Mechanisms Regulating B2AR Sorting to Signaling Microdomains

调节 B2AR 排序至信号微域的机制

基本信息

批准号：
10686148
负责人：
Ian Basil Chronis
金额：
$ 4.01万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10686148
关键词：
Actins Agonist Alanine Binding Biological Assay Biosensor C-terminal Cardiac Myocytes Cardiovascular Diseases Catecholamines Cause of Death Cell membrane Cell surface Cells Complex Cyclic AMP-Dependent Protein Kinases Cysteine Data Drug Prescriptions Drug Targeting Drug usage Endosomes Fluorescence Microscopy Foundations G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Gene Expression Profile Genes Genetic Genetic Transcription Goals Heart Heart failure Human Impairment Intracellular Membranes Label Ligands Measures Mediating Membrane Membrane Microdomains Microscopy Modeling Molecular Molecular Conformation Molecular Genetics Mutate Pathway interactions Pharmaceutical Preparations Phase Phosphorylation Play Post-Translational Protein Processing Process Proteins Receptor Activation Receptor Signaling Receptors, Adrenergic, beta-1 Recycling Regulation Research Resolution Role Serine Signal Transduction Signaling Protein Site Sorting Surface System Tail Testing Time Tubular formation United States Variant beta-2 Adrenergic Receptors cardioprotection clinically relevant experimental study extracellular functional improvement genetic regulatory protein heart function live cell microscopy new therapeutic target novel palmitoylation prevent protein complex receptor receptor recycling response serine receptor socioeconomics sorting nexins trafficking

项目摘要

PROJECT SUMMARY The beta 2 adrenergic receptor (B2AR) is a G Protein Coupled Receptor (GPCR) that plays a significant role in catecholamine signaling in the heart, especially during periods of heart failure. B2AR signaling in heart failure is incompletely understood, with contradictory data indicating both cardioprotective and deleterious impacts. It has become clear in recent years that changes in B2AR localization within the cell are major regulators of signaling and contribute to variation in response to differential stimulation at the same receptor. Once activated, B2AR can signal via the cognate G protein Gs at the cell surface and in intracellular endosomal compartments. The endosomal signaling promotes transcription of particular genes, most of which are not stimulated by B2AR activation at the plasma membrane. Gs activation by B2AR at endosomes is tightly controlled by posttranslational modifications of the B2AR C-terminal tail. Phosphorylation of serines 345 and 346 (SS345/6) on the receptor tail by protein kinase A (PKA) following agonist stimulation is required for B2AR sorting to specific tubular domains from which it can signal via Gs. PKA inhibition or mutating SS345/6 to alanine residues that cannot be phosphorylated prevents B2AR signaling from endosomes. These manipulations also increase the rate at which B2AR recycles to the plasma membrane by allowing the receptor to enter additional bulk recycling tubules which are unavailable to wild type B2AR and from which it cannot signal. The protein interactions governing this regulation are unknown. Phosphorylation of SS345/6 is also regulated by the presence of palmitoylation at B2AR cysteine 341. Abolition of palmitoylation at this site results in a significant increase in basal SS345/6 phosphorylation in the absence of agonist stimulation. This proposal tests the role of specific protein complexes in localizing B2AR to specific intracellular membrane domain, the role of phosphorylation and palmitoylation in this localization, and the functional relevance of this localization to signaling in the heart. I will use fluorescence microscopy and quantitative real-time PCR to determine the impact of candidate proteins on B2AR sorting to endosomal microdomains and signaling from these compartments in both HEK293 cells and primary cardiomyocytes. I will also use novel unbiased proximity labeling approaches to identify and quantify transient interactions of regulatory proteins with wild type, phosphorylation-deficient, and palmitoylation-deficient B2AR. The role of palmitoylation in regulating this process will be examined using functional genetics and microscopy with conformational biosensors that can identify both B2AR localization and signaling. The proposed research, by characterizing pathways with significant impact on the function of cardiomyocytes, will potentially identify new druggable targets that improve the function of the failing heart and alleviate heart failure.

项目摘要 β2肾上腺素能受体（B2AR）是G蛋白偶联受体（GPCR），在心脏中的儿茶酚胺信号传导，尤其是在心力衰竭时期。心力衰竭中的B2AR信号传导是不完全理解的是，矛盾的数据表明心脏保护和有害影响。它有近年来变得清楚，单元内B2AR定位的变化是信号传导的主要调节因子并有助于对同一受体下差异刺激的响应变化。激活后，B2AR 可以通过细胞表面和细胞内内体室中的同源G蛋白GS发出信号。这内体信号传导促进了特定基因的转录，其中大多数不是由B2AR刺激的质膜的激活。 B2AR处于内体的GS激活受到翻译后的紧密控制 B2AR C末端尾部的修改。受体尾巴上丝氨酸345和346（SS345/6）的磷酸化 B2AR对特定管状结构域进行B2AR分类需要蛋白激酶A（PKA）（PKA）（PKA）它可以通过GS发出信号。 PKA抑制或突变为无法是丙氨酸残基的SS345/6 磷酸化可防止内体的B2AR信号传导。这些操作还提高了通过允许受体进入其他大量回收小管，B2AR回收到质膜对于野生型B2AR不可用，无法发出信号。控制这个的蛋白质相互作用调节是未知的。 SS345/6的磷酸化也受B2AR的棕榈酰化的调节半胱氨酸341。废除该地点的棕榈酰化导致基底SS345/6显着增加在没有激动剂刺激的情况下，磷酸化。该建议测试特定蛋白质复合物的作用在将B2AR定位到特定细胞内膜结构域时，磷酸化和棕榈酰化的作用这种本地化以及该定位与心脏信号的功能相关性。我将使用荧光显微镜和定量实时PCR，以确定候选蛋白对B2AR排序的影响 HEK293细胞中这些隔室的内体微区和信号传导心肌细胞。我还将使用新颖的无偏接近标记方法来识别和量化瞬态调节蛋白与野生型，磷酸化缺陷和棕榈酰化缺陷型B2AR的相互作用。棕榈酰化在调节这一过程中的作用将使用功能遗传学和显微镜检查构象生物传感器可以识别B2AR定位和信号传导。拟议的研究，通过表征对心肌细胞功能的显着影响的途径，可能会确定新的可吸毒的目标，可以改善心脏失败的功能并减轻心力衰竭。