Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia.
Sci Signal. 2019 Mar 12;12(572):eaas9485. doi: 10.1126/scisignal.aas9485.
G protein-coupled receptors (GPCRs) convert extracellular stimuli to intracellular responses that regulate numerous physiological processes. Crystallographic and biophysical advances in GPCR structural analysis have aided investigations of structure-function relationships that clarify our understanding of these dynamic receptors, but the molecular mechanisms associated with activation and signaling for individual GPCRs may be more complex than was previously appreciated. Here, we investigated the proposed water-mediated, hydrogen-bonded activation switch between the conserved NPxxY motif on transmembrane helix 7 (TMH7) and a conserved tyrosine in TMH5, which contributes to α-adrenoceptor (α-AR) and β-AR activation. Disrupting this bond by mutagenesis stabilized the α-AR and the β-AR in inactive-state conformations, which displayed decreased agonist potency for stimulating downstream IP and cAMP signaling, respectively. Compared to that for wild-type receptors, agonist-mediated β-arrestin recruitment was substantially reduced or abolished for all α-AR and β-AR inactive-state mutants. However, the inactive-state β-ARs exhibited decreased agonist affinity, whereas the inactive-state α-ARs had enhanced agonist affinity. Conversely, antagonist affinity was unchanged for inactive-state conformations of both α-AR and β-AR. Removing the influence of agonist affinity on agonist potency gave a measure of signaling efficacy, which was markedly decreased for the α-AR mutants but little altered for the β-AR mutants. These findings highlight the pharmacological heterogeneity of inactive-state GPCR conformations, which may facilitate the rational design of drugs that target distinct conformational states of GPCRs.
G 蛋白偶联受体 (GPCR) 将细胞外刺激转化为细胞内反应，从而调节众多生理过程。GPCR 结构分析的晶体学和生物物理学进展有助于研究结构-功能关系，这澄清了我们对这些动态受体的理解，但与单个 GPCR 的激活和信号转导相关的分子机制可能比以前认为的更复杂。在这里，我们研究了跨膜螺旋 7 (TMH7) 上保守的 NPxxY 基序和 TMH5 中保守的酪氨酸之间提议的水介导氢键激活开关，该开关有助于 α-肾上腺素能受体 (α-AR) 和 β-肾上腺素能受体 (β-AR) 的激活。通过突变破坏该键，使 α-AR 和 β-AR 稳定在非活性构象中，这分别导致激动剂刺激下游 IP 和 cAMP 信号的效力降低。与野生型受体相比，激动剂介导的β-arrestin 募集对于所有 α-AR 和 β-AR 非活性状态突变体均大大减少或消除。然而，与野生型受体相比，激动剂结合亲和力降低，而激动剂结合亲和力增强。相反，对于两种类型的 AR 的非活性状态，拮抗剂亲和力保持不变。消除激动剂亲和力对激动剂效力的影响给出了信号转导效率的衡量标准，对于 α-AR 突变体，该效率明显降低，而对于 β-AR 突变体则几乎没有改变。这些发现突出了非活性状态 GPCR 构象的药理学异质性，这可能有助于设计针对 GPCR 不同构象状态的药物。
