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Emergent cellular functions of GPCRs and myosins

GPCR 和肌球蛋白的新兴细胞功能

基本信息

批准号：
10550541
负责人：
Sivaraj Sivaramakrishnan
金额：
$ 47.78万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-01 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10550541
关键词：
Address Binding Biochemical Biological Assay Biological Products Biomimetics Biophysics Biosensor Cell Surface Receptors Cell membrane Cell physiology Cells Cellular biology Chimera organism Collaborations Communication Coupling Crowding Cytoskeleton Diabetes Mellitus Disease Engineering G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Heart failure Homeostasis Integrins Kinetics LDL-Receptor Related Protein 2 Ligands MAP Kinase Gene Maps Measures Mechanics Mediating Membrane Protein Traffic Molecular Molecular Conformation Motor Motor Activity Myosin ATPase Pathway interactions Peptides Permeability Protein Engineering Protein Isoforms Receptor Activation Regulation Research Research Project Grants Role Second Messenger Systems Shapes Signal Transduction Structure Tail Technology cell motility combat cytosolic receptor experimental study genetic approach innovation insight nanobodies neuropsychiatric disorder new technology novel novel therapeutic intervention novel therapeutics optogenetics peptidomimetics plexin programs protein protein interaction receptor scaffold stem trafficking

项目摘要

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.

项目摘要细胞信号传导和膜运输来自动态的、瞬时的蛋白质-蛋白质相互作用的集合（PPI）在拥挤的环境中。传统的结构和生物化学方法大多局限于解剖稳定的、结构化的PPI的功能。为了解决由瞬时PPI引起的紧急功能，我的研究该计划开发创新的蛋白质工程和生物物理技术。我们调查的是 GPCR-G蛋白的选择性和肌球蛋白的细胞表面受体活化的问题。研究将推动 GPCR和肌球蛋白的基础细胞生物学，同时提供新的治疗策略来对抗疾病。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)我们发现并表征了顺序GPCR-G蛋白相互作用的时间耦合，到GPCR信号的变钾调节。我们将通过以下几个方面来剖析变钾调节的结构基础： GPCR的序列趋异的第三胞内环（ICL 3）。使用新型生物传感器和受体嵌合体，我们将确定ICL 3在密切相关的受体亚型中的自动调节和G蛋白选择中的作用。 (2)我们设计了一种简单，无细胞的生物传感器测定来测量GPCR的分子功效配体。我们将使用该检测来鉴定和表征受体亚型选择性生物制剂，包括肽、肽模拟物和纳米抗体/抗体。这些生物制剂将作为探针， GPCR-G蛋白选择性的结构基础，并产生选择性靶向GPCR的细胞渗透策略。 (3)我们成功地整合了计算-实验合作，以揭示GPCR的动态重塑 G蛋白选择的胞浆腔。使用这种策略，我们将映射时间持久性受体- G蛋白相互作用热点跨越GPCR，编码G蛋白的选择性。我们将解剖通过GPCR内残基间通信网络的分散，研究变构调节剂的基础。 (4)我们将运动-货物相互作用动力学和机械刚度确定为两种新的细胞调节因子，细胞骨架马达的机制。我们将使用可编程仿生支架来剖析肌球蛋白的调节通过感受器-适配器和适配器-运动器组合。我们关注的是运动构造以及由包括β1-整联蛋白、丛蛋白D1和LRP 2/megalin的多种细胞表面受体触发的聚集。 (5)我们将通过受体介导的GPCR信号转导研究一种新的时间偏倚机制，肌球蛋白在膜运输期间的接合。我们将描述运动活动的差异调节通过GPCR C尾中的PDZ结合基序。我们将使用光遗传学/化学遗传学策略来引导GPCR 通过第二信使和Akt/MAPK途径的运输和映射时间信号转导谱。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Dynamic multimerization of Dab2-Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization.

DOI：
10.1074/jbc.ra120.012703
发表时间：
2021-01
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Rai A;Vang D;Ritt M;Sivaramakrishnan S
通讯作者：
Sivaramakrishnan S

KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell.

KIF13A电动机由RAB22A调节，以充当细胞内部的弱二聚体。

DOI：
10.1126/sciadv.abd2054
发表时间：
2021-03
期刊：
Science advances
影响因子：
13.6
作者：
Patel NM;Siva MSA;Kumari R;Shewale DJ;Rai A;Ritt M;Sharma P;Setty SRG;Sivaramakrishnan S;Soppina V
通讯作者：
Soppina V

Allosteric modulation of adenosine A1 and cannabinoid 1 receptor signaling by G-peptides.

DOI：
10.1002/prp2.673
发表时间：
2020-12
期刊：
Pharmacology research & perspectives
影响因子：
2.6
作者：
Touma AM;Malik RU;Gupte T;Sivaramakrishnan S
通讯作者：
Sivaramakrishnan S

Optical Mapping of cAMP Signaling at the Nanometer Scale.

在纳米尺度上的cAMP信号传导的光学映射。

DOI：
10.1016/j.cell.2020.07.035
发表时间：
2020-09-17
期刊：
Cell
影响因子：
64.5
作者：
Bock A;Annibale P;Konrad C;Hannawacker A;Anton SE;Maiellaro I;Zabel U;Sivaramakrishnan S;Falcke M;Lohse MJ
通讯作者：
Lohse MJ

Stiffness of Cargo-Motor Linkage Tunes Myosin VI Motility and Response to Load.

货物-电机联动装置的刚度可调节肌球蛋白 VI 的运动性和对负载的响应。