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.

项目概要细胞信号传导和膜运输源自动态、瞬时的蛋白质-蛋白质相互作用的整体（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。为了解决瞬态 PPI 产生的紧急功能，我的研究是稳定的、结构化的 PPI 的功能。计划开发创新的蛋白质工程和生物物理技术。 GPCR-G 蛋白选择性和肌球蛋白细胞表面受体激活的问题研究将推动这一领域的发展。 GPCR 和肌球蛋白的基础细胞生物学，同时提供对抗疾病的新治疗策略。基于我实验室的新技术和概念进步，我们提出了五个并行研究项目。 (1) 我们发现并表征了连续 GPCR-G 蛋白相互作用的时间耦合，导致我们将通过以下方式剖析异体调节的结构基础。 GPCR 的序列不同的第三个细胞内环 (ICL3) 使用新型生物传感器和受体嵌合体，我们将定义 ICL3 在密切相关的受体亚型的自动调节和 G 蛋白选择中的作用。 (2) 我们设计了一种简单、易于使用的无细胞生物传感器测定法来测量 GPCR 的分子功效我们将使用该测定来识别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/亲和体将作为探针来推进。 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-实验协作来揭示GPCR的动态重塑使用这种策略，我们将绘制暂时持久的受体图谱。 GPCR 上的 G 蛋白相互作用热点，编码 G 蛋白选择性。通过 GPCR 内残基间通讯网络的分散，形成变构调节剂的基础。 (4) 我们将汽车-货物相互作用动力学和机械刚度确定为两种新型细胞调节我们将使用可编程仿生支架来剖析肌球蛋白的调节。通过受体-适配器和适配器-运动整体，我们关注运动构象的影响。由多种细胞表面受体（包括 β1-整合素、丛蛋白 D1 和 LRP2/巨蛋白）触发的聚类。 (5) 我们将通过受体介导研究 GPCR 信号传导中一种新的时间偏向机制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。运输并通过第二信使和 Akt/MAPK 途径绘制时间信号传导谱。

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.

Dab2-肌球蛋白 VI 复合物的动态多聚化可调节货物的持续合成能力，同时最大限度地减少皮质肌动蛋白重组。

DOI：
发表时间：
2021-01
期刊：
影响因子：
0
作者：
Rai, Ashim;Vang, Duha;Ritt, Michael;Sivaramakrishnan, Sivaraj
通讯作者：
Sivaramakrishnan, Sivaraj

Minute-scale persistence of a GPCR conformation state triggered by non-cognate G protein interactions primes signaling.

由非同源 G 蛋白相互作用触发的 GPCR 构象状态的分钟级持续性引发信号传导。

DOI：
发表时间：
2019
期刊：
影响因子：
16.6
作者：
Gupte, Tejas M;Ritt, Michael;Dysthe, Matthew;Malik, Rabia U;Sivaramakrishnan, Sivaraj
通讯作者：
Sivaramakrishnan, Sivaraj

Optical Mapping of cAMP Signaling at the Nanometer Scale.

纳米尺度 cAMP 信号传导的光学映射。

DOI：
发表时间：
2020
期刊：
影响因子：
64.5
作者：
Bock, Andreas;Annibale, Paolo;Konrad, Charlotte;Hannawacker, Annette;Anton, Selma E;Maiellaro, Isabella;Zabel, Ulrike;Sivaramakrishnan, Sivaraj;Falcke, Martin;Lohse, Martin J
通讯作者：
Lohse, Martin J

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

KIF13A 马达受 Rab22A 调节，在细胞内作为弱二聚体发挥作用。

DOI：
发表时间：
2021
期刊：
影响因子：
13.6
作者：
Patel, Nishaben M;Siva, Meenakshi Sundaram Aravintha;Kumari, Ruchi;Shewale, Dipeshwari J;Rai, Ashim;Ritt, Michael;Sharma, Prerna;Setty, Subba Rao Gangi;Sivaramakrishnan, Sivaraj;Soppina, Virupakshi
通讯作者：
Soppina, Virupakshi

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。