呤核苷酸结合蛋白对 P 物质受体亲和力的调节。
DOI：
10.1111/j.1471-4159.1989.tb07323.x
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发表时间：
1989
影响因子：
4.7
通讯作者：
Boyd,ND
中科院分区：
文献类型：
--
作者：
Macdonald,SG;Boyd,ND
P 物质 (SP) 与外周组织以及中枢神经系统中的受体的结合受到鸟嘌呤核苷酸的调节。在本报告中，我们提供了直接证据，表明这种效应是由鸟嘌呤核苷酸结合调节蛋白（G 蛋白）介导的，而鸟嘌呤核苷酸结合调节蛋白是 SP 与其受体高亲和力结合所必需的。大鼠颌下腺膜与 SP 和 I25I 标记的 Bolton-Hunter 试剂 (125I-BHSP) 的缀合物结合，具有高亲和力 (KD= 1.2 ± 0.4 × 10−9M) 和对鸟嘌呤核苷酸抑制的敏感性。用碱性缓冲液（pH 11.5）处理膜会导致 125I-BHSP 的高亲和力、GTP 敏感结合丧失，并同时丧失 [35S]鸟苷 5'-(3-O-硫代)三磷酸 ([35S]GTPγS) 结合活性。将来自牛脑的纯化 G 蛋白添加到碱处理的膜中可恢复高亲和力 125 I-BHSP 结合。当 G 蛋白以比 SP 结合位点化学计量过量 30 倍的 GTPγS 结合位点掺入碱处理膜时，重构达到最大。 Go（一种具有 39,000 道尔顿 α 亚基的百日咳毒素敏感 G 蛋白）和 Gi（介导腺苷酸环化酶抑制的 G 蛋白）似乎同样有效，而 Gois 的分离 α 亚基则没有效果。添加的G-蛋白的作用在相同的核苷酸浓度范围内被鸟嘌呤核苷酸特异性逆转，从而降低125 I-BHSP与天然膜的高亲和力结合。尽管我们的结果表明，大鼠颌下腺膜中的 SP 受体与具有与 Go/Gi 类似的核苷酸特异性的 G 蛋白偶联，但相关 G 蛋白在对百日咳毒素治疗的敏感性方面似乎与 Go 和 Giin 不同。所描述的重构方法应该在未来的研究中有用，以纯化这种 G 蛋白并进一步分析其与 SP 受体的相互作用。
The binding of substance P (SP) to receptors in peripheral tissues as well as in the CNS is subject to regulation by guanine nucleotides. In this report, we provide direct evidence that this effect is mediated by a guanine nucleotide‐binding regulatory protein (G‐protein) that is required for high‐affinity binding of SP to its receptor. Rat submaxillary gland membranes bind a conjugate of SP andI25I‐labeled Bolton‐Hunter reagent (125I‐BHSP) with high affinity (KD= 1.2 ± 0.4 × 10−9M) and sensitivity to guanine nucleotide inhibition. Treatment of the membranes with alkaline buffer (pH 11.5) causes a loss of the high‐affinity, GTP‐sensitive binding ofl25I‐BHSP and a parallel loss of [35S]guanosine 5′‐(3‐O‐thio)triphosphate ([35S]GTPγS) binding activity. Addition of purified G‐proteins from bovine brain to the alkaline‐treated membranes restores high‐affinity125I‐BHSP binding. Reconstitution is maximal when the G‐proteins are incorporated into the alkaline‐treated membranes at a 30‐fold stoichiometric excess of GTPγS binding sites over SP binding sites. Both Go(a pertussis toxin‐sensitive G‐protein having a 39,000‐dalton α‐subunit) and Gi(the G‐protein that mediates inhibition of adenylate cyclase) appear to be equally effective, whereas the isolated α‐subunit of Gois without effect. The effects of added G‐proteins are specifically reversed by guanine nucleotides over the same range of nucleotide concentrations that decreases high‐affinity binding of125I‐BHSP to native membranes. Although our results indicate that SP receptors in rat submaxillary gland membranes are coupled to a G‐protein that possesses a nucleotide specificity similar to that of Go/Gi, the relevant G‐protein appears to differ from Goand Giin terms of its sensitivity to pertussis toxin treatment. Reconstitution methods described should be useful in future studies to purify this G‐protein and to analyze further its interaction with the SP receptor.