Mechanisms Regulating B2AR Sorting to Signaling Microdomains

项目总结 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，特别是在心力衰竭期间。心力衰竭中的B2AR信号转导 不完全了解，有相互矛盾的数据表明心脏保护和有害影响。它有 近年来发现，B2AR在细胞内定位的变化是信号的主要调节因素 并导致对同一受体的不同刺激反应的差异。一旦激活，B2AR 可以通过细胞表面和细胞内体隔间的同源G蛋白Gs发出信号。这个 内体信号促进特定基因的转录，其中大部分不受B2AR刺激 质膜上的激活。B2AR在内小体的GS激活受到翻译后的严格控制 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 激动剂刺激后的蛋白激酶A(PKA)需要B2AR分选到特定的管状结构域 从那里它可以通过Gs发出信号。PKA抑制或SS345/6突变为丙氨酸残基不能 磷酸化可以阻止来自内体的B2AR信号。这些操纵也增加了 B2AR通过允许受体进入额外的块状循环小管而循环到质膜 对于野生型B2AR是不可用的，并且它不能从那里发出信号。主宰这一过程的蛋白质相互作用 监管是未知的。SS345/6的磷酸化也受到B2AR棕榈酰化的调节 半胱氨酸341取消该位点的棕榈酰化导致基础SS345/6显著增加 在没有激动剂刺激的情况下磷酸化。这项提议测试了特定蛋白质复合体的作用。 在将B2AR定位于特定的细胞膜结构域过程中，磷酸化和棕榈酰化在 这种定位，以及这种定位与心脏信号的功能相关性。我会用荧光 显微镜和实时定量聚合酶链式反应确定候选蛋白对B2AR分选的影响 HEK293细胞和原代细胞的内体微区及其信号传导 心肌细胞。我还将使用新的无偏见的邻近标记方法来识别和量化瞬变 调节蛋白与野生型、磷酸化缺陷和棕榈酰化缺陷的B2AR的相互作用。 棕榈酰化在调节这一过程中的作用将用功能遗传学和显微镜来检验。 具有构象生物传感器，可以识别B2AR定位和信号。拟议的研究， 通过表征对心肌细胞功能有重大影响的途径，可能会发现新的 改善衰竭心脏功能和缓解心力衰竭的可用药靶点。

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.