GPCRs, Pain and Inflammation Team, UMR7242, CNRS-University of Strasbourg, LabEx Medalis, 67412 Illkirch, France.
J Cell Sci. 2014 Feb 15;127(Pt 4):773-87. doi: 10.1242/jcs.136432. Epub 2013 Dec 11.
The mTORC1 kinase promotes cell growth in response to growth factors by activation of receptor tyrosine kinase. It is regulated by the cellular energy level and the availability of nutrients. mTORC1 activity is also inhibited by cellular stresses through overexpression of REDD1 (regulated in development and DNA damage responses). We report the identification of REDD1 in a fluorescent live-imaging screen aimed at discovering new proteins implicated in G-protein-coupled receptor signaling, based on translocation criteria. Using a sensitive and quantitative plasma membrane localization assay based on bioluminescent resonance energy transfer, we further show that a panel of endogenously expressed GPCRs, through a Ca(2+)/calmodulin pathway, triggers plasma membrane translocation of REDD1 but not of its homolog REDD2. REDD1 and REDD2 share a conserved mTORC1-inhibitory motif characterized at the functional and structural level and differ most in their N-termini. We show that the N-terminus of REDD1 and its mTORC1-inhibitory motif participate in the GPCR-evoked dynamic interaction of REDD1 with the plasma membrane. We further identify REDD1 as a novel effector in GPCR signaling. We show that fast activation of mTORC1 by GPCRs correlates with fast and maximal translocation of REDD1 to the plasma membrane. Overexpression of functional REDD1 leads to a reduction of mTORC1 activation by GPCRs. By contrast, depletion of endogenous REDD1 protein unleashes mTORC1 activity. Thus, translocation to the plasma membrane appears to be an inactivation mechanism of REDD1 by GPCRs, which probably act by sequestering its functional mTORC1-inhibitory motif that is necessary for plasma membrane targeting.
mTORC1 激酶通过激活受体酪氨酸激酶对生长因子做出反应，促进细胞生长。它受细胞能量水平和营养物质可用性的调节。mTORC1 活性也通过 REDD1（发育和 DNA 损伤反应中的调节因子）的过表达被细胞应激抑制。我们报告了 REDD1 的鉴定，该鉴定是在荧光活细胞成像筛选中发现的，该筛选旨在根据易位标准发现新的与 G 蛋白偶联受体信号转导相关的蛋白质。我们使用基于生物发光共振能量转移的灵敏和定量质膜定位测定法进一步表明，一组内源性表达的 GPCR 通过 Ca(2+)/钙调蛋白途径触发 REDD1 但不是其同源物 REDD2 的质膜易位。REDD1 和 REDD2 具有保守的 mTORC1 抑制基序，在功能和结构水平上具有特征性，并且在其 N 末端差异最大。我们表明 REDD1 的 N 末端及其 mTORC1 抑制基序参与 REDD1 与质膜之间的 GPCR 诱导的动态相互作用。我们进一步将 REDD1 鉴定为 GPCR 信号转导中的一种新型效应物。我们表明，GPCR 快速激活 mTORC1 与 REDD1 快速且最大程度地易位到质膜相关。功能性 REDD1 的过表达导致 GPCR 对 mTORC1 的激活减少。相比之下，内源性 REDD1 蛋白的耗竭会释放 mTORC1 活性。因此，向质膜的易位似乎是 GPCR 使 REDD1 失活的机制，这可能通过隔离其功能上的 mTORC1 抑制基序来发挥作用，该基序对于质膜靶向是必需的。
