PDF(Pubmed)
Abstract：
The concept of agonist-independent signalling that can be attenuated by inverse agonists is a fundamental element of the cubic ternary complex model of G protein-coupled receptor (GPCR) activation. This model shows how a GPCR can exist in two conformational states in the absence of ligands; an inactive R state and an active R* state that differ in their affinities for agonists, inverse agonists, and G-protein alpha subunits. The proportion of R* receptors that exist in the absence of agonists determines the level of constitutive receptor activity. In this study we demonstrate that mechanical stimulation can induce β2-adrenoceptor agonist-independent Gs-mediated cAMP signalling that is sensitive to inhibition by inverse agonists such as ICI-118551 and propranolol. The size of the mechano-sensitive response is dependent on the cell surface receptor expression level in HEK293G cells, is still observed in a ligand-binding deficient D113A mutant β2-adrenoceptor and can be attenuated by site-directed mutagenesis of the extracellular N-glycosylation sites on the N-terminus and second extracellular loop of the β2-adrenoceptor. Similar mechano-sensitive agonist-independent responses are observed in HEK293G cells overexpressing the A2A-adenosine receptor. These data provide new insights into how agonist-independent constitutive receptor activity can be enhanced by mechanical stimulation and regulated by inverse agonists.
摘要：
可以通过反向激动剂减弱的非激动剂依赖性信号传导的概念是G蛋白偶联受体（GPCR）激活的立方三元复合物模型的基本要素。该模型显示了在不存在配体的情况下，GPCR如何以两种构象状态存在；非活性R状态和活性R*状态对激动剂的亲和力不同，反向激动剂，和G蛋白α亚基。在不存在激动剂的情况下存在的R*受体的比例决定了组成型受体活性的水平。在这项研究中，我们证明了机械刺激可以诱导β2-肾上腺素受体激动剂非依赖性Gs介导的cAMP信号传导，该信号对反向激动剂如ICI-118551和普萘洛尔的抑制敏感。机械敏感反应的大小取决于HEK293G细胞中细胞表面受体的表达水平，在配体结合缺陷型D113A突变体β2-肾上腺素受体中仍然观察到,并且可以通过定点诱变β2-肾上腺素受体的N-末端和第二胞外环上的胞外N-糖基化位点而减弱。在过表达A2A-腺苷受体的HEK293G细胞中观察到类似的机械敏感性激动剂非依赖性应答。这些数据提供了关于不依赖激动剂的组成型受体活性如何通过机械刺激增强和通过反向激动剂调节的新见解。
