Mechanisms Regulating B2AR Sorting to Signaling Microdomains

调节 B2AR 排序至信号微域的机制

• 批准号: 10543061
• 负责人: Ian Basil Chronis
• 金额: $ 3.91万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 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/10543061
• 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 - Cell Movement; Surface; System; Tail; Testing; Time; Tubular formation; United States; Variant; beta-2 Adrenergic Receptors; cardioprotection; clinically relevant; experimental study; extracellular; genetic regulatory protein; heart function; improved functioning; live cell microscopy; new therapeutic target; novel; palmitoylation; prevent; protein complex; receptor; receptor recycling; response; serine receptor; socioeconomics; sorting nexins; trafficking

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.

项目概要 β2 肾上腺素能受体 (B2AR) 是一种 G 蛋白偶联受体 (GPCR)，在 心脏中的儿茶酚胺信号传导，尤其是在心力衰竭期间。心力衰竭中的 B2AR 信号传导是 尚不完全了解，相互矛盾的数据表明有心脏保护作用和有害影响。它有 近年来人们逐渐清楚，细胞内 B2AR 定位的变化是信号传导的主要调节因素 并导致同一受体对不同刺激的反应发生变化。一旦激活，B2AR 可以通过细胞表面和细胞内内体区室中的同源 G 蛋白 Gs 发出信号。这 内体信号传导促进特定基因的转录，其中大多数基因不受 B2AR 刺激 质膜处的激活。 B2AR 在内体上的 Gs 激活受到翻译后严格控制 B2AR C 末端尾部的修饰。受体尾部丝氨酸 345 和 346 (SS345/6) 的磷酸化 B2AR 分选特定管状结构域需要激动剂刺激后的蛋白激酶 A (PKA) 它可以通过 Gs 发出信号。 PKA 抑制或将 SS345/6 突变为无法被识别的丙氨酸残基 磷酸化可阻止来自内体的 B2AR 信号传导。这些操纵也增加了 B2AR 通过允许受体进入额外的大量回收管而回收到质膜 对于野生型 B2AR 来说是不可用的，并且它不能从中发出信号。控制这一点的蛋白质相互作用 监管未知。 SS345/6 的磷酸化也受到 B2AR 处棕榈酰化的调节 半胱氨酸 341。取消该位点的棕榈酰化会导致基础 SS345/6 显着增加 在没有激动剂刺激的情况下磷酸化。该提案测试了特定蛋白质复合物的作用 在将 B2AR 定位到特定的细胞内膜结构域中，磷酸化和棕榈酰化的作用 这种定位，以及这种定位与心脏信号传导的功能相关性。我将使用荧光 显微镜和定量实时 PCR 以确定候选蛋白对 B2AR 分选的影响 HEK293 细胞和原代细胞中的内体微结构域和来自这些区室的信号传导 心肌细胞。我还将使用新颖的无偏邻近标记方法来识别和量化瞬态 调节蛋白与野生型、磷酸化缺陷型和棕榈酰化缺陷型 B2AR 的相互作用。 将使用功能遗传学和显微镜检查棕榈酰化在调节这一过程中的作用 具有可以识别 B2AR 定位和信号传导的构象生物传感器。拟议的研究， 通过表征对心肌细胞功能具有重大影响的途径，将有可能发现新的 改善衰竭心脏功能并缓解心力衰竭的药物靶标。