Thompson J B, Wade S M, Harrison J K, Salafranca M N, Neubig R R
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-0632, USA.
J Pharmacol Exp Ther. 1998 Apr;285(1):216-22.
Peptides from the intracellular regions of G protein-coupled receptors are useful probes of receptor-G protein coupling mechanisms. As a first step toward the genetic delivery of such "G protein inhibitors," we describe inhibition of angiotensin II (AII) receptor responses by expressed fragments of the second and third intracellular loops of the AT1a receptor (AT1a/i2 and AT1a/i3). Transient transfection of human embryonic kidney 293 cells with DNA encoding the rat AT1a receptor resulted in AII-dependent increases of inositol phosphates (maximum 4.5-fold). Cotransfection of AT1a/i2 and AT1a/i3 fragments raised the EC50 for AII stimulation of phospholipase C activity 5-fold (from 0.18 nM to 0.99 nM, n = 12, P < .001) and 3-fold (from 0.38 nM to 1.2 nM, n = 8, P < .002), respectively. The combined effect of AT1a/i2 and AT1a/3 was additive, and transfection of an alpha-1b adrenergic receptor third intracellular loop (alpha1b/i3) fragments also increased the EC50 for AII. Neither AT1a/i1 nor C-terminus (AT1a/Ct) constructs had significant effects on angiotensin responses. These data confirm a role for the second and third intracellular loops in angiotensin receptor responses and show the potential of this approach to blocking multiple phospholipase C-linked receptors.
来自G蛋白偶联受体细胞内区域的肽是研究受体-G蛋白偶联机制的有用探针。作为向这种“G蛋白抑制剂”进行基因递送的第一步，我们描述了通过AT1a受体（AT1a/i2和AT1a/i3）的第二和第三细胞内环的表达片段对血管紧张素II（AII）受体反应的抑制作用。用编码大鼠AT1a受体的DNA瞬时转染人胚肾293细胞，导致AII依赖性的肌醇磷酸增加（最大增加4.5倍）。共转染AT1a/i2和AT1a/i3片段分别使AII刺激磷脂酶C活性的EC50提高了5倍（从0.18 nM提高到0.99 nM，n = 12，P <.001）和3倍（从0.38 nM提高到1.2 nM，n = 8，P <.002）。AT1a/i2和AT1a/3的联合作用是相加的，并且转染α-1b肾上腺素能受体第三细胞内环（α1b/i3）片段也增加了AII的EC50。AT1a/i1和C末端（AT1a/Ct）构建体对血管紧张素反应均无显著影响。这些数据证实了第二和第三细胞内环在血管紧张素受体反应中的作用，并显示了这种方法阻断多个磷脂酶C偶联受体的潜力。
