Emergent cellular functions of GPCRs and myosins

项目摘要 细胞信号和膜交通来自一系列动态的、瞬时的蛋白质-蛋白质相互作用 (PPI)在拥挤的环境中。传统的结构和生化方法大多局限于解剖。 稳定、结构化的PPI的功能。为了解决由瞬时PPI产生的紧急功能，我的研究 该计划开发创新的蛋白质工程和生物物理技术。我们调查杰出的 肌球蛋白gpr-G蛋白选择性和细胞表面受体激活的问题。研究将推动 GPCRs和肌球蛋白的基础细胞生物学，同时提供抗击疾病的新治疗策略。 在我实验室的新技术和概念进步的基础上，我们提出了五个平行的研究项目。 (1)我们发现并表征了序列gpr-G蛋白相互作用的时间耦合，导致 对gpr信号的同种异位开尔酸调节。我们将通过以下方式剖析异能凯酸调制的结构基础 GPCRs序列发散的第三胞内环(ICL3)。使用新的生物传感器和受体嵌合体， 我们将在密切相关的受体亚型中确定ICL3在自我调节和G蛋白选择中的作用。 (2)我们设计了一种简单易用的无细胞生物传感器方法来测量gpr的分子效力。 配基。我们将使用这一检测方法来鉴定和表征受体异构体选择性生物制品，包括 多肽、多肽模拟物和纳米体/亲和体。这些生物制品将作为探针，推动 GPCR-G蛋白选择性的结构基础和产生选择性靶向GPCRs的细胞通透性策略。 (3)我们成功地集成了计算-实验协作来揭示GPCR的动态重塑 G蛋白选择的细胞质空腔。使用这一策略，我们将映射时间上持续的受体- 跨GPCRs的G蛋白相互作用热点，编码G蛋白选择性。我们将解剖结构 通过在GPCRs内分散残基间通信网络，为变构调节剂奠定基础。 (4)确定了马达-货物相互作用动力学和机械刚度是两种新的细胞调控机制。 细胞骨架马达的机制。我们将使用可编程的仿生支架来剖析肌球蛋白的调控。 通过受体-适配器和适配器-运动组合。我们关注的是马达构型的影响 不同的细胞表面受体包括β1整合素、网织蛋白D1和LRP2/megalin触发的聚集。 (5)我们将通过受体介导，研究gpr信号中的一种新的时间偏差机制。 膜运输过程中肌球蛋白的结合。我们将描述运动活动的不同调节 通过GPCRC-Tail中的PDZ结合基序。我们将使用光遗传/化学遗传策略来引导gpr。 通过第二信使和Akt/MAPK通路绘制时间信号图谱。

Project Summary Cell signaling and membrane traffic emerge from an ensemble of dynamic, transient protein-protein interactions (PPIs) in a crowded milieu. Traditional structural and biochemical approaches are mostly limited to dissecting the function of stable, structured PPIs. To address emergent function stemming from transient PPIs, my research program develops innovative protein engineering and biophysical technologies. We investigate outstanding questions in GPCR-G protein selectivity and cell surface receptor activation of myosins. Studies will advance the fundamental cell biology of GPCRs and myosins, while delivering new therapeutic strategies to combat disease. Building on new technologies and conceptual advances from my lab, we propose five parallel research projects. (1) We discovered and characterized the temporal coupling of sequential GPCR-G protein interactions, leading to allokairic modulation of GPCR signaling. We will dissect the structural basis of allokairic modulation through the GPCR’s sequence-divergent third intracellular loop (ICL3). Using novel biosensors and receptor chimeras, we will define roles for ICL3 in autoregulation and G protein selection in closely related receptor isoforms. (2) We engineered a simple, accessible cell-free biosensor assay to measure the molecular efficacy of GPCR ligands. We will use this assay to identify and characterize receptor isoform-selective biologics, including peptides, peptide-mimetics, and nanobodies/affibodies. These biologics will serve as probes to advance the structural basis of GPCR-G protein selectivity and yield cell-permeable strategies to selectively target GPCRs. (3) We successfully integrated a computation-experiment collaboration to reveal the dynamic reshaping of GPCR cytosolic cavities underlying G protein selection. Using this strategy, we will map temporally persistent receptor- G protein interaction hot-spots across GPCRs, that encode G protein selectivity. We will dissect the structural basis of allosteric modulators through the dispersal of inter-residue communication networks within GPCRs. (4) We identified motor-cargo interaction kinetics and mechanical stiffness as two novel cellular regulatory mechanisms of cytoskeletal motors. We will use programmable biomimetic scaffolds to dissect myosin regulation through both receptor-adaptor and adaptor-motor ensembles. We focus on the impact of motor conformation and clustering triggered by diverse cell surface receptors including β1-integrin, plexin D1, and LRP2/megalin. (5) We will investigate a novel temporal bias mechanism in GPCR signaling, through receptor-mediated engagement of myosins during membrane traffic. We will characterize the differential regulation of motor activity through PDZ-binding motifs in the GPCR C-tail. We will use optogenetic/chemogenetic strategies to steer GPCR trafficking and map the temporal signaling profile through second messenger and Akt/MAPK pathways.