Martini Lene, Hastrup Hanne, Holst Birgitte, Fraile-Ramos Alberto, Marsh Mark, Schwartz Thue W
Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, University of Copenhagen, Denmark.
Mol Pharmacol. 2002 Jul;62(1):30-7. doi: 10.1124/mol.62.1.30.
Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.
抑制蛋白是一种胞质蛋白，在七跨膜（7TM）受体受到刺激时，通过与受体结合、取代G蛋白并将受体靶向网格蛋白包被小窝来终止信号传导。β-抑制蛋白1与神经激肽NK1受体的C末端融合产生了一种嵌合蛋白，该蛋白在细胞表面有一定程度的表达，但也独立于激动剂刺激而在转铁蛋白标记的回收内体中积累。正如预期的那样，融合蛋白在通过正常的Gq/G11和Gs途径的激动剂诱导信号传导方面几乎完全沉默。NK1-β-抑制蛋白1融合构建体以增加的亲和力结合非肽拮抗剂，但令人惊讶的是，它也以高亲和力和正常亲和力结合两种激动剂，即P物质和神经激肽A。在野生型NK1受体中，神经激肽A（NKA）与P物质竞争结合，尤其是与表观亲和力比功能测定和同源结合测定中确定的低多达1000倍的拮抗剂竞争结合。当NK1受体与G蛋白紧密融合时，这种激动剂之间的现象被消除，但激动剂仍以低亲和力与拮抗剂竞争。相比之下，在NK1-β-抑制蛋白1融合蛋白中，所有配体以相似的亲和力结合，与放射性配体的选择无关，且希尔系数接近1。我们得出结论，与抑制蛋白复合的NK1受体处于高亲和力、稳定的激动剂结合形式，可能最适合进行结构分析，并且当受体被迫仅与单个细胞内蛋白伴侣复合时，它可以表现出几乎理论上理想的结合特性。
